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All sourcesNASA NTRSDOE OSTI
NASA NTRS2009

Bone Biomarkers on the Pathway to Effective Spaceflight Countermeasures

Osteocyte cells are the most abundant yet least understood bone cell type in the human body. However, recent discovers in osteocyte cell biology have shed light on their importance as key mechanosensing cells regulating the bone remodeling process. Thus, we propose the first ever in vitro gene expression evaluation of osteocytes exposed to simulated microgravity to determine mechanistic pathways of their gravity sensing ability. Improved understanding of the fundamental mechanisms at the osteocyte cellular level may lead to improved treatment options to mitigate the effects of bone loss encountered by astronauts on long duration space missions and provide tailored treatment options for maintaining bone strength of immobilized/partially paralyzed patients here on Earth. Aim 1: Characterize the gene expression patterns and protein levels following exposure of murine osteocytelike cell line (MLO-Y4) to simulated microgravity using the NASA Rotating Wall Vessel (RWV) Bioreactor. Osteocytes are theorized to be the mechanosensors and transducers of mechanical load for bones, yet the biological mechanism of this action remains elusive. We propose to investigate the genetic regulation of the mechanism of the MLO-Y4 cell in the NASA Bioreactor as it is the accepted ground-based analog for simulating vector averaged microgravity.

Spatz, Jordan↗
NASA NTRS2016

Biospecimen Retrieval from NASA's Rodent Research-1: Maximizing Science Return from Flight Missions

Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed to support long duration missions on the International Space Station. After 37 days in microgravity twenty mice were euthanized and frozen on orbit. Upon return to Earth the carcasses were dissected and yielded 32 different types of tissues from each mouse and over 3200 tissue aliquots. Many tissues were distributed to the Space Life and Physical Sciences (SLPS) Biospecimen Sharing Program (BSP) Principal Investigators (PIs) through the Ames Life Science Data Archive (ALSDA). A second round of dissections was performed to collect additional tissues from the remaining carcasses thawed for a second time for additional BSP PIs. Tissues retrieved included vaginal walls, aorta, pelvis, brown adipose tissue, tail, spine and forearms. Although the analyses are still in progress, some of the PIs have reported that the quality of the tissues was acceptable for their study. In a separate experiment we tested the RNA quality of the tissues that were dissected from frozen carcasses that were subjected to euthanasia, freezing, first and second thaw dissections. Timelines simulated the on-orbit RR-1 procedures to assess the quality of the tissues retrieved from the second thaw dissections. We analyzed the RIN values of select tissues including kidney, brain, white adipose tissue (WAT) and brown adipose tissue (BAT). Overall the RIN values from the second thaw were lower compared to those from the first by about a half unit; however, the tissues yielded RNA that are acceptable quality for some quantitative gene expression assays. Interestingly, RIN values of brain tissues were 8.4+/-0.6 and 7.9+/-0.7 from first and second round dissections, respectively (n=5). Kidney and WAT yielded RIN values less than 8 but they can still be used for qPCR. BAT yielded higher quality RNA (8.2+/-0.5) than WAT (5.22+/-0.9), possibly due to the high fat content. Together, these data show that select tissues can be utilized for gene expression studies even if they are retrieved from carcasses that were subjected to at least two freezing and thawing processes; this further expands science return from valuable and infrequent rodent experiments in space.

