Microgravity leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast is the multinucleated bone resorbing cell. In this study we used NASA developed ground based Rotary Wall Vessel Bioreactor (RWV) Rotary Cell Culture System (RCCS) to simulate microgravity (uXg) conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to ground based control (Xg) mouse bone marrow cultures. We further determined the gene expression profiling of RAW 264.7 osteoclast progenitor cells in microgravity by agilent microarray analysis. Gene expression pattern was functional group clustered by transcriptome analysis using gene ontology tree machine (GOTM) for cell proliferation/survival differentiation and function. We confirm the microgravity modulated gene expression critical for osteoclast differentiation by real-time RT-PCR and Western blot analysis in murine bone marrow cultures. We identify transcription factors such as c-Jun c-Fos PU-1 critical for osteoclast differentiation is up-regulated in microgravity conditions. In addition microgravity resulted in 2.3 and 2.0-fold increase in the level of cathepsin K and MMP-9 matrix metalloproteinase expression in preosteoclast cells involved in the bone resorption process respectively. We also demonstrate a significant increase in the expression levels of M-CSF receptor c-Fms and PLCy2 and S100A8 molecules that play an important role in Ca2+ signaling essential for osteoclast function. Further microgravity stimulated preosteoclast cells showed elevated cytosolic Ca2+ levels compared to ground based control cells. Thus microgravity regulated gene expression profiling in preosteoclast cells provide new insights in to molecular mechanisms and therapeutic targets of osteoclast differentiation/activation responsible for bone loss and fracture risk in astronauts during space flight mission. Microgravity associated with space flight is a challenge for normal bone homeostasis. Astronauts experience 10-15% bone loss during a space flight mission. We aimed to determine the effect of simulated microgravity on osteoclast preosteoclasts cells. RAW264.7 cells (1.5 x 106 /ml) were loaded in RCCS with DMEM containing 10% FBS for 24 h. The cells were stimulated with RANKL (80ng/ml) for 24 h to obtain preosteoclasts in parallel with ground based control cells. Total RNA was isolated using RNAzol reagent (Biotecx Labs Houston TX) from control (Xg) and microgravity (uXg) subjected cells and hybridized with Agilent whole mouse genome 4x44K array system. Slides were washed and scanned on an Agilent G2565 microarray scanner. Data obtained were analyzed with Agilent feature extraction and GeneSpring GX v7.3.1 software packages (Genus biosystem Inc. Northbrook IL USA).

The effect of microgravity on gene expression in C.elegans was comprehensively analysed by DNA microarray. This is the first DNA microarray analysis for C.elegans grown under microgravity. Hyper gravity and clinorotation experiments were performed as reference against the flight experiment.

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in microgravity (mg). Immunosuppression during spaceflight is a major barrier to safe long-term human space habitation and travel. The goals of these experiments were to prove that mg was the cause of impaired T cell activation during spaceflight as well as understand the mechanisms controlling early T cell activation. T cells from 4 human donors were stimulated with concanavalin A (ConA) and anti-CD28 onboard the International Space Station (ISS). An onboard centrifuge was used to generate a 1g simultaneous control to isolate the effects of mg from other variables of spaceflight. Microarray expression analysis after 1.5 hours of activation demonstrated that mg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly differentially down-regulated in mg. Importantly several key immediate early genes were inhibited in mg. T cells were isolated from human volunteers. T cells from each donor were kept separate and loaded into individual chambers in separate cassettes for the following treatments: mg non-activated mg activated and 1g activated. Therefore samples represent biological triplicates. Experimental units were launched into space and placed into the KUBIK facility onboard the International Space Station. The 1g units were placed in the central centrifuge positions and centrifuged with an applied 1g force. The mg units were place in the static positions for continued mg exposure. After 30 minutes of pre-incubation mg non-activated units were fixed by addition of RNALater (QIAGEN Valencia CA) removed from the incubator and stored in 4 xc2 xb0C. The mg and 1g activated units were injected with final concentration 10mg/ml Con A and 4mg/ml anti-CD28. These cassettes were replaced into KUBIK on either the centrifuge or static positions and activated for 1.5 hours. Activation was stopped with the addition of RNALater and the units were then moved to 4 xc2 xb0C storage. All units were returned to Earth for analysis.

Cell cycle and cell proliferation are decoupled under altered gravity conditions. We have previously shown that semisolid cell cultures of Arabidopsis suffer overall genome changes in response to altered gravity and also that cell cycle stages duration is altered. By using synchronized cell cultures we will demonstrate the precise alterations in cell cycle duration and also the transcriptional signature in any of them. Experiments consists on exposures of Arabidopsis cell cultures to 1g control/simulated microgravity (RPM) conditions. Asynchronous cells exposed for 14 h + Syncronous populations choosen to have an enrichment of cell cycle phases were used (being T7/T10 samples on G2 phase T14/T16 samples on G1 phase). 6 dye-swap - time course treated vs untreated comparison.

In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a ground-based Rotating Wall Vessel (RWV) bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1g-incubated ones. Among these miR-9-5p miR-9-3p miR-155-5p miR-150-3p and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichments in the biological processes of immune/inflammatory response signal transduction regulation of response to stress regulation of programmed cell death and regulation of cell proliferation. We identified the correlation between miR-9-3p miR-155-5p miR-150-3p and miR-378-3p expression with that of genes involved in immune/inflammatory response (eg. IFNG and IL17F) apoptosis (eg. PDCD4 and PTEN) and cell proliferation (eg. NKX3-1 and GADD45A). Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation. Gene expression profiling was carried out in MMG-incubated PBLs vs. 1g-incubated PBLs on total RNA extracted from the same PBL samples assayed for miRNA profiling. We used the Whole Human Genome Oligo Microarray (Agilent) consisting of ~41.000 (60-mer) oligonucleotide probes which span conserved exons across the transcripts of the targeted full-length genes.

Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions two spaceflight-analogue culture systems i.e. the rotating wall vessel (RWV) and the random position machine (RPM) were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG) revealed a regulatory role for AlgU (RpoE). Specifically P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore cultivation in LSMMG increased heat and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public.

Transcriptional crosstalk between mammary gland liver and adipose tissue Experiment Overall Design: Pregnant and Lactating rats exposed to 3 gravity conditions

Articular and sterno-costal cartilages were isolated from skeletally-mature mice flown for 30 days on the BION-M1 mission. Samples were characterized histologically for proteoglycan loss and at the gene expression levels using Affymetrix gene arrays.

Here we present a whole-genome survey of the murine transcriptomic response to physiologically-relevant radiation doses 2 and 8 Gy. There are 18 distinct biological samples here. Mice were exposed to ionizing radiation (Cesium-138 source) and whole blood was collected by cardiac puncture 6 hours post treatment. Doses were 0 (7 samples) 2 (5 samples) and 8 (6 samples) gy.

Transcriptional crosstalk between mammary gland liver and adipose tissue Experiment Overall Design: Pregnant and Lactating rats exposed to 3 gravity conditions