المؤلفون: María Arroyo, Florian D. Hastert, Andreas Zhadan, Florian Schelter, Susanne Zimbelmann, Cathia Rausch, Anne K. Ludwig, Thomas Carell, M. Cristina Cardoso

المصدر: Nature Communications, Vol 13, Iss 1, Pp 1-28 (2022)

مصطلحات موضوعية: Science

الوصف: A short isoform of the Tet1 enzyme (Tet1s) that oxidizes the DNA 5-methylcytosine (5mC) mark is overexpressed in tumors. Here the authors show Tet1s, but not full length Tet1, changes localization over the cell cycle upon ubiquitination and Uhrf1 interaction and is targeted to heterochromatin during S-phase. This leads to 5mC oxidation and loss of DNA methylation in heterochromatin.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2041-1723Test

الوصول الحر: https://doaj.org/article/4bd2bce040994572835707652c0ebd76Test

المؤلفون: María Arroyo, Florian D. Hastert, Andreas Zhadan, Florian Schelter, Susanne Zimbelmann, Cathia Rausch, Anne K. Ludwig, Thomas Carell, M. Cristina Cardoso

المصدر: Nature Communications, Vol 14, Iss 1, Pp 1-2 (2023)

مصطلحات موضوعية: Science

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2041-1723Test

الوصول الحر: https://doaj.org/article/f3d86993de65494b9a523388754a4850Test

المؤلفون: Christin Schmidt, Florian D. Hastert, Julia Gerbeth, Tim Beissert, Ugur Sahin, Mario Perkovic, Barbara S. Schnierle

المصدر: Vaccines, Vol 10, Iss 9, p 1374 (2022)

مصطلحات موضوعية: chikungunya virus, Ross River virus, alphavirus, RNA vaccine, replicon, taRNA, Medicine

الوصف: Alphaviruses such as the human pathogenic chikungunya virus (CHIKV) and Ross River virus (RRV) can cause explosive outbreaks raising public health concerns. However, no vaccine or specific antiviral treatment is yet available. We recently established a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). This novel system consists of a replicase-encoding mRNA and a trans-replicon (TR) RNA encoding the antigen. The TR-RNA is amplified by the replicase in situ. We were interested in determining whether multiple TR-RNAs can be amplified in parallel and if, thus, a multivalent vaccine candidate can be generated. In vitro, we observed an efficient amplification of two TR-RNAs, encoding for the CHIKV and the RRV envelope proteins, by the replicase, which resulted in a high antigen expression. Vaccination of BALB/c mice with the two TR-RNAs induced CHIKV- and RRV-specific humoral and cellular immune responses. However, antibody titers and neutralization capacity were higher after immunization with a single TR-RNA. In contrast, alphavirus-specific T cell responses were equally potent after the bivalent vaccination. These data show the proof-of-principle that the taRNA system can be used to generate multivalent vaccines; however, further optimizations will be needed for clinical application.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2076-393X/10/9/1374Test; https://doaj.org/toc/2076-393XTest

الوصول الحر: https://doaj.org/article/1248d33fb49a4a7cb963048bb93ccafeTest

المؤلفون: Florian D. Hastert, Sascha Hein, Christine von Rhein, Nuka Ivalu Benz, Younes Husria, Doris Oberle, Thorsten J. Maier, Eberhard Hildt, Barbara S. Schnierle

المصدر: Vaccines, Vol 10, Iss 5, p 794 (2022)

مصطلحات موضوعية: SARS-CoV-2, Delta, Omicron, vaccine, neutralization, Medicine

الوصف: The SARS-CoV-2 variant Omicron has spread world-wide and is responsible for rapid increases in infections, including in populations with high vaccination rates. Here, we analysed in the sera of vaccinated individuals the antibody binding to the receptor-binding domain (RBD) of the spike protein and the neutralization of wild-type (WT), Delta (B.1.617.2), and Omicron (B.1.1.529; BA.1) pseudotyped vectors. Although sera from individuals immunized with vector vaccines (Vaxzevria; AZ and COVID-19 Janssen, Ad26.COV2.S; J&J) were able to bind and neutralize WT and Delta, they showed only background levels towards Omicron. In contrast, mRNA (Comirnaty; BNT) or heterologous (AZ/BNT) vaccines induced weak, but detectable responses against Omicron. While RBD-binding antibody levels decreased significantly six months after full vaccination, the SARS-CoV-2 RBD-directed avidity remained constant. However, this still coincided with a significant decrease in neutralization activity against all variants. A third booster vaccination with BNT significantly increased the humoral immune responses against all tested variants, including Omicron. In conclusion, only vaccination schedules that included at least one dose of mRNA vaccine and especially an mRNA booster vaccination induced sufficient antibody levels with neutralization capacity against multiple variants, including Omicron.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2076-393X/10/5/794Test; https://doaj.org/toc/2076-393XTest

