TY - JOUR A1 - Sivarajan, Rinu A1 - Oberwinkler, Heike A1 - Roll, Valeria A1 - König, Eva-Maria A1 - Steinke, Maria A1 - Bodem, Jochen T1 - A defined anthocyanin mixture sourced from bilberry and black currant inhibits Measles virus and various herpesviruses JF - BMC Complementary Medicine and Therapies N2 - Background Anthocyanin-containing plant extracts and carotenoids, such as astaxanthin, have been well-known for their antiviral and anti-inflammatory activity, respectively. We hypothesised that a mixture of Ribes nigrum L. (Grossulariaceae) (common name black currant (BC)) and Vaccinium myrtillus L. (Ericaceae) (common name bilberry (BL)) extracts (BC/BL) with standardised anthocyanin content as well as single plant extracts interfered with the replication of Measles virus and Herpesviruses in vitro. Methods We treated cell cultures with BC/BL or defined single plant extracts, purified anthocyanins and astaxanthin in different concentrations and subsequently infected the cultures with the Measles virus (wild-type or vaccine strain Edmonston), Herpesvirus 1 or 8, or murine Cytomegalovirus. Then, we analysed the number of infected cells and viral infectivity and compared the data to non-treated controls. Results The BC/BL extract inhibited wild-type Measles virus replication, syncytia formation and cell-to-cell spread. This suppression was dependent on the wild-type virus-receptor-interaction since the Measles vaccine strain was unaffected by BC/BL treatment. Furthermore, the evidence was provided that the delphinidin-3-rutinoside chloride, a component of BC/BL, and purified astaxanthin, were effective anti-Measles virus compounds. Human Herpesvirus 1 and murine Cytomegalovirus replication was inhibited by BC/BL, single bilberry or black currant extracts, and the BC/BL component delphinidin-3-glucoside chloride. Additionally, we observed that BC/BL seemed to act synergistically with aciclovir. Moreover, BC/BL, the single bilberry and black currant extracts, and the BC/BL components delphinidin-3-glucoside chloride, cyanidin-3-glucoside, delphinidin-3-rutinoside chloride, and petunidin-3-galactoside inhibited human Herpesvirus 8 replication. Conclusions Our data indicate that Measles viruses and Herpesviruses are differentially susceptible to a specific BC/BL mixture, single plant extracts, purified anthocyanins and astaxanthin. These compounds might be used in the prevention of viral diseases and in addition to direct-acting antivirals, such as aciclovir. KW - anthocyanin KW - astaxanthin KW - bilberry KW - black currant KW - herpesvirus KW - measels virus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301423 VL - 22 ER - TY - JOUR A1 - Geiger, Nina A1 - König, Eva-Maria A1 - Oberwinkler, Heike A1 - Roll, Valeria A1 - Diesendorf, Viktoria A1 - Fähr, Sofie A1 - Obernolte, Helena A1 - Sewald, Katherina A1 - Wronski, Sabine A1 - Steinke, Maria A1 - Bodem, Jochen T1 - Acetylsalicylic acid and salicylic acid inhibit SARS-CoV-2 replication in precision-cut lung slices JF - Vaccines N2 - Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds. KW - acetylsalicylic acid KW - salicylic acid KW - antiviral activity KW - aspirin KW - SARS-CoV-2 KW - precision-cut lung slices Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-289885 SN - 2076-393X VL - 10 IS - 10 ER - TY - JOUR A1 - Geiger, Nina A1 - Diesendorf, Viktoria A1 - Roll, Valeria A1 - König, Eva-Maria A1 - Obernolte, Helena A1 - Sewald, Katherina A1 - Breidenbach, Julian A1 - Pillaiyar, Thanigaimalai A1 - Gütschow, Michael A1 - Müller, Christa E. A1 - Bodem, Jochen T1 - Cell type-specific anti-viral effects of novel SARS-CoV-2 main protease inhibitors JF - International Journal of Molecular Sciences N2 - Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner. KW - SARS-CoV-2 KW - protease inhibitors KW - cell