rodent↗
NASA NTRS2001

Rapid evolution of cis-regulatory sequences via local point mutations

Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

Non-NASA Center↗
NASA NTRSDate not supplied

MULTI-OMICS ANALYSIS OF THE IMPACT OF CHRONIC LOW-DOSE RADIATION AND HINDLIMB SUSPENSION ON MURINE BRAIN AND RETINA

The space environment includes hazards like radiation and microgravity which can adversely affect biological systems. We assessed multi-omics multi-tissue NASA GeneLab datasets where 6-month-old female mice were gamma irradiated (IR) and/or hindlimb unloaded (HLU) for 21 days. Whole transcriptome shotgun sequencing (RNA-Seq) and reduced representation bisulfite sequencing (RRBS) of brain and retina samples collected at 4 months post-exposure was performed to better characterize the retinal and neurological responses to spaceflight. We compared epigenomic and transcriptomic profiles within each exposure group for both tissue types to identify correlation (Pearson’s correlation test; p-value < 0.05) between gene expression and DNA methylation levels that may be related to transcriptional regulation. We then obtained genes with methylation-expression correlation that also showed differences in mean expression or dispersion between exposed and control groups (adjusted p-value < 0.25; relaxed to denote ‘hypothesis’) in the brain (37 genes in HLU, 4 in IR, and 156 in HLU+IR) or retina (92 genes in HLU, 1 in IR, and 55 in HLU+IR). Enriched Gene Ontology (GO) terms for these genes are listed in Table 1 for HLU and HLU+IR for both tissue types. No enriched terms and only a few genes were detected with IR-only exposure in the brain (Chmp1a, Limd1, Rab40b, Ubc) and retina (retinoblastoma binding protein Rbbp7). Cellular components related to synapse were enriched in both tissue types. Previous analysis of differentially expressed genes in the retina after 1 month of HLU+IR showed enrichment in the somatodendritic compartment of the neuron, which was also observed in the brain 4 months post-exposure. Interestingly, genes related to ubiquitination showed correlation between methylation and expression and were differentially expressed or dispersed in different exposure groups indicating that this pathway may play an important role in multi-stressor response (Figure 1). The current multi-omics and multi-tissue analysis interrogates the epigenomic and transcriptomic impacts of radiation and hindlimb unloading, in isolation and in combination, on the retina and the brain. The results provide insight into the adaptive response to individual spaceflight hazard analogs and their interplay, as well as hypotheses to be further tested for understanding spaceflight-induced neurological and vision effects.

Prachi Kothiyal↗
NASA NTRS2016

Hindlimb Suspension (HLS) in Rodents for the Study of Intracranial Pressure, Molecular and Histologic Changes in the Eye, and CSF Production Regulation and Resorption: A Status Report of Two Studies

This status report corresponds to two studies tied to an animal experiment being executed at the University of California Davis (Charles Fuller's laboratory). The animal protocol uses the well-documented rat hindlimb suspension (HLS) model, to examine the relationship between cephalic fluid shifts and the regulation of intracranial (ICP) and intraocular (IOP) pressures as well as visual system structure and function. Long Evans rats are subjected to HLS durations of 7, 14, 28 and 90 days. Subgroups of the 90-day animals are studied for recovery periods of 7, 14, 28 or 90 days. All HLS subjects have age-matched cage controls. Various animal cohorts are planned for this study: young males, young females and old males. In addition to the live measures (ICP by telemetry, IOP and retinal parameters by optical coherence tomography) which are shared with the Fuller study, the specific outcomes for this study include: -Gene expression analysis of the retina -Histologic analysis - Analysis of the microvasculature of retina flat mounts by NASA's VESsel GENeration Analysis (VESGEN) Software. To date, the young male and female cohorts are being completed. Due to the need to keep technical variation to a minimum, the histologic and genomic analyses have been delayed until all samples from each cohort are available and can be processed in a single batch per cohort. The samples received so far correspond to young males sacrificed at 7,14, 28 and 90 days of HLS and at 90 days of recovery; and from young females sacrificed at 7, 14 and 28 of HLS. A complementary study titled: "A gene expression and histologic approach to the study of cerebrospinal fluid (CSF) production and outflow in hindlimb suspended rats" seeks to study the molecular components of CSF production and outflow modulation as a result of HLS, bringing a molecular and histologic approach to investigate genome wide expression changes in the arachnoid villi and choroid plexus of HLS rats compared to rats in normal posture.

