The present study concerns the effects of 21 days of sustained recumbence (bedrest) and hypoxia alone and in combination on skeletal muscle microRNA expression. 14 male subjects participated in 3 experimental campaigns in a counterbalanced fashion: normoxic bedrest (NBR) hypoxic bedrest (HBR) and hypoxic ambulatory confinement (HAMB) both hypoxic conditions with FO2 = 0.141 and PIO2 = 90 xc2 xb1 0.4 mmHg equivalent to an altitude of xe2 x89 x88 4000 m). Each intervention (bedrest or ambulatory confinement) lasted 21 days and the interventions were separated by a 4-month wash-out/recovery period. The order in which each subject undertook the interventions is denoted Intervention_order Throughout both bedrest interventions each subject remained in a horizontal position at all times. He was allowed one pillow for the head and to occasionally lean on an elbow while eating or being transferred to a gurney. Muscular exercise was prohibited. During the HAMB confinement each subject was allowed to move freely within the hypoxic area. To mimic a normal level of physical activity subjects performed 30 minutes of low-to-moderate-intensity exercise twice daily. Muscle samples from Vastus lateralis.

Proper interpretation of RNA sequencing data requires an understanding of assay sensitivity and sources of variability. To this end the External RNA Control Consortium (ERCC) developed a standard set of 92 poly-adenylated RNA transcripts that are orthogonal to mammalian RNA that can be added to RNA extracts before library generation and sequencing. The presence of these RNA standards at known ratios improves interpretation of RNA sequencing datasets. To test the utility of the ERCC RNA controls total RNA extracted from mouse livers from the Rodent Research 1 (flight ground control basal control and vivarium control groups) and Rodent Research 3 (flight ground control and basal control groups) missions were sequenced with and without the ERCC control RNA (RR-3 liver samples without ERCC control have been sequenced previously and the dataset is released in the GLDS-137). To allow comparison within and between groups ERCC Mix 1 or Mix 2 were added to half of the samples from each group respectively.

Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms underpinning the effects of microgravity on the immune system. The goal was to validate the Drosophila model for understanding alterations of innate immune responses in humans due to spaceflight. Five containers of flies with ten female and five male fruit flies in each container were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours with a new generation reared in microgravity. RNA was extracted on the day of shuttle landing from whole body animals (3rd instar larvae and adults) hybridized to Drosophila 2.0 Affymetrix genome arrays and the expression level of all genes was normalized against the gene expression level from the corresponding developmental stage animals raised on ground. Spaceflight altered the expression of larval genes involved in the maturation of plasmatocytes (macrophages) and their phagocytic response as well as the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria and of lysozymes antimicrobial peptide pathway and immune stress genes hallmarks of humoral immunity. Larval microarrays (FL 6 samples) are based on RNA extracted from 6 independent sets of 50 mid 3rd instar larvae reared in microgravity and collected on the day of landing after 12 days and 18.5 hours on the space shuttle and the same number of control larvae raised on ground (GL 6 samples). Adults microarrays (F1 3 samples) are based on RNA from 3 sets of 20 adult females each that emerged during spaceflight and within 4 hours of landing and the same number of adult females from the corresponding ground control containers (G1 3 samples).

Spaceflight is known to affect immune cell populations. In particular splenic B-cell numbers decrease during spaceflight and in ground-based physiological models. Although antibody isotype changes have been assessed during and after spaceflight an extensive characterization of the impact of spaceflight on antibody composition has not been conducted in mice. Next Generation Sequencing and bioinformatic tools are now available to assess antibody repertoires. We can now identify immunoglobulin gene- segment usage junctional regions and modifications that contribute to specificity and diversity. Due to limitations on the International Space Station alternate sample collection and storage methods must be employed. Our group compared Illumina MiSeq xc3 x82 xc2 xae sequencing data from multiple sample preparation methods in normal C57Bl/6J mice to validate that sample preparation and storage would not bias the outcome of antibody repertoire characterization. In this report we also compared sequencing techniques and a bioinformatic workflow on the data output when we assessed the IgH and Ig xc3 x8e xc2 xba variable gene usage. Our bioinformatic workflow has been optimized for Illumina HiSeq xc3 x82 xc2 xae and MiSeq xc3 x82 xc2 xae datasets and is designed specifically to reduce bias capture the most information from Ig sequences and produce a data set that provides other data mining options.