line specificity pyridyl indole carboxylates KW - azapeptide nitriles KW - peptidomimetics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304034 SN - 1422-0067 VL - 24 IS - 4 ER - TY - THES A1 - König, Eva-Maria T1 - Pathogenese von Kraniosynostosen T1 - Pathogenesis of Craniosynostoses N2 - Das humane Schädeldach besteht aus fünf Schädelplatten, die durch intramembranöse Ossifikation entstehen. Wenn diese in der Embryonalentwicklung aufeinandertreffen, bilden sich Schädelnähte aus, die eine Fusion der Schädelplatten verhindern und damit ein Schädelwachstum parallel zu Gehirnentwicklung ermöglichen. Für diesen Prozess ist eine Balance aus Zellproliferation und Differenzierung nötig, deren Aufrechterhaltung wiederum durch eine komplexe Regulation von verschiedenen Signalwegen gewährleistet wird. Störungen in diesem regulatorischen System können zu einer vorzeitigen Fusion der Schädelplatten, Kraniosynostose genannt, führen. Die Kraniosynostose ist eine der häufigsten kraniofazialen Fehlbildungen beim Menschen. Durch kompensatorisches Wachstum an den nicht fusionierten Suturen entstehen charakteristische Schädeldeformationen, die sekundär einen erhöhten intrakranialen Druck zur Folge haben können. Eine vorzeitige Fusion der Suturen kann sowohl isoliert als auch syndromal zusammen mit weiteren klinischen Auffälligkeiten vorliegen. Bisher sind über 150 verschiedene Kraniosynostose Syndrome beschrieben und insgesamt 25-30% aller Kraniosynostose Patienten sind von einer syndromalen Form betroffen. Da die klinischen Merkmale der Kraniosynostose Syndrome variabel sind und zum Teil überlappen, ist eine klare klinische Diagnose häufig erschwert. Sowohl Umwelteinflüsse als auch genetische Veränderungen können die Ursache für Kraniosynostosen sein. Vor allem bei syndromalen Kraniosynostosen wurden genetische Veränderungen, wie beispielsweise Mutationen in den Genen FGFR2, FGFR3, TWIST1 und EFNB1, identifiziert. Darüber hinaus wurden chromosomale Veränderungen wie partielle Monosomien von 7p, 9p oder 11p sowie partielle Trisomien von 5q, 13q oder 15q mit Kraniosynostose assoziiert. Trotzdem ist in über 50% der Fälle die genetische Ursache unbekannt und die Pathogenese von Kraniosynostosen noch nicht vollständig geklärt. Ziel dieser Arbeit war es neue genetische Ursachen bei Kraniosynostose Patienten zu identifizieren und so zur Aufklärung der Pathogenese beizutragen. Es wurde die genomische DNA von 83 Patienten molekulargenetisch durch Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) oder durch ein speziell entworfenes Next Generation Sequencing (NGS) Genpanel untersucht. Bei 30% der Patienten konnte eine potentiell pathogene Veränderung identifiziert werden. Davon waren 23% chromosomale Aberrationen wie unbalancierte Translokationen, isolierte interstitielle Verluste und ein Zugewinn an genomischen Material. Bei zwei Patienten wurden unbalancierte Translokationen mit partieller 5q Trisomie nachgewiesen. Das Gen MSX2 liegt innerhalb des duplizierten Bereichs, sodass möglicherweise eine MSX2 Überexpression vorliegt. Für ein normales Schädelwachstum ist jedoch die richtige Menge an MSX2 kritisch. Des Weiteren wurde eine partielle Deletion von TCF12 detektiert, die in einer Haploinsuffizienz von TCF12 resultiert. TCF12 Mutationen sind mit Koronarnahtsynosten assoziiert. In einem anderen Fall lag das Gen FGF10 innerhalb der duplizierten 5p15.1-p12 Region. Das Gen kodiert für einen Liganden des FGF Signalwegs und wurde bisher noch nicht mit Kraniosynostose assoziiert. Aufgrund dessen wurden Analysen im Tiermodell Danio rerio durchgeführt. Eine simulierte Überexpression durch Injektion der fgf10a mRNA in das 1-Zell Stadium führte zu schweren Gehirn-, Herz- und Augendefekten. Mittels NGS wurden 77% der potentiell pathogenen genetischen Veränderungen identifiziert. Hierfür