TY - JOUR A1 - Nagy, Magdolna A1 - van Geffen, Johanna P. A1 - Stegner, David A1 - Adams, David J. A1 - Braun, Attila A1 - de Witt, Susanne M. A1 - Elvers, Margitta A1 - Geer, Mitchell J. A1 - Kuijpers, Marijke J. E. A1 - Kunzelmann, Karl A1 - Mori, Jun A1 - Oury, Cécile A1 - Pircher, Joachim A1 - Pleines, Irina A1 - Poole, Alastair W. A1 - Senis, Yotis A. A1 - Verdoold, Remco A1 - Weber, Christian A1 - Nieswandt, Bernhard A1 - Heemskerk, Johan W. M. A1 - Baaten, Constance C. F. M. J. T1 - Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis JF - Frontiers in Cardiovascular Medicine N2 - Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis. KW - arterial thrombus formation KW - bleeding KW - collagen KW - glycoprotein VI KW - platelets KW - microfluidics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232194 VL - 6 ER - TY - JOUR A1 - Nguyen, Minh-Thu A1 - Saising, Jongkon A1 - Tribelli, Paula Maria A1 - Nega, Mulugeta A1 - Diene, Seydina M. A1 - François, Patrice A1 - Schrenzel, Jacques A1 - Spröer, Cathrin A1 - Bunk, Boyke A1 - Ebner, Patrick A1 - Hertlein, Tobias A1 - Kumari, Nimerta A1 - Härtner, Thomas A1 - Wistuba, Dorothee A1 - Voravuthikunchai, Supayang P. A1 - Mäder, Ulrike A1 - Ohlsen, Knut A1 - Götz, Friedrich T1 - Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus JF - Frontiers in Microbiology N2 - Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent. KW - antibiotic KW - Gram-positive bacteria KW - rhodomyrtone KW - Staphylococcus KW - membrane active Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224117 VL - 10 ER - TY - JOUR A1 - Lang, Isabell A1 - Füllsack, Simone A1 - Wajant, Harald T1 - Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies JF - Frontiers in Immunology N2 - Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF. KW - binding studies KW - Gaussia princeps luciferase fusion protein KW - progranulin KW - tumor necrosis factor KW - tumor necrosis factor receptor-1 KW - tumor necrosis factor receptor-2 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236373 VL - 9 ER - TY - JOUR A1 - Klotz, Peter A1 - Higgins, Paul G. A1 - Schaubmar, Andreas R. A1 - Failing, Klaus A1 - Leidner, Ursula A1 - Seifert, Harald A1 - Scheufen, Sandra A1 - Semmler, Torsten A1 - Ewers, Christa T1 - Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany JF - Frontiers in Microbiology N2 - Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans. KW - ESKAPE KW - Acinetobacter baumannii KW - antimicrobial susceptibility KW - MLST KW - cattle KW - epidemiology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325927 VL - 10 ER - TY - JOUR A1 - Kervarrec, Thibault A1 - Samimi, Mahtab A1 - Guyétant, Serge A1 - Sarma, Bhavishya A1 - Chéret, Jérémy A1 - Blanchard, Emmanuelle A1 - Berthon, Patricia A1 - Schrama, David A1 - Houben, Roland A1 - Touzé, Antoine T1 - Histogenesis of Merkel Cell Carcinoma: A Comprehensive Review JF - Frontiers in Oncology N2 - Merkel cell carcinoma (MCC) is a primary neuroendocrine carcinoma of the skin. This neoplasia features aggressive behavior, resulting in a 5-year overall survival rate of 40%. In 2008, Feng et al. identified Merkel cell polyomavirus (MCPyV) integration into the host genome as the main event leading to MCC oncogenesis. However, despite identification of this crucial viral oncogenic trigger, the nature of the cell in which MCC oncogenesis occurs is actually unknown. In fact, several hypotheses have been proposed. Despite the large similarity in phenotype features between MCC tumor cells and physiological Merkel cells (MCs), a specialized subpopulation of the epidermis acting as mechanoreceptor of the skin, several points argue against the hypothesis that MCC derives directly from MCs. Alternatively, MCPyV integration could occur in another cell type and induce acquisition of an MC-like phenotype. Accordingly, an epithelial as well as a fibroblastic or B-cell origin of MCC has been proposed mainly based on phenotype similarities shared by MCC and these potential ancestries. The aim of this present review is to provide a comprehensive review of the current knowledge of the histogenesis of MCC. KW - merkel cell polyomavirus (MCPyV) KW - epithelial KW - fibroblast KW - B cell KW - Merkel cell carcinoma (MCC) KW - histogenesis KW - origin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325733 VL - 9 ER - TY - JOUR A1 - Kasaragod, Vikram B. A1 - Schindelin, Hermann T1 - Structure–Function Relationships of Glycine and GABAA Receptors and Their Interplay With the Scaffolding Protein Gephyrin JF - Frontiers in Molecular Neuroscience N2 - Glycine and γ-aminobutyric acid (GABA) are the major determinants of inhibition in the central nervous system (CNS). These neurotransmitters target glycine and GABAA receptors, respectively, which both belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGICs). Interactions of the neurotransmitters with the cognate receptors result in receptor opening and a subsequent influx of chloride ions, which, in