Theriot, C. A.↗
NASA NTRS2000

Effects of spaceflight and simulated weightlessness on longitudinal bone growth

Indirect measurements have suggested that spaceflight impairs bone elongation in rats. To test this possibility, our laboratory measured, by the fluorochrome labeling technique, bone elongation that occurred during a spaceflight experiment. The longitudinal growth rate (LGR) in the tibia of rats in spaceflight experiments (Physiological Space Experiments 1, 3, and 4 and Physiological-Anatomical Rodent Experiment 3) and in two models of skeletal unloading (hind-limb elevation and unilateral sciatic neurotomy) were calculated. The effects of an 11 day spaceflight on gene expression of cartilage matrix proteins in rat growth plates were also determined by northern analysis and are reported for the first time in this study. Measurements of longitudinal growth indicate that skeletal unloading generally did not affect LGR, regardless of age, strain, gender, duration of unloading, or method of unloading. There was, however, one exception with 34% suppression in LGR detected in slow-growing, ovariectomized rats skeletally unloaded for 8 days by hind-limb elevation. This detection of reduced LGR by hind-limb elevation is consistent with changes in steady-state mRNA levels for type II collagen (-33%) and for aggrecan (-53%) that were detected in rats unloaded by an 11 day spaceflight. The changes detected in gene expression raise concern that spaceflight may result in changes in the composition of extracellular matrix, which could have a negative impact on conversion of growth-plate cartilage into normal cancellous bone by endochondral ossification.

manned↗
NASA NTRS1997

Analysis of myosin heavy chain mRNA expression by RT-PCR

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

Non-NASA Center↗
NASA NTRSDate not supplied

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

Many microbial pathogens have repeatedly exhibited unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, host-pathogen interactions, and virulence. Notably, the classic foodborne pathogen Salmonella enterica serovar Typhimurium displayed increased virulence in animals when cultured in either the spaceflight analogue or true spaceflight environment. Recently, Serratia marcescens also was shown to increase virulence when cultured in the spaceflight environment. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system at multiple levels, which suggests an increased risk of infectious diseases. Moreover, astronauts have some degree of clinical infectious disease incidence. However, the contribution of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. The goal of this study is to gain insight into the breadth of other medically significant microbial pathogens that may exhibit altered virulence and pathogenesis-related responses when cultured in spaceflight analogue conditions. Specifically, we are characterizing the effect of spaceflight analogue culture (Low Shear Modeled Microgravity/LSMMG) on microbial pathogenesis-related stress responses, in vitro host-pathogen interactions, gene expression, and virulence potential in animals using five important model bacterial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. The information to date is providing a better understanding into the potential impact of microgravity on alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C M Ott↗
NASA NTRSDate not supplied

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

Many microbial pathogen shave repeatedly exhibited unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, host-pathogen interactions, and virulence. Notably, the classic foodborne pathogen Salmonella enterica serovar Typhimurium displayed increased virulence in animals when cultured in either the spaceflight analogue or true spaceflight environment. Recently, Serratia marcescens also was shown to increase virulence when cultured in the spaceflight environment. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system at multiple levels, which suggests an increased risk of infectious diseases. Moreover, astronauts have some degree of clinical infectious disease incidence. However, the contribution of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. The goal of this study is to gain insight into the breadth of other medically significant microbial pathogens that may exhibit altered virulence and pathogenesis-related responses when cultured in space flight analogue conditions. Specifically, we are characterizing the effect of spaceflight analogue culture (Low Shear Modeled Microgravity/LSMMG) on microbial pathogenesis-related stress responses, in vitro host-pathogen interactions, gene expression, and virulence potential in animals using five important model bacterial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. Herein, we present data from one of these pathogens, the foodborne bacterium, S. enterica Enteritidis, which is closely related to S. enterica Typhimurium. Phenotypes evaluated included growth profiles, environmental stress responses(acid, oxidative, bile, and thermal stresses),and in vitro colonization of3-D biomimetic cultures of human intestinal tissue containing immune cells. Transcriptomic profiling and virulence studies are ongoing. We show that S. Enteritidis exhibited key alterations in pathogenic responses to LSMMG culture that suggest increased infection risk, including several responses which were different from those observed in the closely related pathovar S. Typhimurium. This information will provide critical mechanistic insight into the potential impact of microgravity on alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C M Ott↗
NASA NTRS2024

