RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embr

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Aptamer-medierter Transport von microRNAs in Zellen des kardiovaskulären Systems

Ischemic injuries of the cardiovascular system are still the leading cause of death worldwide. They are often accompanied by loss of cardiomyocytes (CM) and their replacement by non-functional heart tissue. Cardiac fibroblasts (CF) play a major role in the recovery after ischemic injury and in the scar formation. In the last few years researchers were able to reprogram fibroblasts into CM in vitro and in murine models of myocardial infarction using various protocols including a cocktail of microRNAs (miRs). These miRs can target hundreds of messenger RNAs and inhibit their translation into proteins, potentially regulating multiple cellular signaling pathways. Because of this, there has been a rising interest in the use of miRs for therapeutic purposes. However, as different miRs have different effects in different cells, there is the danger of causing serious side effects. These could be alleviated by enacting a cell-specific transport of miRs, for example by using aptamers. Aptamers are usually short strands of DNA or RNA, which can fold into a specific three-dimensional confirmation which allows them to bind specifically to target molecules. Aptamers are commonly selected from a large library for their ability to bind to target molecules using a procedure called SELEX. Aptamers have already been used to transport miRs into cancer cells. In this thesis, we first established the transport of miRs into cells of the cardiovascular system using aptamers. MiR-126 is an important part of the signaling in endothelial cells (EC), protects from atherosclerosis and supports angiogenesis, which is why we chose it as a candidate to transport into the vasculature. We first tested two aptamers for their ability to internalize into EC and fibroblasts. Both the aptamer for the ubiquitously expressed transferrin receptor (TRA) and a general internalizing RNA motif, but not a control construct, could internalize efficiently into all cell types tested. We then designed three chimeras (Ch) using different strategies to connect TRA to miR-126. While all chimeras could internalize efficiently, only Ch3, which connects TRA to Pre-miR-126 using a sticky bridge structure, had functional effects in EC. Ch3 reduced the protein expression of VCAM-1 in EC and increased the VEGF induced sprouting of EC in a spheroid-sprouting assay. Treatment of breast cancer cells with Ch3 emulated the effects of treatment with classical miR-126-3p and miR-126-5p mimics. In the SK-BR3 cell line Ch3 and miR-126-3p reduce the viability of the cells while they reduce recruitment of EC by the MCF7 cell line. miR-126-5p had no apparent effect in the SK-BR3 line, but increased viability of MCF7 cells, as did Ch3. This implies that Ch3 can be processed to both functional miR-126-3p and miR-126-5p in treated cells. We were unable to achieve a reprogramming of adult murine cardiac fibroblasts into cells resembling CM using the cocktail of 4 miRs. This indicates that the miR-mediated transdifferentiation is only possible in neonatal fibroblasts. The effects in mice after an AMI might possibly be caused by an enhanced plasticity of fibroblasts in and close to the infarcted area. We also screened to find aptamers specifically binding to cells of the cardiovascular system. We used two oligonucleotide libraries in a cell-SELEX to select candidates which bind to CF, but not EC. We observed that only the library which contains two randomized regions of 26 bases showed an enrichment of species binding to fibroblasts. We then sequenced rounds 5-7 of the SELEX and analyzed the data bioinfomatically to select 10 candidate aptamers. All candidates showed a strong binding not only to CF, but also EC. This indicates that the selection pressure against species binding to EC was not high enough and would have to be increased to find true CF-aptamers. Four promising candidates were also analyzed for their potential to be internalized and we surprisingly found that all of them were internalized by EC and CF more efficiently than TRA. The similar behavior of the candidates implies that they possibly share a ligand, which is expressed both by EC and CF, but more prominently by the latter. This work demonstrates the possibility of using aptamers to transport miRs into cells of the cardiovascular system. It also shows that it is possible to select aptamers for non-cancerous mammalian cells, which has not been done before. It is reasonable to assume that a refinement of the cell-SELEX will allow selection of cell-specific aptamers. Due to the failure of reprogramming of adult fibroblasts into induced cardiomyocytes we were unable to test whether a miR-mediated reprogramming might be inducible using aptamer transported-miRs. Ultimately, aptamer mediated transport of miRs is a feasible and promising therapeutic option for the treatment of cardiovascular diseases and other disorders like cancer.Ischämische Erkrankungen des Herz-Kreislaufsystems sind immer noch die führende Todesursache in den Ländern der westlichen Welt. Ein dabei häufig auftretendes Problem ist der Verlust funktioneller