No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction

Microorganisms perform countless tasks on Earth and they are expected to be essential for human space exploration. Despite the interest in the responses of bacteria to space conditions, the findings on the effects of microgravity have been contradictory, while the effects of Martian gravity are nearly unknown. We performed the ESA BioRock experiment on the International Space Station to study microbe-mineral interactions in microgravity, simulated Mars gravity and simulated Earth gravity, as well as in ground gravity controls, with three bacterial species: Sphingomonas desiccabilis, Bacillus subtilis, and Cupriavidus metallidurans. To our knowledge, this was the first experiment to study simulated Martian gravity on bacteria using a space platform. Here, we tested the hypothesis that different gravity regimens can influence the final cell concentrations achieved after a multi-week period in space. Despite the different sedimentation rates predicted, we found no significant differences in final cell counts and optical densities between the three gravity regimens on the ISS. This suggests that possible gravityrelated effects on bacterial growth were overcome by the end of the experiment. The results indicate that microbial-supported bioproduction and life support systems can be effectively performed in space (e.g., Mars), as on Earth

Mechanisms of CDE-dependent mRNA decay

The cytokine TNFα is a potent effector of inflammation, and is causally involved in toxic shock syndrome and rheumatoid arthritis. It is vital to maintain continuous control over TNFα levels under continuous control, which can be achieved in one way by regulating the decay rate of the TNFα mRNA. One cis-element, the AU-rich element (ARE) in the TNFα 3’UTR confers to rapid degradation of this message. When the element is knocked out in mice, TNFα is overproduced resulting in the corresponding pathology. We recently described a second cis-element, the constitutive decay element (CDE), that is unique to TNFα and, compared to the ARE, is unresponsive to TNFα RNA stabilizing signals such as lipopolysaccharide (LPS) or phorbol esters. Furthermore a CDE-bearing reporter was degraded in Slow C cells that are defective in ARE dependent decay, implying that CDE mediated degradation is performed by a different mechanism. Through deletion mutation we were able to define a 40 nucleotide (nt) stretch (fragment O), located 42 nt downstream of the ARE, that is the minimal functional CDE. Any mutation to this minimal sequence was not only inactive in promoting decay, but also exhibited differences in protein binding compared to fragment O. The CDE binding proteins were assayed by UV-crosslinking assays, purified by biochemical fractionation and identified by mass spectrometry. The RNA-binding protein nucleolin was the prime candidate. We produced full length recombinant nucleolin with the baculovirus system, and could demonstrate that recombinant nucleolin binds in vitro to fragment O but not its mutated sequences by electrophoretic mobility shift assay. The nucleolin-CDE association was also observed with band shift and super shift assays in THP-1 human macrophage cytoplasmic extracts. Furthermore we could show by RNA immunoprecipitation assays in THP-1 cells, that nucleolin is specifically associated with endogenous TNFα mRNA in vivo, but not with the transcripts of IL-6 and IL-10, or GAPDH. To assess whether down regulation of nucleolin by RNA interference would affect CDE mediated decay, we transfected nucleolin or control siRNAs into a human HT1080 GFP-CDE reporter cell line. By FACS we observed an increase in GFP protein levels with the nucleolin specific siRNAs but not with the control siRNAs, indicative of elevated CDE-reporter mRNA levels. Actinomycin D chase experiments confirmed stabilization of the CDE reporter with a 40% increase in reporter mRNA half-life from 96+2 min to 135+11 min, that was statistically significant. We conclude that nucleolin is associated with the CDE in vitro and in vivo and is be functionally involved in the CDE-mediated degradation pathway

A Constitutive Decay Element Promotes Tumor Necrosis Factor Alpha mRNA Degradation via an AU-Rich Element-Independent Pathway