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

Many microbial pathogen shave repeatedly exhibited unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, host-pathogen interactions, and virulence. Notably, the classic foodborne pathogen Salmonella enterica serovar Typhimurium displayed increased virulence in animals when cultured in either the spaceflight analogue or true spaceflight environment. Recently, Serratia marcescens also was shown to increase virulence when cultured in the spaceflight environment. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system at multiple levels, which suggests an increased risk of infectious diseases. Moreover, astronauts have some degree of clinical infectious disease incidence. However, the contribution of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. The goal of this study is to gain insight into the breadth of other medically significant microbial pathogens that may exhibit altered virulence and pathogenesis-related responses when cultured in space flight analogue conditions. Specifically, we are characterizing the effect of spaceflight analogue culture (Low Shear Modeled Microgravity/LSMMG) on microbial pathogenesis-related stress responses, in vitro host-pathogen interactions, gene expression, and virulence potential in animals using five important model bacterial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. Herein, we present data from one of these pathogens, the foodborne bacterium, S. enterica Enteritidis, which is closely related to S. enterica Typhimurium. Phenotypes evaluated included growth profiles, environmental stress responses(acid, oxidative, bile, and thermal stresses),and in vitro colonization of3-D biomimetic cultures of human intestinal tissue containing immune cells. Transcriptomic profiling and virulence studies are ongoing. We show that S. Enteritidis exhibited key alterations in pathogenic responses to LSMMG culture that suggest increased infection risk, including several responses which were different from those observed in the closely related pathovar S. Typhimurium. This information will provide critical mechanistic insight into the potential impact of microgravity on alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C.M. Ott↗
NASA NTRSDate not supplied

Space Flown Rodent Liver RNA Sequencing Data for Machine Learning in Space Biology Research

High-throughput nucleic acid sequencing (DNA-seq, RNA-seq) has become widespread in biomedical research due to the growing availability and affordability of these assays. Data analysis has been accelerated in recent years by the adoption of artificial intelligence (AI) and machine learning (ML) techniques by biomedical researchers. In space biology research, RNAseq datasets from space-flown experimental samples are critical for characterizing the gene expression aberrations associated with exposure to spaceflight stressors. However, space biological experiments tend to be very low sample size, so identifying proper AI/ML algorithms for sequencing data analysis is an ongoing challenge since these algorithms typically require large sample size. The NASA Science Mission Directorate (SMD) has started the “Benchmark Initiative for AI/ML”, focused on creating datasets meant for three main applications: 1) scientific benchmarking, which finds the best algorithm for a specific problem; 2) application benchmarking, which measures algorithm performance against a set of parameters; and 3) system benchmarking, which evaluates performance of hardware and software architecture. These scientific benchmarks consist of an AI-ready dataset and a reference implementation on a specific scientific question. In this work, we focused on generating standardized datasets to allow the scientific community to benchmark AI/ML algorithms in the domain of space biology. We present here a standardized, AI-ready, publicly available benchmark dataset for space biology RNA-seq data as a collaboration between the NASA AI4LS (Artificial Intelligence for Life Sciences) working group. and NASA’s SMD. This dataset consists of space-flown and ground control mouse liver found in the NASA GeneLab omics database. However, to amplify the small sample number (n=112 samples) for ML purposes, we employ Gaussian noise and a generative adversarial network to extend this dataset to 6,000 synthetic samples, matching the original gene expression characteristics.

James Casaletto↗
NASA NTRS2015

Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in space has little effect on the gene and miRNA expression. Gene and miRNA expression changes were observed in cells that were confluent, but still proliferating slowly. The faster growth in the flown cells was associated with the activation of NF(sub kappa)B pathways which triggers the expression of several growth factors and the suppression of the cell cycle checkpoint.