wurde in dieser Arbeit ein Genpanel erstellt, das 68 Gene umfasst. Es wurden sowohl bekannte Kraniosynostose- als auch Kandidaten-Gene sowie Gene, die mit der Ossifikation assoziiert sind, in die Analyse eingeschlossen. Das Genpanel wurde durch die Sequenzierung von fünf Kontrollproben mit bekannten Mutationen erfolgreich validiert. Anschließend wurde die genomische DNA von 66 Patienten analysiert. Es konnten 20 (potentiell) pathogene Varianten identifiziert werden. Neben bereits bekannten Mutationen in den Genen FGFR1, FGFR2, FGFR3 und TWIST1, konnten zusätzlich 8 neue, potentiell pathogene Varianten in den Genen ERF, MEGF8, MSX2, PTCH1 und TCF12 identifiziert werden. Die Ergebnisse dieser Arbeit tragen dazu bei das Mutationsspektrum dieser Gene zu erweitern. Bei zwei der Varianten handelte es sich um potentielle Spleißvarianten. Für diese konnte in einem in vitro Spleißsystem gezeigt werden, dass sie eine Änderung des Spleißmusters bewirken. Der Nachweis von zwei seltenen Varianten in den Genen FGFR2 und HUWE1 hat außerdem dazu beigetragen die Pathogenität dieser spezifischen Varianten zu bekräftigen. Eine Variante in POR, die aufgrund bioinformatischer Analysen als potentiell pathogen bewertet wurde, wurde nach der Segregationsanalyse als wahrscheinlich benigne eingestuft. Zusammenfassend konnten bei etwa einem Drittel der Patienten, die mit dem NGS Genpanel analysiert wurden, eine genetische Ursache identifiziert werden. Dieses Genpanel stellt somit ein effizientes diagnostisches Tool dar, das zukünftig in der genetischen Routine-Diagnostik von Kraniosynostose-Patienten eingesetzt werden kann. Die Ergebnisse dieser Arbeit zeigen, dass sowohl eine Untersuchung auf CNVs als auch auf Sequenzänderungen bei Kraniosynostose Patienten sinnvoll ist. N2 - Cranial bones are formed by intramembranous ossification. During development, the cranial bones are separated by fibrous sutures, which function as bone growth sites and therefore, the cranial sutures need to remain patent to allow the expansion of the skull during brain development. Thus, there must be a balance of cell proliferation and differentiation within the suture. This complex process requires a tight regulation of gene expression and interacting signal pathways. Imbalances or dysfunction of the involved factors can result in abnormal skull growth. One of the most common congenital craniofacial disorders by affecting approximately one in 2500 newborns is craniosynostosis. It is defined as the premature ossification of one or more calvarial sutures. Compensatory growth of the skull leads to a characteristic dysmorphic cranial vault and facial asymmetry. Premature ossification of the cranial sutures can occur either as isolated malformation or as part of a syndrome. Isolated craniosynostoses are more frequent, nevertheless, 25-30% of all cases are syndromic craniosynostoses with more than 150 syndromes reported. There is a high intra- and interfamilial variability and clinical overlap of the different syndromes. Environmental influences as well as genetic defects like mutations and chromosomal aberrations are known to cause craniosynostosis. So far genetic causes have been identified mainly for syndromic craniosynostoses, i.e. mutations in FGFR2, FGFR3, TWIST1, and EFNB1. Furthermore, chromosomal rearrangements like i.e. partial monosomy of 7p, 9p, and 11p as well as partial trisomy of 5q, 13q, and 15q, have been reported in 11-15% of the syndromic craniosynostosis cases. However, in more than 50% of the cases the underlying genetic cause remains unknown. Furthermore, the pathogenesis of craniosynostoses is still not fully understood. In this project 83 craniosynostosis patients were analysed either by microarray-based comparative genomic hybridisation (array-CGH) or gene panel based next generation sequencing (NGS) to