Tumor necrosis factor alpha (TNF-α) expression is regulated by transcriptional as well as posttranscriptional mechanisms, the latter including the control of mRNA decay through an AU-rich element (ARE) in the 3′ untranslated region (UTR). Using two mutant cell lines deficient for ARE-mediated mRNA decay, we provide evidence for a second element, the constitutive decay element (CDE), which is also located in the 3′ UTR of TNF-α. In stably transfected RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS), the CDE continues to target a reporter transcript for rapid decay, whereas ARE-mediated decay is blocked. Similarly, the activation of p38 kinase and phosphatidylinositol 3-kinase in NIH 3T3 cells inhibits ARE-mediated but not CDE-mediated mRNA decay. The CDE was mapped to an 80-nucleotide (nt) segment downstream of the ARE, and point mutation analysis identified within the CDE a conserved sequence of 15 nt that is required for decay activity. We propose that the CDE represses TNF-α expression by maintaining the mRNA short-lived, thereby preventing excessive induction of TNF-α after LPS stimulation. Thus, CDE-mediated mRNA decay is likely to be an important mechanism limiting LPS-induced pathologic processes

MASON: Betonerhärtung in Schwerelosigkeit – Simulationen mit dem Klinostaten

Im Rahmen des Projekts MASON (Material Science on Solidification of Concrete) wird der Einfluss der Erdgravitation auf die Erhärtung von Beton untersucht. Ziel dabei ist es, einerseits Rückschlüsse auf die Eignung von Beton für die Verwendung auf extraterrestrischen Himmelskörpern zu ziehen und andererseits mögliche Erkenntnisse hinsichtlich eines ressourcenschonenderen Einsatzes auf der Erde zu gewinnen. Hierzu wurden vom deutschen ESA-Astronauten Matthias Maurer im Rahmen der Mission „Cosmic Kiss“ auf der Internationalen Raumstation (ISS) 64 Betonproben in Schwerelosigkeit hergestellt. Diese werden verglichen sowohl mit konventionell unter terrestrischer Schwerkraft (1g) ausgehärten Proben als auch mit Probekörpern, die während der Erstarrungszeit in einem Klinostaten rotiert wurden. Durch die Klinostatrotation wird die Schwerelosigkeit simuliert und mögliche Auftriebs- oder Sedimentationsvorgänge im Frischbeton werden minimiert. Das Funktionsprinzip, ein Ansatz, die zutreffende Rotationsgeschwindigkeit abzuschätzen, sowie Ergebnisse einer Studie mit verschiedenen Mischungen und unterschiedlichen Rotationsgeschwindigkeiten werden nachfolgend vorgestellt

Habitate auf Himmelskörpern? – Ein Beitrag: Betonerhärtung unter Schwerelosigkeit.

Die Errichtung von Habitaten auf Himmelskörpern wie dem Mond oder auch dem Mars ist aufgrund der Fortschritte in der Raumfahrt in greifbare Nähe gerückt. Hinsichtlich der Identifikation geeigneter Konstruktionsmaterialien steht neben verschiedenen anderen Werkstoffen auch Beton zur Auswahl. Im Projekt MASON wird untersucht, welche Einflüsse die Mikrogravitation auf die Erhärtung und die Eigenschaften von Beton hat. Hierzu wurden Experimente auf der internationalen Raumstation (ISS) in realer Schwerelosigkeit durchgeführt. Auf der Erde wurden parallel Proben unter Erdgravitation und unter simulierter Schwerelosigkeit hergestellt. Hierfür kamen, erstmalig in der Betonentwicklung, Klinostaten und Random Positioning Machines zum Einsatz - Gerätschaften, die normalerweise in den Lebenswissenschaften verwendet werden. Aus den Ergebnissen der Untersuchungen sollen jedoch nicht nur Erkenntnisse zum Bauen unter Schwerelosigkeit abgeleitet werden, sondern es soll auch festgestellt werden, ob die Charakterisierung von Betonen, die ohne äußere Einflüsse wie Auftrieb oder Sedimentation erhärten, tiefere Einblicke in das Erstarrungsverhalten ermöglichen. Ziel ist dabei, sowohl für das Bauen auf der Erde als auch auf dem Mond Rückschlüsse hinsichtlich eines reduzierten Zementanteils im Beton zu ziehen und damit ein wirtschaftlicheres und nachhaltigeres Bauen zu ermöglichen. Nachfolgend wird über das Projekt MASON, den eigens hierfür entwickelten MASON Concrete Mixer, den Einsatz von Klinostaten in der Betonforschung und die Untersuchungen an den hiermit hergestellten Betonprobekörpern berichtet

Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿ †

CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3′ untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3′ UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3′ UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis