The general objective of the study was to determine modulation of gene expression by environmental factors with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. This experiment was designed as a new type of gravitational experiment which we like to call relative microgravity referring to the fact that the larvae first grow in hyper gravity for 5 days and are then returned to 1g normal gravity for 1 day. Zebrafish embryos were placed on a Large Diameter Centrifuge at 3 hpf brought to a gravitational force of 3 g until 5 dpf. Reference embryos were kept in parallel at 1g (Inc). At 5dpf one batch was left at 3g (3g) one batch was returned to 1g (3g>1g) while a third batch was returned to 1g but left on the axis of the centrifuge (Axe; 3g>Axe). The experiment was repeated 4 times each time with 4 batches of 60 larvae.

The general objective of the study was to determine modulation of gene expression by environmental factors specifically simulation of microgravity with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a clinostat (CLINO) for 24 hours which was shown to simulate microgravity by the specific rotational movements it generates. We show that CLINO exposure causes a clear decrease of bone formation as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red by morphometric analysis of the resulting images. Thus a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.

The general objective of the study was to determine modulation of gene expression by environmental factors specifically simulation of microgravity with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a clinostat (CLINO) for 24 hours which was shown to simulate microgravity by the specific rotational movements it generates. We show that CLINO exposure causes a clear decrease of bone formation as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red by morphometric analysis of the resulting images. Thus a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.

The general objective of the study was to determine modulation of gene expression by environmental factors, with a special emphasis on bone formation. For this reason, the specific period of treatment was chosen between 5-6 days post-fertilization (dpf), when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a Large Diameter Centrifuge and brought to a gravitational force of 3g or 5g for 24 hours. We show that this treatment causes a clear increase of bone formation, as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red, by morphometric analysis of the resulting images and by gene expression studies of selected genes. Thus, a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.

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. microRNA expression profiling were carried out on total RNA extracted from PBLs of twelve healthy donors at the end of 24h-incubation time in MMG and in 1g conditions. Analyses were performed by using the Human miRNA Microarray kit (V2) (Agilent Technologies) that allows the detection of 723 known human (miRBase v.10.1) and 76 human viral miRNAs. By comparing the expression profile of MMG-incubated vs. 1g-incubated PBLs of the same donor we found 42 differentially expressed miRNAs 25 up-regulated and 17 down-regulated.

Microgravity alters the immune response to in vitro LPS assault engineered in spaceflight: A multi-omics study Microgravity can facilitate creation of a potent environment for opportunistic infection by augmenting virulence and suppressing the host defense. Presumably extraterrestrial infection may trigger potentially novel bionetworks different from the terrestrial equivalent which could only be probed by investigating the host-pathogen relationship with minimum terrestrial bias. Towards this objective we strategically engineered a cell culture module equipped with a feedback controlled semi-automated platform to expose human endothelial cells to lipopolysaccharide (LPS). The assay was carried out in the STS-135 space shuttle and a concurrent ground study constituted the baseline. Transcriptomic investigation revealed an immune blunting in microgravity; Lbp MyD88 and MD-2 failed to encode proteins responsible for early LPS uptake. Longer exposure results implied that there was a delayed response potentially ineffectual in preventing pathogens from opportunistically modulating the infection network. Lack of recruitment of growth factors and a debilitated apoptosome supported this potential explanation. Certain cytokines such as IL-6 and IL-8 surged in response to LPS insult in microgravity. Contrasting expressions of B2M TIMP-1 and VEGRs suggested impaired pro-survival adaptation and healing mechanisms. The susceptibility of oxidative stress and immune regulation to microgravity compelled further investigation of the respective microRNA modulators such as miR-200a and miR-146b. These miRNAs were expressed differently in response to LPS assaults in different gravitational limits. In conclusion despite a serious drawback attributed to the small sample size we delineated some of the important aspects of the extraterrestrial etiology; more comprehensive follow up studies are warranted. Present study though compromised by the small sample size was able to shade lights on several aspects of immunological responses to the endotoxic assault mediated by uG. Implementing the host-pathogen interactions in the spaceflight and subsequently lysing the cells onboard presented the critical distinguishing features of the present study from the past reports. We identified the CCM of Tissue Genesis Inc. HI as the suitable hardware system to carry out the experiment in the spaceflight. CCM is an automated feedback controlled module that can concurrently support 24 bioreactors following protocols exclusively programmed for individual bioreactor. For this experiment we use samples EA41 EA 47 EA45 and EA155 that were exposed to LPS for 4 hours. Samples EA123 EA165 EA127 EA126 were exposed to LPS for 8Hrs. Samples EA33 EA 125 EA79 and EA 39 were controls in this experiment.