Herzmuskelzellen und deren Ersatz mit nicht-funktionellem Narbengewebe. Kardiale Fibroblasten spielen eine wichtige Rolle bei dieser Narbenbildung. In den letzten Jahren konnten Forscher erfolgreich eine Reprogrammierung dieser Fibroblasten zu Herzmuskelzellen in Kultur und auch in in Mausmodellen von Herzinfarkt bewerkstelligen, unter anderem mit Hilfe von MicroRNAs (miRs). Diese miRs können potentiell hunderte verschiedene messenger RNAs ansteueren und deren Translation in Eiweiße inhibieren, wodurch miRs ganze zelluläre Signalwege regulieren können. Darum kam in den letzten Jahren ein immer größeres Interesse auf, miRs für die Therapie von vielen Krankheiten, unter anderem vaskuläre Erkrankungen und Krebs, einzusetzen. Auf Grund der unterschiedlichen Effekte von miRs, die abhängig von den verschiedene Zell-oder Gewebetypen sind, besteht allerdings ein großes Risiko von unerwünschten Nebenwirkungen. Diese könnten durch einen zielspezischen Transport von miRs, z.B. durch die Verwendung von Aptameren, verhindert werden. Aptamere sind meist kurze Stränge aus DNA oder RNA, welche sich zu drei-dimensionalen Strukturen falten, durch welche sie spezifisch an Zielmoleküle binden. Aptamere werden mittels einem Verfahren namens SELEX aus einer großen Bibliothek von möglichen Sequenzen auf ihre Fähigkeit, an Zielmoleküle zu binden, ausgewählt. Einige miRs wurden bereits mittels Aptamere in Krebszellen transportiert. In dieser Doktorarbeit wurde zuerst der Transport von funktionellen miRs in Zellen des kardiovaskulären Systems etabliert. MiR-126 ist ein wichtiger Bestandteil der Signalwege von Endotheelzellen (EZ), schützt vor Atherosklerose und unterstützt die Angiogenese, weshalb wir es als Kandidat für den Transport mittels Aptamer in Zellen auswählten. Zuerst wurden zwei Aptamere auf Ihre Fähigkeit getestet, in EZ und Fibroblasten zu gelangen. Sowohl der Aptamer für den ubiquitär exprämierten Transferrin Rezeptor (TRA) als auch das generelle internalisierende RNA Motif, aber nicht ein Kontrollkonstrukt von gleicher Länge, internalisierten effizient in allen genutzten Zelltypen. Danach wurden drei Chimären getestet, welche verschieden Strategien nutzen um TRA und miR-126 zu verbinden. Obschon alle drei Chimären effizient in EZ internalisieren, zeigte nur die Chimäre 3 (Ch3) in welcher das TRA und der Precursor von miR-126 mittels einer klebrigen Brückensequenz verbunden sind funktionelle Effekte. Ch3 verringerte die Eiweißexpression von VCAM-1 in EZ und verstärkte außerdem die VEGF-induzierte Sproßung von EC in einem Sphäroid-Sproßungs-Assay. Des Weiteren zeigte die Behandlung von Bruskrebszellen mit Ch3 dieselben Effekte wie eine Behandlung mit klassischen Mimetika von miR-126-3p und 5p. In der SK-BR3 Zellinie verursachte Ch3 und miR-126-3p eine Abnahme der Vitalität während in der MCF7 Zellinie die Rekrutierung von EZ zu den Krebzellen abnahm. MiR-126-5p hatte keinen offensichtlichen Effekt auf die SK-BR3 Zellinie, verursachte aber ebenso wie Ch3 eine Zunahme der Vitalität in der MCF7-Linie. Dies impliziert, dass Ch3 in Zellen sowohl zu funktioneller miR-126-3p als auch miR-126-5p prozessiert werden kann. Es war uns nicht möglich eine Reprogrammierung von kardialen Fibroblasten aus erwachsenen Mäusen zu herzmuskelzellähnlichen Zellen mittels miRs zu erwirken. Dies deutet darauf hin, dass miR gesteuerte Reprogrammierung nur in Fibroblasten von neugeborenen Mäusen in Zellkultur sowie in erwachsenen Mäusen nach einem Infarkt erfolgen kann. Außerdem haben wir ein Screening durchgeführt um Aptamere zu finden, welche an Zellen aus dem kardiovaskulären System, im speziellen kardiale Fibroblasten, binden. Wir benutzten zwei verschiedene Aptamer-Bibliotheken in einem Zell-SELEX Verfahren um Oligonukleotide anzureichern, welche an kardiale Fibroblasten aber nicht an EC binden. Wir sahen, dass lediglich die Bibliothek mit zwei Bereichen aus 26 randomisierten Nukleotiden zu einer Anreicherung von Spezies führt, die an Fibroblasten binden. Die letzten drei Runden dieser Zell-SELEX wurden im Anschluß sequenziert. Nach einer ausgiebigen bioinformatischen Analyse wurden zehn mögliche Kandidaten auf Ihre Fähigkeit, an kardiale Fibroblasten zu binden, untersucht. Alle Kandidaten konnten mit unterschiedlicher Effizienz sowohl an kardiale Fibroblasten als auch EC binden. Daraufhin wurden die vier vielversprechendsten Kandidaten auf ihre Fähigkeit zur Internalisierung in Fibroblasten und EC überprüft und waren alle in der Lage, in beide Zelltypen zu internalisieren. Hierbei zeigten sie sogar mehr Effizienz als TRA bei gleicher Konzentration. Zusammen genommen demonstriert diese Doktorarbeit, dass Aptamere genutzt werden können um funktionelle microRNAs in das kardiovaskuläre System zu transportieren. Sie zeigt auch, dass es möglich ist Aptamere für gesunde Säugetier-Zellen zu selektieren, was zuvor noch nicht beschrieben wurde. Da wir keine Reprogrammierung von Fibroblasten durch miRs hervorrufen konnten war es uns leider nicht möglich, zu testen ob Aptamere als Träger im Rahmen einer solchen Reprogrammierung geeignet sind. Dennoch deuten unsere Daten darauf hin, dass Aptamer medierter Transport von microRNAs eine mögliche und vielvesprechende Option für die Behandlung von kardiovaskulären und anderen Erkrankungen ist