turn, leads to hyperpolarization of the membrane potential, thus counteracting excitatory stimuli. The majority of glycine receptors and a significant fraction of GABAA receptors (GABAARs) are recruited and anchored to the post-synaptic membrane by the central scaffolding protein gephyrin. This ∼93 kDa moonlighting protein is structurally organized into an N-terminal G-domain (GephG) connected to a C-terminal E-domain (GephE) via a long unstructured linker. Both inhibitory neurotransmitter receptors interact via a short peptide motif located in the large cytoplasmic loop located in between transmembrane helices 3 and 4 (TM3-TM4) of the receptors with a universal receptor-binding epitope residing in GephE. Gephyrin engages in nearly identical interactions with the receptors at the N-terminal end of the peptide motif, and receptor-specific interaction toward the C-terminal region of the peptide. In addition to its receptor-anchoring function, gephyrin also interacts with a rather large collection of macromolecules including different cytoskeletal elements, thus acting as central scaffold at inhibitory post-synaptic specializations. Dysfunctions in receptor-mediated or gephyrin-mediated neurotransmission have been identified in various severe neurodevelopmental disorders. Although biochemical, cellular and electrophysiological studies have helped to understand the physiological and pharmacological roles of the receptors, recent high resolution structures of the receptors have strengthened our understanding of the receptors and their gating mechanisms. Besides that, multiple crystal structures of GephE in complex with receptor-derived peptides have shed light into receptor clustering by gephyrin at inhibitory post-synapses. This review will highlight recent biochemical and structural insights into gephyrin and the GlyRs as well as GABAA receptors, which provide a deeper understanding of the molecular machinery mediating inhibitory neurotransmission. KW - glycine receptors KW - GABAA receptors KW - gephyrin KW - moonlighting protein KW - inhibitory post-synaptic specialization KW - cytoskeletal proteins Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325607 VL - 11 ER - TY - JOUR A1 - John, Cathy N. A1 - Abrantes, Pedro M. D. S. A1 - Prusty, Bhupesh K. A1 - Ablashi, Dharam V. A1 - Africa, Charlene W. J. T1 - K21 Compound, a Potent Antifungal Agent: Implications for the Treatment of Fluconazole-Resistant HIV-Associated Candida Species JF - Frontiers in Microbiology N2 - Background/Objectives: With mucocutaneous candidiasis being highly prevalent in HIV patients, the emergence of fluconazole-resistant Candida species forms a major challenge in treating and eradicating these infections. The objective of this study was to establish the antifungal activity of K21, a membrane-rupturing antimicrobial compound derived from a silica quaternary ammonium compound (SiQAC) with tetraethoxysilane (TEOS). Methods: The study sample included 81 Candida species of which 9 were type strains and 72 were clinical isolates. Minimum inhibitory concentrations, synergy, fractional inhibitory concentration index (FICI), and time kill assays were determined by broth microdilution. Electron microscopy (EM) was used to determine the qualitative changes brought about after treatment with K21. Results: K21 inhibited the growth of all fluconazole-resistant and susceptible Candida strains with only 2 h of exposure required to effectively kill 99.9% of the inoculum, and a definite synergistic effect was observed with a combination of K21 and fluconazole. EM demonstrated the presence of two forms of extracellular vesicles indicative of biofilm formation and cell lysis. Conclusion: The study established the efficacy of K21 as an antifungal agent and with fluconazole-resistant candidiasis on the increase, the development of K21 can provide a promising alternative to combat acquired drug resistance. KW - HIV-associated candidiasis KW - antimicrobial compounds KW - Candida KW - fluconazole KW - antifungal susceptibility KW - broth microdilution Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323505 VL - 10 ER - TY - JOUR A1 - Gryszel, Maciej A1 - Schlossarek, Tim A1 - Würthner, Frank A1 - Natali, Mirco A1 - Głowacki, Eric Daniel T1 - Water‐soluble cationic perylene diimide dyes as stable photocatalysts for H\(_2\)O\(_2\) evolution JF - ChemPhotoChem N2 - Photocatalytic generation of hydrogen peroxide, H\(_2\)O\(_2\), has gained increasing attention in recent years, with applications ranging from solar energy conversion to biophysical research. While semiconducting solid‐state materials are normally regarded as the workhorse for photogeneration of H\(_2\)O\(_2\), an intriguing alternative for on‐demand H\(_2\)O\(_2\) is the use of photocatalytic organic dyes. Herein we report the use of water‐soluble dyes based on perylene diimide molecules which behave as true molecular catalysts for the light‐induced conversion of dissolved oxygen