Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1

Background: Multicellular tumor spheroids (MCTS) formed scaffold-free under microgravity are of high interest for research and medicine. Their formation mechanism can be studied in space in real microgravity or on Earth using ground-based facilities (GBF), which simulate microgravity. On Earth, these experiments are more cost-efficient and easily performable. However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells. Results: FTC-133 human thyroid cancer cells were cultivated on a 2D clinostat (CN) and a random positioning machine (RPM) and compared with corresponding 1 g control cells. Harvested cell samples were investigated by microscopy, quantitative realtime-PCR and Multi-Analyte Profiling. Spheroid formation and growth occurred during 72 h of cultivation on both devices. Cytokine secretion and gene activation patterns frequently altered in different ways, when the cells were cultured either on the RPM or the CN. A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines. Conclusion: The development of MCTS proceeds similarly on the RPM and the CN resembling the situation observed under real microgravity conditions, while no MCTS formation was observed at 1 g under identical experimental conditions. Simultaneously, changes in the regulation of CTGF and CAV1 appeared in a comparable manner on both machines. A relationship between these molecules and MCTS formation is discussed

ARADISH - Development of a Standardized Plant Growth Chamber for Experiments in Gravitational Biology Using Ground-Based Facilities

Plant development strongly relies on environ- mental conditions. Growth of plants in Biological Life Support Systems (BLSS), which are a necessity to allow human survival during long-term space exploration missions, poses a particular problem for plant growth, as in addition to the traditional environmental factors, microgravity (or reduced gravity such as on Moon or Mars) and limited gas exchange hamper plant growth. Studying the effects of reduced gravity on plants requires real or simulated microgravity experiments under highly standardized conditions, in order to avoid the influence of other environmental factors. Analysis of a large number of biological replicates, which is necessary for the detection of subtle phenotypical differences, can so far only be achieved in Ground-Based Facilities (GBF). Besides different experimental conditions, the usage of a variety of different plant growth chambers was a major factor that led to a lack of reproducibility and comparability in previous studies. We have developed a flexible and customizable plant growth chamber, called ARAbidopsis DISH (ARADISH), which allows plant growth from seed to seedling, being realized in a hydroponic system or on Agar. By developing a special holder, the ARADISH can be used for experiments with Arabidopsis thaliana or a plant with a similar habitus on common GBF hardware, including 2D clinostats and Random Positioning Machines (RPM). The ARADISH growth chamber has a controlled illumination system of red and blue light emitting diodes (LED), which allows the user to apply defined light conditions. As a proof of concept we tested a prototype in a proteomic experiment in which plants were exposed to simulated microgravity or a 90◦ stimulus. We optimized the design and performed viability tests after several days of growth in the hardware that underline the utility of ARADISH in microgravity research

Analysis of dynamic gene expression responses to altered gravity in the wildtype and auxin efflux carrier mutants of the model plant Arabidopsis thaliana

Plant roots are among most intensively studied biological systems in gravity research. Altered gravity induces asymmetric cell growth leading to root bending. Differential distribution of the phytohormone auxin underlies root's responses to gravity being coordinated by auxin efflux transporters from the PIN family. The objective of this study was to compare early transcriptomic changes in roots of Arabidopsis thaliana using experiments on board of parabolic flights, suborbital and orbital flights, and ground-based facilities for simulated microgravity conditions to correlate these changes to auxin distribution. By comparing immediate and initial responses of the gene expression to the different gravitational forces identified primary gravity regulated genes and resolved time-effects in gene expression leading to an understanding of the underlying physiological responses and adaptive processes. High-resolution imaging in combination with computational approaches further resolved phenotypic changes initiated by altered gravity at the cellular level. Our study provides important insights towards understanding signal transduction processes in altered gravity conditions by combining experimental platforms with the analysis of different genetic mutants in the model Arabidopsis

2-D Clinostat for Simulated Microgravity Experiments with Arabidopsis Seedlings

Ground-based simulators of microgravity such as fast rotating 2-D clinostats are valuable tools to study gravity related processes. We describe here a versatile g-value-adjustable 2-D clinostat that is suitable for plant analysis. To avoid seedling adaptation to 1 g after clinorotation, we designed chambers that allow rapid fixation. A detailed protocol for fixation, RNA isolation and the analysis of selected genes is described. Using this clinostat we show that mRNA levels of LONG HYPOCOTYL 5 (HY5), MIZU-KUSSEI 1 (MIZ1) and microRNA MIR163 are down- regulated in 5-day-old Arabidopsis thaliana roots after 3 min and 6 min of clinorotation using a maximal reduced g-force of 0.02 g, hence demonstrating that this 2-D clinostat enables the characterization of early transcriptomic events during root response to microgravity. We further show that this 2-D clinostat is able to compensate the action of gravitational force as both gravitropic-dependent statolith sedimentation and subsequent auxin redistribution (monitoring DR5rev :: GFP reporter) are abolished when plants are clinorotated. Our results demonstrate that 2-D clinostats equipped with interchangeable growth chambers and tunable rotation velocity are suitable for studying how plants perceive and respond to simulated microgravity

Linkage and LOH studies in 19 cylindromatosis families show no evidence of genetic heterogeneity and refine the CYLD locus on chromosome 16q12-q13

Familial cylindromatosis is an autosomal dominant predisposition to multiple neoplasms of the skin appendages. The susceptibility gene has previously been mapped to chromosome 16q12-q13 and has features of a recessive oncogene/tumour suppressor gene. We have now evaluated 19 families with this disease by a combination of genetic linkage analysis and loss of heterozygosity in cylindromas from affected individuals. All 15 informative families show linkage to this locus, providing no evidence for genetic heterogeneity. Recombinant mapping has placed the gene in an interval of approximately 1 Mb. There is no evidence, between families, of haplotype sharing that might be indicative of common founder mutations