Project description:Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood, and the fact that different genotypes adapt differently is recognized. However, the basic question of whether these spaceflight responses are required for survival has yet to be posed, and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes, Col-0 and WS, as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS, phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0, suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed and even simpler spaceflight transcriptome response in the ambient light of the ISS, more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce, or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS, the WS genotype actually mounted a larger transcriptome response than Col-0, suggesting that the in-space light environment affects physiological adaptation, which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

Project description:The Heat Shock Factor A2 (HsfA2), as a part of the HSF network, is essential to the plant’s response to almost any environmental stress and to the cellular homeostatic control mechanisms. Plant cell cultures disabled in HsfA2 function were grown aboard the International Space Station (ISS) in order to ascertain whether or not they use the same terrestrially effective systems to adapt to the novel environment of spaceflight. Cultured lines of Arabidopsis thaliana derived from wild type (WT) cultivar Col-0 and from a knock-out line deficient in the gene encoding HSFA2 (HSFA2 KO) were launched to the ISS on SpaceX-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC17 spaceflight mission and were fixed in-flight after 10 days on orbit. Microarray gene expression data were analyzed using a two-part comparative approach. First, differentially expressed genes were identified between the environments (spaceflight to ground) within cells of the same genotype, which represented physiological adaptation to the spaceflight environment. Second, gene expression profiles were identified between the genotypes (HSFA2 KO to WT) within the same environment, defining genes uniquely required by the two genotypes in the ground and spaceflight adapted states. The physiological state of the cells as a result of disabling a gene has tremendous control over the mechanisms induced to adapt to the environment of spaceflight. The HsfA2 demonstrated a role in the physiological adaptation to the spaceflight environment since the cells disabled in the HsfA2 gene used substantially different genes to achieve the spaceflight adapted state than the WT cells. The endoplasmic reticulum (ER) stress and unfolded protein response (UPR) define the HSFA2 KO cells’ physiological state regardless of the environment and likely result from the deficiency in the chaperone-mediated protein folding machinery. HsfA2 seems to have a universal stress response role but also specific roles in the physiological adaptation to spaceflight through cell wall remodeling, signal perception and transduction and starch biosynthesis. Implementation of knock-out cells identified a set of genes with a required expression level in order for a cell to achieve a spaceflight-adapted state. The HSFA2 KO cells helped to unravel the HsfA2-dependent genes of the adaption of wild type cells to the environment of spaceflight.

Project description: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 4M-BM-0C. 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 4M-BM-0C storage. All units were returned to Earth for analysis.

Project description:Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic.

Project description: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 Î¼G. 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.

Project description:Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic. This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end, P. aeruginosa cultures were grown in space, and the expression profile was compared with ground control samples (both in biological triplicate). Two RWV samples also examined (did not re-analyze them, only compared the outputs).

Dataset Information

Characterizing Epigenetic Changes in Methylation Mutants (elp2-5 and met1-7) in Response to Spaceflight. [RNA-Seq]

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