TY - GEN A1 - Neuschäfer-Rube, Frank A1 - Pathe-Neuschäfer-Rube, Andrea A1 - Püschel, Gerhard Paul T1 - Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1257 KW - cell-based assay KW - genetically modified BoNT KW - BoNT/B uptake Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-558032 SN - 1866-8372 SP - 1 EP - 11 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - GEN A1 - Michaud Schjeide, Brit-Maren A1 - Schenke, Maren A1 - Seeger, Bettina A1 - Püschel, Gerhard Paul T1 - Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1269 KW - CRISPR editing validation KW - copy number analyses KW - homology-directed repair KW - homologous recombination deficiency Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-561755 SN - 1866-8372 SP - 1 EP - 14 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - GEN A1 - Schäfer, Marjänn Helena A1 - Kakularam, Kumar Reddy A1 - Reisch, Florian A1 - Rothe, Michael A1 - Stehling, Sabine A1 - Heydeck, Dagmar A1 - Püschel, Gerhard Paul A1 - Kuhn, Hartmut T1 - Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1295 KW - eicosanoids KW - lipid peroxidation KW - oxidative stress KW - polyenoic fatty acids KW - erythropoiesis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-576491 SN - 1866-8372 IS - 1295 ER - TY - GEN A1 - Pathe-Neuschäfer-Rube, Andrea A1 - Neuschäfer-Rube, Frank A1 - Püschel, Gerhard Paul T1 - Cell-based reporter release assay to determine the activity of calcium-dependent neurotoxins and neuroactive pharmaceuticals T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1139 KW - cell-based assay KW - neurotoxins KW - muscarinic acetylcholine receptor KW - voltage-dependent calcium channels KW - VGCC Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-503225 SN - 1866-8372 IS - 1139 ER - TY - GEN A1 - Schenke, Maren A1 - Schjeide, Brit-Maren A1 - Püschel, Gerhard Paul A1 - Seeger, Bettina T1 - Analysis of motor neurons differentiated from human induced pluripotent stem cells for the use in cell-based Botulinum neurotoxin activity assays T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1083 KW - Botulinum neurotoxin KW - motor neurons KW - cell-based in vitro assay KW - potency assessment KW - induced pluripotent stem cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-472071 SN - 1866-8372 IS - 1083 ER - TY - GEN A1 - Henkel, Janin A1 - Buchheim-Dieckow, Katja A1 - Castro, José Pedro A1 - Laeger, Thomas A1 - Wardelmann, Kristina A1 - Kleinridders, André A1 - Jöhrens, Korinna A1 - Püschel, Gerhard Paul T1 - Reduced Oxidative Stress and Enhanced FGF21 Formation in Livers of Endurance-Exercised Rats with Diet-Induced NASH T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Non-alcoholic fatty liver diseases (NAFLD) including the severe form with steatohepatitis (NASH) are highly prevalent ailments to which no approved pharmacological treatment exists. Dietary intervention aiming at 10% weight reduction is efficient but fails due to low compliance. Increase in physical activity is an alternative that improved NAFLD even in the absence of weight reduction. The underlying mechanisms are unclear and cannot be studied in humans. Here, a rat NAFLD model was developed that reproduces many facets of the diet-induced NAFLD in humans. The impact of endurance exercise was studied in this model. Male Wistar rats received control chow or a NASH-inducing diet rich in fat, cholesterol, and fructose. Both diet groups were subdivided into a sedentary and an endurance exercise group. Animals receiving the NASH-inducing diet gained more body weight, got glucose intolerant and developed a liver pathology with steatosis, hepatocyte hypertrophy, inflammation and fibrosis typical of NAFLD or NASH. Contrary to expectations, endurance exercise did not improve the NASH activity score and even enhanced hepatic inflammation. However, endurance exercise attenuated the hepatic cholesterol overload and the ensuing severe oxidative stress. In addition, exercise improved glucose tolerance possibly in part by induction of hepatic FGF21 production. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 807 KW - NAFLD KW - NASH KW - endurance exercise KW - FGF21 KW - glucose intolerance KW - cholesterol KW - oxidative stress Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-442384 SN - 1866-8372 IS - 807 ER - TY - GEN A1 - Pathe-Neuschäfer-Rube, Andrea A1 - Neuschäfer-Rube, Frank A1 - Haas, Gerald A1 - Langoth-Fehringer, Nina A1 - Püschel, Gerhard Paul T1 - Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins T2 - Toxins N2 - Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 473 KW - botulinum toxin KW - BoNT KW - tetanus toxin KW - RRR KW - replacement Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-418141 ER - TY - GEN A1 - Henkel, Janin A1 - Coleman Mac Gregor of Inneregny, Charles Dominic A1 - Schraplau, Anne A1 - Jöhrens, Korinna A1 - Weiss, Thomas Siegfried A1 - Jonas, Wenke A1 - Schürmann, Annette A1 - Püschel, Gerhard Paul T1 - Augmented liver inflammation in a microsomal prostaglandin E synthase 1 (mPGES-1)-deficient diet-induced mouse NASH model T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - In a subset of patients, non-alcoholic fatty liver disease (NAFLD) is complicated by cell