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here,we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8+ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Primeboost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19

Pathogen-induced tissue-resident memory T(H)17 (T(RM)17) cells amplify autoimmune kidney disease

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (T-RM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether T-RM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4(+) T-RM cells with a T(H)17 signature (termed T(RM)17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal T(RM)17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney T(RM)17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced T(RM)17 cells have a previously unrecognized function in aggravating autoimmune disease.erman Research Foundation (DFG) SFB1192 SFB1286 SFBTR57 Deutsche Nierenstiftung Deutsche Gesellschaft fur Nephrologie Werner Otto Stiftung eMed Consortia "Fibromap" from the Federal Ministry of Education and Research Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) AFB 170004 Conicyt/FONDEQUIP/EQM140016 Else Kroner-Fresenius Foundatio

Pulmonary impairment independently determines mortality in critically ill patients with acute-on-chronic liver failure

Background & Aims: In ACLF patients, an adequate risk stratification is essential, especially for liver transplant allocation, since ACLF is associated with high short-term mortality. The CLIF-C ACLF score is the best prognostic model to predict outcome in ACLF patients. While lung failure is generally regarded as signum malum in ICU care, this study aims to evaluate and quantify the role of pulmonary impairment on outcome in ACLF patients. Methods: In this retrospective study, 498 patients with liver cirrhosis and admission to IMC/ICU were included. ACLF was defined according to EASL-CLIF criteria. Pulmonary impairment was classified into three groups: unimpaired ventilation, need for mechanical ventilation and defined pulmonary failure. These factors were analysed in different cohorts, including a propensity score-matched ACLF cohort. Results: Mechanical ventilation and pulmonary failure were identified as independent risk factors for increased short-term mortality. In matched ACLF patients, the presence of pulmonary failure showed the highest 28-day mortality (83.7%), whereas mortality rates in ACLF with mechanical ventilation (67.3%) and ACLF without pulmonary impairment (38.8%) were considerably lower (p < .001). Especially in patients with pulmonary impairment, the CLIF-C ACLF score showed poor predictive accuracy. Adjusting the CLIF-C ACLF score for the grade of pulmonary impairment improved the prediction significantly. Conclusions: This study highlights that not only pulmonary failure but also mechanical ventilation is associated with worse prognosis in ACLF patients. The grade of pulmonary impairment should be considered in the risk assessment in ACLF patients. The new score may be useful in the selection of patients for liver transplantation

Das Lagemanagement des Robert Koch-Instituts während der COVID-19-Pandemie und der Austausch zwischen Bund und Ländern

The Robert Koch Institute (RKI) plays a central role in Germany in the management of health hazards of biological origin. The RKI's crisis management aims to contribute to protecting the health of the population in Germany in significant epidemic situations and to maintain the RKI's working ability over a long period of time even under high load. This article illustrates the crisis management of the RKI in general as well as during the COVID-19 pandemic. The generic RKI crisis management structures and the setup of the RKI emergency operations centre (EOC), their operationalisation in the context of the COVID-19 pandemic and the resulting challenges as of 31 October 2020 are described in this paper. The exchange between the federal and state governments during the pandemic is also described.The COVID-19 pandemic has led to extraordinary circumstances. During the epidemic situation, good communication and coordination has been essential, both within the RKI and with other federal or state authorities and expert groups. Under great pressure, the RKI produces and regularly updates recommendations, statements and assessments on various topics. To provide operational support for all COVID-19 related activities, an EOC was activated at the RKI. During the COVID-19 pandemic, there are various challenges regarding personnel and structures. It became apparent that good preparation (e.g. existing task descriptions and premises) has an important positive impact on crisis management