الوصول الحر: https://doaj.org/article/cb303763eb034c6cbff83ad987d871b3Test

المؤلفون: Florian D. Hastert, Lisa Henss, Christine von Rhein, Julia Gerbeth, Imke Wieters, Frauke Borgans, Yascha Khodamoradi, Kai Zacharowski, Gernot Rohde, Maria J.G.T. Vehreschild, Barbara S. Schnierle

المصدر: Viruses, Vol 14, Iss 5, p 882 (2022)

مصطلحات موضوعية: SARS-CoV-2, Delta, Omicron, SARS-CoV-1, NL63, neutralization, Microbiology, QR1-502

الوصف: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has now been continuing for more than two years. The infection causes COVID-19, a disease of the respiratory and cardiovascular system of variable severity. Here, the humoral immune response of 80 COVID-19 patients from the University Hospital Frankfurt/Main, Germany, was characterized longitudinally. The SARS-CoV-2 neutralization activity of serum waned over time. The neutralizing potential of serum directed towards the human alpha-coronavirus NL-63 (NL63) also waned, indicating that no cross-priming against alpha-coronaviruses occurred. A subset of the recovered patients (n = 13) was additionally vaccinated with the mRNA vaccine Comirnaty. Vaccination increased neutralization activity against SARS-CoV-2 wild-type (WT), Delta, and Omicron, although Omicron-specific neutralization was not detectable prior to vaccination. In addition, the vaccination induced neutralizing antibodies against the more distantly related SARS-CoV-1 but not against NL63. The results indicate that although SARS-CoV-2 humoral immune responses induced by infection wane, vaccination induces a broad neutralizing activity against multiple SARS-CoVs, but not to the common cold alpha-coronavirus NL63.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1999-4915/14/5/882Test; https://doaj.org/toc/1999-4915Test

الوصول الحر: https://doaj.org/article/3f798f13f58343bd8df7d2b98e4b6083Test

المؤلفون: Marie-Luise Herrlein, Sascha Hein, Tobias Zahn, Ines Mhedhbi, Jan Raupach, Younes Husria, Nuka Ivalu Benz, Jonathan Eisert, Daniela Bender, Vanessa Haberger, Florian D. Hastert, Lisa Henss, Barbara S. Schnierle, Julia C. Stingl, Michael Dreher, Eberhard Hildt

المصدر: Viruses, Vol 14, Iss 2, p 410 (2022)

مصطلحات موضوعية: COVID-19, SARS-CoV-2, convalescent sera, ARDS, neutralization assay, humoral immune response, Microbiology, QR1-502

الوصف: In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen–antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera—as in cases of non-ARDS sera—combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen–antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1999-4915/14/2/410Test; https://doaj.org/toc/1999-4915Test

الوصول الحر: https://doaj.org/article/6308a186ab2d442dac3f373702a5e91fTest

المؤلفون: Malini Rajan, Oliver Mortusewicz, Ulrich Rothbauer, Florian D. Hastert, Katrin Schmidthals, Alexander Rapp, Heinrich Leonhardt, M. Cristina Cardoso

المصدر: FEBS Open Bio, Vol 5, Iss 1, Pp 779-788 (2015)

مصطلحات موضوعية: Chromobodies, DNA repair, Post-translational modifications, Live cell microscopy, Alpaca heavy chain antibodies, Laser microirradiation, Biology (General), QH301-705.5

الوصف: Post‐translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single‐chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ‐H2AX.In vitro andin vivo characterization showed the specificity of the γ‐H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2211-5463Test

