Entwicklung und Validierung eines Bioassays zum Nachweis induzierter Genaktivität für den Einsatz auf der Internationalen Raumstation

Neben der fehlenden Schwerkraft stellt die Strahlenbelastung des Astronauten einen limitierenden Faktor für den Langzeitaufenthalt des Menschen im Weltraum dar. Experimente zur Untersuchung einer möglichen Interaktion zwischen Mikrogravitation und der zellulären Strahlenantwort können auf der Erde nur unzureichend bearbeitet werden. Die Internationale Raumstation bietet als derzeit wichtigste Forschungsplattform eine Möglichkeit, dieser Fragestellung nachzugehen. Im Weltraumexperiment Cellpath soll eine geänderte Genexpression schadensrelevanter Gene unter Verwendung von indikativen Bioassays analysiert werden. Dazu sollte ein auf dem Rezeptor-Reporter-Prinzip basierendes, zelluläres Testsystem konzipiert werden. Um einen für diesen Zweck geeigneten Promotor- oder Enhancer zu finden, der die Rolle eines regulatorischen Elements übernimmt, wurde die Expression verschiedener Gene untersucht, deren Genprodukte in unterschiedlichen DNA-Reparaturwege involviert sind. Anhand einer in verschiedenen humanen Zelllinien (A549, MCF-7, AGS, HEK und NHF) durchgeführten strahlenbiologischen Charakterisierung wurden A549-Zellen als geeignete Wirtslinie für das Testsystem identifiziert. Die Ergebnisse der mittels quantitativer Real Time RT-PCR durchgeführten Genexpressionsstudien wiesen das p53R2 Gen als geeigneten Kandidaten für die Etablierung eines induzierbaren Bioassays aus. Für dessen Entwicklung wurde ein Vektorsystem konstruiert, das die Expression des Reportermoleküls EGFP unter der Kontrolle der p53-abhängigen Form der Ribonukleotid Reduktase (p53R2) als Sensor ermöglicht. Dazu wurde die Bindestelle für den Transkriptionsfaktor p53 aus dem 1. Intron des p53R2 Gens in den promotorlosen EGFP-Vektor kloniert und stabil in A549 Zellen transfiziert. Die Induzierbarkeit der rekombinanten Zelllinie A549-RRM2b wurde in biologischen Experimenten mit Strahlung unterschiedlicher Qualität überprüft. Die nach Exposition mit Röntgenstrahlen, beschleunigten Kohlenstoff- und Argon-Ionen erhaltenen Ergebnisse zeigen, dass der rekombinanten Zelllinie eine hohe Bedeutsamkeit für ein Weltraumexperiment zukommt

Dual PI3K/mTOR inhibitor NVP-BEZ235 enhances radiosensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines due to suppressed Double-Strand Break (DSB) repair by non-homologous end joining

The PI3K/Akt/mTOR pathway is frequently altered in human papillomavirus (HPV)-positive and negative squamous cell carcinoma of the head and neck (HNSCC) and overstimulation is associated with poor prognosis. PI3K drives Akt activation and constitutive signaling acts pro-proliferative, supports cell survival, DNA repair, and contributes to radioresistance. Since the small molecule NVP-BEZ235 (BEZ235) is a potent dual inhibitor of this pathway, we were interested whether BEZ235 could be an efficient radiosensitizer. The 50 nM BEZ235 was found to abrogate endogenous and irradiation-induced phosphorylation of Akt (Ser473). The anti-proliferative capacity of the drug resulted in an increase in G1-phase cells. Repair of radiation-induced DNA double-strand breaks (DSBs) was strongly suppressed. Reduction in DSB repair was only apparent in G1- but not in G2-phase cells, suggesting that BEZ235 primarily affects non-homologous end joining. This finding was confirmed using a DSB repair reporter gene assay and could be attributed to an impaired phosphorylation of DNA-PKcs (S2056). Cellular radiosensitivity increased strongly after BEZ235 addition in all HNSCC cell lines used, especially when irradiated in the G0 or G1 phase. Our data indicate that targeting the PI3K/Akt/mTOR pathway by BEZ235 with concurrent radiotherapy may be considered an effective strategy for the treatment of HNSCC, regardless of the HPV and Akt status

A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest

Nascent polypeptides can induce ribosome stalling, regulating downstream genes. Stalling of ErmBL peptide translation in the presence of the macrolide antibiotic erythromycin leads to resistance in Streptococcus sanguis. To reveal this stalling mechanism we obtained 3.6-angstrom-resolution cryo-EM structures of ErmBL-stalled ribosomes with erythromycin. The nascent peptide adopts an unusual conformation with the C-terminal Asp10 side chain in a previously unseen rotated position. Together with molecular dynamics simulations, the structures indicate that peptide-bond formation is inhibited by displacement of the peptidyl-tRNA A76 ribose from its canonical position, and by non-productive interactions of the A-tRNA Lys11 side chain with the A-site crevice. These two effects combine to perturb peptide-bond formation by increasing the distance between the attacking Lys11 amine and the Asp10 carbonyl carbon. The interplay between drug, peptide and ribosome uncovered here also provides insight into the fundamental mechanism of peptide-bond formation

Einheit von Park und Architektur : der Park am IG Hochhaus

Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, a

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Assessment of space environmental factors by cytotoxicity bioassays

Cellular bioassays for detection of cyto- and genotoxicity are useful in the risk assessment of space environmental factors. Such bioassay systems have the potential complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. The experiment Cellular Responses to Radiation in Space (CERASP) has been selected by NASA/ESA to be performed on the International Space Station. It will supply basic information on the cellular response to radiation applied in microgravity. One of the biological endpoints under investigation will be survival reflected by radiation-dependent reduction of constitutive expression of the enhanced variant of green fluorescent protein (EGFP), originally isolated from the bioluminescent jellyfish Aequorea victoria. In this ground based study, the usefulness of this approach in comparison to standard techniques (colony forming ability test, MTT test) is shown

Gene expression in mammalian cells after exposure to 95MeV/amu argon ions

High LET radiations, such as heavy ions or neutrons, have an increased biological effectiveness compared to X-rays for gene mutation, genomic instability and carcinogenesis. Estimating the biological risks from space radiation encountered by cosmonauts will continue to influence long term duration in space, such as the planned mission to Mars. The human radiation responsive genes CDKN1A (p21/WAF), GADD45a (GADD45), GADD45b (MyD118), RRM2b (p53R2) and BRCA2 (FancD1), involved in cell cycle control or damage repair, were screened for gene expression changes in MCF-7 cells by quantitative real-time reverse transcription PCR (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after irradiation with accelerated doses of 36-argon ions and X-rays. Examination of the expression profiles 2 and 12 h after exposure reveals a pattern consistent with a population of cells in the early response to DNA damage and invoking cell stress responses. Interesting new data showing different expression patterns according to the gene and the type of ionizing radiation used could be obtained. Results show, that the signaling and repair activities induced after heavy ion or X-ray exposure are not the same and gene expression patterns may become useful indicators for distinguishing different types of radiation in relation to their biological effects