further investigate the pathogenesis of craniosynostosis. In a total of 30% of the patients a potential genetic cause was identified. Among those 23% had chromosomal rearrangements which are likely to cause the observed phenotypes, i.e. unbalanced translocations affecting several genes as well as interstitial deletions and an isolated duplication have been detected. Two patients had unbalanced translocations with partial 5q trisomies encompassing MSX2. MSX2 gene dosage is critical for normal growth of the cranial bone plates as loss-of-function mutations lead to delayed and incomplete ossification of the parietal bones. Furthermore, we identified a partial TCF12 deletion which is likely to result in TCF12 haploinsufficiency. TCF12 mutations frequently lead to premature fusion of the coronal sutures, although its pathogenesis is still not fully understood. In another case isolated duplication of 5p15.1-p12 includes FGF10 which is a known ligand of the FGF signalling pathway. So far, no association of FGF10 with craniosynostosis has been made. To investigate its potential role during development and in the pathogenesis of craniosynostosis functional experiments were performed in Danio rerio, an animal model for craniosynostosis. Simulation of fgf10a overexpression by injection of fgf10a RNA at 1-cell stage resulted in severe anomalies of the brain, heart and eyes. In addition, 77% of the identified genetic causes were detected by NGS. For this study a gene panel was designed comprising 68 genes of known and candidate craniosynostosis genes as well as genes associated with bone development. Performance of the NGS gene panel was validated by sequencing five control patients with known mutations. Subsequently, genomic DNA of 66 patients was analysed by the designed craniosynostosis panel. 20 (potential) pathogenic variants were detected. Although, in most of the cases hot spot sequencing of one or more common craniosynostosis genes was performed prior to including the patients in the study, we determined 9 known mutations in the genes FGFR1, FGFR2, FGFR3, and TWIST1. In addition, 8 novel, potentially disease-causing variants in the genes ERF, MEGF8, MSX2, PTCH1, and TCF12 were identified. This work contributed to extend the mutational spectrum within those genes. Two of those variants were predicted to affect splice sites. Analysis by an in vitro splice assay revealed that those variants result in aberrant splicing. Furthermore, the detection of two rare variants of FGFR2 and HUWE1 adds support to their pathogenicity. An additional variant within POR had to be classified as likely benign after segregation analysis. Overall, in nearly one third of the analysed cases an underlying genetic cause could be identified by the designed gene panel. Thus, the NGS panel presents as an efficient tool for genetic diagnostics of craniosynostoses. The data of this work clearly show both copy number variant and single nucleotide variant analysis should be considered in genetic diagnostics of craniosynostosis patients. KW - Kraniosynostose KW - Genetik KW - Next Generation Sequencing (NGS) KW - Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175181 ER - TY - JOUR A1 - Geiger, Nina A1 - Kersting, Louise A1 - Schlegel, Jan A1 - Stelz, Linda A1 - Fähr, Sofie A1 - Diesendorf, Viktoria A1 - Roll, Valeria A1 - Sostmann, Marie A1 - König, Eva-Maria A1 - Reinhard, Sebastian A1 - Brenner, Daniela A1 - Schneider-Schaulies, Sibylle A1 - Sauer, Markus A1 - Seibel, Jürgen A1 - Bodem, Jochen T1 - The acid ceramidase is a SARS-CoV-2 host factor JF - Cells N2 - SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor. KW - SARS-CoV-2 KW - ceramides KW - ceramidase KW - fluoxetine KW - acid sphingomyelinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286105 SN - 2073-4409 VL - 11 IS - 16 ER -