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem we designed installed and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA s C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights xe2 x80 x94four consecutive missions flown over the United States (US) from 30 October to 2 November 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause we collected air during aircraft Ascent/Descent (0.3 to 11 km) as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes including Bacillus sp. Micrococcus sp. Arthrobacter sp. and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Some taxa were more abundant in the flight samples than the ground samples including OTUs from families Lachnospiraceae Ruminococcaceae and Erysipelotrichaceae as well as the following genera: Clostridium Mogibacterium Corynebacterium Bacteroides Prevotella Pseudomonas and Parabacteroides. Surprisingly our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US as suggested by modeled back trajectories and satellite measurements. However the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

Comparative gene expression in skin between mice maintained in microgravity (0g) and normogravity (1g) environment. Six male C57Bl/J10 mice were housed for 91 days in the specially designed Mouse Drawer System in weightlessness aboard the International Space Station. Three wild-type mice (WT) and three transgenic mice overexpressing the osteogneic factor PTN/OSF1 under the control of the human bone specific ostecalcin promoter (Tg) were used in the experiment. During the 3-month stay on the ISS 3 mice unfortunately died leaving 2 Tg and 1 WT. MDS tissue sharing program allowed several teams to study various tissues from these mice. Our aim was to investigate the effect of such a long period of microgravity on skin physiology by morphological biochemical and genomewide analyses by comparison to similar mice on ground. Gene expression in the skin of 3 space mice and of 3 ground mice was analyzed by microarray. As this unique experiment performed on 3 mice limits the power of statistical analyis as the transgene PTN/OSF1 was not overexpressed in skin and as a pair wise Pearson s correlation rates between the individual levels of expressed transcripts in the WT and the Tg mice were not significantly different from each other in one experimental group (space or ground) data from the 3 mice were combined to compare results from the space an ground groups.

Aspergillus fumigatus is a saprophytic filamentous fungus that is ubiquitous outdoors (soil decaying vegetation) and indoors (hospitals simulated closed habitats etc.). A. fumigatus can adapt to various environmental conditions and form airborne conidia that are the inoculum for a variety of diseases (e.g. non- and invasive pulmonary infections allergic bronchopulmonary aspergillosis etc.) in immunocompromised hosts. In an on-going Microbial Observatory Experiments on the International Space Station (ISS) molecular phylogeny of several fungal isolates were characterized. Two strains ISSF 21 and IF1SW-F4 were isolated from the HEPA filter and the surface of the Cupola of the ISS respectively. Using primers targeting the internal transcribed spacers ITS1 and 2 both isolates were identified as A. fumigatus. The whole genome sequence analysis of ISSF 21 revealed increased number of single nucleotide polymorphisms (SNPs) when compared to the reference A. fumigatus 293. Knowing that A. fumigatus is an opportunistic pathogen and microgravity highly influences the antibiotic susceptibility and pathogenicity of microorganisms we examined pathogenicity of both ISS isolates using the zebrafish larval model. The space station isolates (ISSF-021 and IF1SW-F4) were more virulent than two clinical strains (Af293 and CEA10) whose pathogenicity was highly characterized. Here the whole genome sequences of ISSF-021 strain are being deposited.

The purpose of this study was to search for microgravity-sensitive genes specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. Experiment Overall Design: Two-group design with paired samples i.e. one 1G and one MMG culture came from the same donor. Therefore 6 samples came from 3 different donors. Experiment Overall Design: Donor 1 :GSM96146,GSM96147 Experiment Overall Design: Donor 2: GSM96148 GSM96149 Experiment Overall Design: Donor 3: GSM96150,GSM96151 Experiment Overall Design: Total RNA was submitted to and then labeled hybridized and data generated by the Baylor College Medicine Microarray Core Facility (333E One Baylor Plaza Houston TX 77030).

Results from C-20A science flights (30 October to 2 November 2017) collecting airborne microorganisms in the troposphere and lower stratosphere.

The purpose of this study was to search for microgravity-sensitive genes specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. Experiment Overall Design: Two-group design with paired samples i.e. one 1G and one MMG culture came from the same donor. Therefore 6 samples came from 3 different donors. Experiment Overall Design: Donor 1 :GSM96146,GSM96147 Experiment Overall Design: Donor 2: GSM96148 GSM96149 Experiment Overall Design: Donor 3: GSM96150,GSM96151 Experiment Overall Design: Total RNA was submitted to and then labeled hybridized and data generated by the Baylor College Medicine Microarray Core Facility (333E One Baylor Plaza Houston TX 77030).