Microgravity alters the immune response to in vitro LPS assault engineered in spaceflight: A multi-omics study Microgravity can facilitate creation of a potent environment for opportunistic infection by augmenting virulence and suppressing the host defense. Presumably extraterrestrial infection may trigger potentially novel bionetworks different from the terrestrial equivalent which could only be probed by investigating the host-pathogen relationship with minimum terrestrial bias. Towards this objective we strategically engineered a cell culture module equipped with a feedback controlled semi-automated platform to expose human endothelial cells to lipopolysaccharide (LPS). The assay was carried out in the STS-135 space shuttle and a concurrent ground study constituted the baseline. Transcriptomic investigation revealed an immune blunting in microgravity; Lbp MyD88 and MD-2 failed to encode proteins responsible for early LPS uptake. Longer exposure results implied that there was a delayed response potentially ineffectual in preventing pathogens from opportunistically modulating the infection network. Lack of recruitment of growth factors and a debilitated apoptosome supported this potential explanation. Certain cytokines such as IL-6 and IL-8 surged in response to LPS insult in microgravity. Contrasting expressions of B2M TIMP-1 and VEGRs suggested impaired pro-survival adaptation and healing mechanisms. The susceptibility of oxidative stress and immune regulation to microgravity compelled further investigation of the respective microRNA modulators such as miR-200a and miR-146b. These miRNAs were expressed differently in response to LPS assaults in different gravitational limits. In conclusion despite a serious drawback attributed to the small sample size we delineated some of the important aspects of the extraterrestrial etiology; more comprehensive follow up studies are warranted. Present study though compromised by the small sample size was able to shade lights on several aspects of immunological responses to the endotoxic assault mediated by uG. Implementing the host-pathogen interactions in the spaceflight and subsequently lysing the cells onboard presented the critical distinguishing features of the present study from the past reports. We identified the CCM of Tissue Genesis Inc. HI as the suitable hardware system to carry out the experiment in the spaceflight. CCM is an automated feedback controlled module that can concurrently support 24 bioreactors following protocols exclusively programmed for individual bioreactor. For this experiment we use samples EA41 EA 47 EA45 and EA155 that were exposed to LPS for 4 hours. Samples EA123 EA165 EA127 EA126 were exposed to LPS for 8Hrs. Samples EA33 EA 125 EA79 and EA 39 were controls in this experiment.

The general objective of the study was to determine modulation of gene expression by environmental factors with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a Large Diameter Centrifuge and brought to a gravitational force of 3g or 5g for 24 hours. We show that this treatment causes a clear increase of bone formation as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red by morphometric analysis of the resulting images and by gene expression studies of selected genes. Thus a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.

Here in this study we systematically examined the patterns of DNA methylation and hydroxy-methylation with its functional implications in gene regulation for the cultured TK6 lymphoblastoid cells upon exposure to micro-gravity conditions. The results reported here indicate that simulated microgravity alters methylation patterns in a limited way and subsequently the expression of genes involved in stress response like ATF3 FBXO17 MAP3K13 and VCL in TK6 cells. Overall design: Examination of RNA-seq with 2 replicates each for 1 cell type

Here in this study we systematically examined the patterns of DNA methylation and hydroxy-methylation with its functional implications in gene regulation for the cultured TK6 lymphoblastoid cells upon exposure to micro-gravity conditions. The results reported here indicate that simulated microgravity alters methylation patterns in a limited way and subsequently the expression of genes involved in stress response like ATF3 FBXO17 MAP3K13 and VCL in TK6 cells. Overall design: Examination of 2 different DNA modifications with 2 replicates each for 1 cell type.