death and inflammation resulting in non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and subsequent organ failure. Apart from cytokines, prostaglandins, in particular prostaglandin E-2 (PGE(2)), play a pivotal role during inflammatory processes. Expression of the key enzymes of PGE(2) synthesis, cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), was increased in human NASH livers in comparison to controls and correlated with the NASH activity score. Both enzymes were also induced in NASH-diet-fed wild-type mice, resulting in an increase in hepatic PGE(2) concentration that was completely abrogated in mPGES-1-deficient mice. PGE(2) is known to inhibit TNF-alpha synthesis in macrophages. A strong infiltration of monocyte-derived macrophages was observed in NASH-diet-fed mice, which was accompanied with an increase in hepatic TNF-alpha expression. Due to the impaired PGE(2) production, TNF-alpha expression increased much more in livers of mPGES-1-deficient mice or in the peritoneal macrophages of these mice. The increased levels of TNF-alpha resulted in an enhanced IL-1 beta production, primarily in hepatocytes, and augmented hepatocyte apoptosis. In conclusion, attenuation of PGE(2) production by mPGES-1 ablation enhanced the TNF-alpha-triggered inflammatory response and hepatocyte apoptosis in diet-induced NASH. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 483 KW - suppress VLDL secretion KW - mice lacking KW - nonalcoholic steatohepatthis KW - insulin-resistance KW - rat hepatocytes KW - kupffer cells KW - E-2 KW - disease KW - expression KW - accumulation Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-420879 SN - 1866-8372 IS - 483 ER - TY - GEN A1 - Henkel, Janin A1 - Alfine, Eugenia A1 - Saín, Juliana A1 - Jöhrens, Korinna A1 - Weber, Daniela A1 - Castro, José Pedro A1 - König, Jeannette A1 - Stuhlmann, Christin A1 - Vahrenbrink, Madita A1 - Jonas, Wenke A1 - Kleinridders, André A1 - Püschel, Gerhard Paul T1 - Soybean Oil-Derived Poly-Unsaturated Fatty Acids Enhance Liver Damage in NAFLD Induced by Dietary Cholesterol T2 - Nutrients N2 - While the impact of dietary cholesterol on the progression of atherosclerosis has probably been overestimated, increasing evidence suggests that dietary cholesterol might favor the transition from blunt steatosis to non-alcoholic steatohepatitis (NASH), especially in combination with high fat diets. It is poorly understood how cholesterol alone or in combination with other dietary lipid components contributes to the development of lipotoxicity. The current study demonstrated that liver damage caused by dietary cholesterol in mice was strongly enhanced by a high fat diet containing soybean oil-derived ω6-poly-unsaturated fatty acids (ω6-PUFA), but not by a lard-based high fat diet containing mainly saturated fatty acids. In contrast to the lard-based diet the soybean oil-based diet augmented cholesterol accumulation in hepatocytes, presumably by impairing cholesterol-eliminating pathways. The soybean oil-based diet enhanced cholesterol-induced mitochondrial damage and amplified the ensuing oxidative stress, probably by peroxidation of poly-unsaturated fatty acids. This resulted in hepatocyte death, recruitment of inflammatory cells, and fibrosis, and caused a transition from steatosis to NASH, doubling the NASH activity score. Thus, the recommendation to reduce cholesterol intake, in particular in diets rich in ω6-PUFA, although not necessary to reduce the risk of atherosclerosis, might be sensible for patients suffering from non-alcoholic fatty liver disease. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 479 KW - non-alcoholic fatty liver disease (NAFLD) KW - NASH KW - cholesterol KW - PUFA KW - inflammation KW - oxidative stress Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-419773 ER - TY - GEN A1 - Camargo, Rodolfo Gonzalez A1 - Riccardi, Daniela Mendes dos Reis A1 - Ribeiro, Henrique Quintas Teixeira A1 - Carnevali Junior, Luiz Carlos A1 - Matos-Neto, Emidio Marques de A1 - Enjiu, Lucas A1 - Neves, Rodrigo Xavier A1 - Lima, Joanna Darck Carola Correia A1 - Figuerêdo, Raquel Galvão A1 - Alcântara, Paulo Sérgio Martins de A1 - Maximiano, Linda A1 - Otoch, José A1 - Batista Jr., Miguel Luiz A1 - Püschel, Gerhard Paul A1 - Seelaender, Marilia T1 - NF-kappa Bp65 and expression of its pro-inflammatory target genes are upregulated in the subcutaneous adipose tissue of cachectic cancer patients N2 - Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB). We have examined the gene expression of the subunits NF-κBp65 and NF-κBp50, as well as NF-κBp65 and NF-κBp50 binding, the gene expression of pro-inflammatory mediators under NF-κB control (IL-1β, IL-6, INF-γ, TNF-α, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-κBp65 and its target genes expression (TNF-α, IL-1β, MCP-1 and IκB-α) were significantly higher in cachectic cancer patients. Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-κB pathway plays a role in the promotion of WAT inflammation during cachexia. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 349 KW - cancer cachexia KW - inflammation KW - white adipose tissue KW - NF-κB KW - IκB Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-400163 ER -