to hydrogen peroxide. In particular, we address how to obtain visible‐light photocatalysts which are stable with respect to aggregation and photochemical degradation. We report on the factors affecting efficiency and stability, including variable electron donors, oxygen partial pressure, pH, and molecular catalyst structure. The result is a perylene diimide derivative with unprecedented peroxide evolution performance using a broad range of organic donor molecules and operating in a wide pH range. KW - hydrogen peroxide KW - oxygen reduction reaction KW - perylene KW - photocatalysis KW - dyes/pigments Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-370250 SN - 2367-0932 VL - 7 IS - 9 ER - TY - JOUR A1 - Jarick, Katja J. A1 - Mokhtari, Zeinab A1 - Scheller, Lukas A1 - Hartweg, Julia A1 - Thusek, Sina A1 - Le, Duc-Dung A1 - Ranecky, Maria A1 - Shaikh, Haroon A1 - Qureischi, Musga A1 - Heinze, Katrin G. A1 - Beilhack, Andreas T1 - Photoconversion of Alloreactive T Cells in Murine Peyer’s Patches During Acute Graft-Versus-Host Disease: Tracking the Homing Route of Highly Proliferative Cells In Vivo JF - Frontiers in Immunology N2 - The regulation of immune cell migration throughout the body is essential to warrant immunosurveillance and to maintain immune homeostasis. Marking and tracking of these cells has proven important to study mechanisms of immune cell trafficking and cell interaction in vivo. Photoconversion is a well-suited technique for intravital application because it enables contactless time- and location-specific marking of cells in the tissue without surgically manipulating the microenvironment of the cells in question. However, in dividing cells the converted fluorescent protein may decline quickly. Here, we provide a detailed description of the photoconversion technique and its applicability to tracking highly proliferating T cells from the priming site of T cell activation to peripheral target organs of effector function in a preclinical model. Dendra2+ T cells were photoconverted in the Peyer’s patches during the initiation phase of acute graft-versus-host disease (GvHD) and tracked through the mesenteric lymph nodes and the peripheral blood to the small intestine with flow cytometry and intravital two-photon microscopy. Photoconverted alloreactive T cells preserved the full proliferative capacity, homing, and migration of alloreactive T cells in the intestinal lamina propria. We conclusively proved that photoconversion of highly proliferative alloreactive T cells in the Peyer’s patches is an effective tool to study trafficking of alloreactive T cells under physiologic conditions and to GvHD target tissues. This technique can also be applied to the study of immune cell tracking under inflammatory and non-inflammatory conditions. KW - T cell migration KW - acute graft-versus-host disease KW - mouse models KW - photoconversion KW - Dendra2 KW - Peyer's patch KW - in vivo cell tracking KW - lymphocyte homing Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323309 VL - 9 ER - TY - JOUR A1 - Herster, Franziska A1 - Bittner, Zsofia A1 - Codrea, Marius Cosmin A1 - Archer, Nathan K. A1 - Heister, Martin A1 - Löffler, Markus W. A1 - Heumos, Simon A1 - Wegner, Joanna A1 - Businger, Ramona A1 - Schindler, Michael A1 - Stegner, David A1 - Schäkel, Knut A1 - Grabbe, Stephan A1 - Ghoreschi, Kamran A1 - Miller, Lloyd S. A1 - Weber, Alexander N. R. T1 - Platelets Aggregate With Neutrophils and Promote Skin Pathology in Psoriasis JF - Frontiers in Immunology N2 - Psoriasis is a frequent systemic inflammatory autoimmune disease characterized primarily by skin lesions with massive infiltration of leukocytes, but frequently also presents with cardiovascular comorbidities. Especially polymorphonuclear neutrophils (PMNs) abundantly infiltrate psoriatic skin but the cues that prompt PMNs to home to the skin are not well-defined. To identify PMN surface receptors that may explain PMN skin homing in psoriasis patients, we screened 332 surface antigens on primary human blood PMNs from healthy donors and psoriasis patients. We identified platelet surface antigens as a defining feature of psoriasis PMNs, due to a significantly increased aggregation of neutrophils and platelets in the blood of psoriasis patients. Similarly, in the imiquimod-induced experimental in vivo mouse model of psoriasis, disease induction promoted PMN-platelet aggregate formation. In psoriasis patients, disease incidence directly correlated with blood platelet counts and platelets were detected in direct contact with PMNs in psoriatic but not healthy skin. Importantly, depletion of circulating platelets in mice in vivo ameliorated disease severity significantly, indicating that both PMNs and platelets may be relevant for psoriasis pathology and disease severity. KW - psoriasis KW - neutrophil KW - platelet KW - platelet-neutrophil complexes (PNCs) KW - imiquimod Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320175 VL - 10 ER -