الوصول الحر: https://doaj.org/article/ba209d7079364f15884bd6637543f620Test

المؤلفون: Bingqian Qu, Csaba Miskey, André Gömer, Dylan Potmus, Maximillian Nocke, Robin D.V. Kleinert, Tabitha K. Itotia, Lara Valder, Janice Brückmann, Sebastian Höck, Florian D. Hastert, Christine von Rhein, Christoph Schürmann, Aileen Ebenig, Marek Widera, Sandra Ciesek, Stephanie Pfaender, Zoltán Ivics, Barbara S. Schnierle, Alexander W. Tarr, Christine Goffinet, Michael D. Mühlebach, Daniel Todt, Richard J.P. Brown

الوصف: SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::3754bd3ba15fb507050b9e65e13df0a8Test
https://doi.org/10.1101/2022.08.05.502936Test

المؤلفون: Stefan Canzar, Vigo Heissmeyer, Nicholas B. Angstman, Mark Helm, Aloys Schepers, Franziska Weichmann, Kaouthar Slama, Regina Feederle, Julian König, Robert Hett, Gunter Meister, Florian D. Hastert, M. Cristina Cardoso, Taku Ito-Kureha, Stefan Hüttelmaier, Andrew Flatley, Christoph Dieterich

المصدر: RNA

مصطلحات موضوعية: chemistry.chemical_classification, Regulation of gene expression, 0303 health sciences, Messenger RNA, biology, Nucleotides, medicine.drug_class, 030302 biochemistry & molecular biology, Method, Computational biology, Ribonucleotides, Monoclonal antibody, Antibodies, 03 medical and health sciences, Low affinity, chemistry, biology.protein, medicine, RNA, Nucleotide, RNA, Messenger, Antibody, Molecular Biology, 030304 developmental biology

الوصف: Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2′-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::774ba88d110b3d78cd7df016f58b05c2Test
https://doi.org/10.1261/rna.076026.120Test

المؤلفون: Florian D. Hastert, Ana Cañuelo, J. Alberto Marchal, María Arroyo, Antonio Sánchez, Jesús Calahorra, Duncan J. Clarke

المصدر: FEBS J

مصطلحات موضوعية: 0301 basic medicine, Immunoblotting, Regulator, Mitosis, Cell Cycle Proteins, Protein Serine-Threonine Kinases, Biochemistry, PLK1, Article, Cell Line, 03 medical and health sciences, 0302 clinical medicine, Proto-Oncogene Proteins, Humans, Topoisomerase II Inhibitors, CHEK1, Molecular Biology, Loss function, biology, Chemistry, Kinase, Topoisomerase, Cell Biology, G2-M DNA damage checkpoint, Flow Cytometry, Cell biology, G2 Phase Cell Cycle Checkpoints, Cytoskeletal Proteins, DNA Topoisomerases, Type II, HEK293 Cells, 030104 developmental biology, 030220 oncology & carcinogenesis, Checkpoint Kinase 1, Biocatalysis, biology.protein, biological phenomena, cell phenomena, and immunity, HeLa Cells

الوصف: Catalytic inhibition of topoisomerase II during G2 phase delays onset of mitosis due to the activation of the so-called decatenation checkpoint. This checkpoint is less known compared with the extensively studied G2 DNA damage checkpoint and is partially compromised in many tumor cells. We recently identified MCPH1 as a key regulator that confers cells with the capacity to adapt to the decatenation checkpoint. In the present work, we have explored the contributions of checkpoint kinase 1 (Chk1) and polo-like kinase 1 (Plk1), in order to better understand the molecular basis of decatenation checkpoint. Our results demonstrate that Chk1 function is required to sustain the G2 arrest induced by catalytic inhibition of Topo II. Interestingly, Chk1 loss of function restores adaptation in cells lacking MCPH1. Furthermore, we demonstrate that Plk1 function is required to bypass the decatenation checkpoint arrest in cells following Chk1 inhibition. Taken together, our data suggest that MCPH1 is critical to allow checkpoint adaptation by counteracting Chk1-mediated inactivation of Plk1. Importantly, we also provide evidence that MCPH1 function is not required to allow recovery from this checkpoint, which lends support to the notion that checkpoint adaptation and recovery are different mechanisms distinguished in part by specific effectors.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::97240d987ac698c05774c1c430750b8aTest
https://doi.org/10.1111/febs.15280Test