Zhang, Ye↗
NASA NTRS2001

Characterization of the yeast copper-inducible promoter system in Arabidopsis thaliana

Inducible promoters or gene-switches are used to both spatially and temporally regulate gene expression. Such regulation can provide information concerning the function of a gene in a developmental context as well as avoid potential harmful effects due to overexpression. A gfp construct under the control of a copper-inducible promoter was introduced into Arabidopsis thaliana (L.) Heynh. and the regulatory parameters of this inducible promoter were determined. Here, we describe the time-course of up- and down-regulation of GFP expression in response to copper level, the optimal regulatory levels of copper, and the tissue specificity of expression in three transgenic lines. We conclude that the copper-inducible promoter system may be useful in regulating the time and location of gene expression in A. thaliana.

Non-NASA Center↗
NASA NTRS1993

Molecular Basis of the Increase in Invertase Activity Elicited by Gravistimulation of Oat-Shoot Pulvini

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oatshoot pulvini starting within 3h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly(A)(+)RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the Polymerase Chain Reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52 % deduced amino-acid sequence homology to that of carrot beta-fructosi- dase and a 48 % homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

Wu, Liu-Lai↗
NASA NTRS2011

Response of Human Prostate Cancer Cells to Mitoxantrone Treatment in Simulated Microgravity Environment

This study explores the changes in growth of human prostate cancer cells (LNCaP) and their response to the treatment of antineoplastic agent, mitoxantrone, under the simulated microgravity condition. In comparison to static 1g, microgravity and simulated microgravity have been shown to alter global gene expression patterns and protein levels in various cultured cell models or animals. However, very little is known about the effect of altered gravity on the responses of cells to drugs, especially chemotherapy drugs. To test the hypothesis that zero gravity would result in altered regulation of cells in response to antineoplastic agents, we cultured LNCaP cells for 96 hr either in a High Aspect Ratio Vessel (HARV) bioreactor at the rotating condition to model microgravity in space or in the static condition as a control. 24 hr after the culture started, mitoxantrone was introduced to the cells at a final concentration of 1 M. The mitoxantrone treatment lasted 72 hr and then the cells were collected for various measurements. Compared to static 1g controls, the cells cultured in the simulated microgravity environment did not show significant differences in cell viability, growth rate, or cell cycle distribution. However, in response to mitoxantrone (1uM), a significant proportion of bioreactor cultured cells (30%) was arrested at G2 phase and a significant number of these cells were apoptotic in comparison to their static controls. The expressions of 84 oxidative stress related genes were analyzed using Qiagen PCR array to identify the possible mechanism underlying the altered responses of bioreactor culture cells to mitoxantrone. Nine out of 84 genes showed higher expression at four hour post mitoxantrone treatment in cells cultured at rotating condition compared to those at static. Taken together, the results reported here indicate that simulated microgravity may alter the responses of LNCaP cells to mitoxantrone treatment. The alteration of oxidative stress pathways in cells cultured under simulated microgravity conditions may be one of the mechanisms to cause such changes of sensitivity of LNCaP cells to mitoxantrone treatment.

Zhang, Ye↗
NASA NTRSDate not supplied

Preparation for and Performance of a Pseudomonas aeruginosa Biofilm Experiment On Board the International Space Station

Biofilms are a problem on Earth given their ability to degrade the materials upon which they grow and due to their relevance to infections. Remarkably, 65% and 80% of infections and chronic diseases on Earth are associated with biofilms, respectively. In space, these problems' impact is higher because the crew's lives and mission success depend on nominal operation of mechanical systems. Furthermore, the isolated confined environment nature of spaceflight may increase the rates of disease transmission. In the case of the International Space Station (ISS), biofilms are an identified problem on the Environmental Control and Life Support System (ECLSS), namely on the water processor assembly (WPA). In late 2019, the Space Biofilms experiment launched towards ISS to (i) characterize the mass, thickness, morphology, and gene expression of biofilms formed in space with respect to matched Earth controls, (ii) interrogate the expression of antimicrobial resistance genes, and (iii) test novel materials as potential biofilm control strategies for future ECLSS components. For this, 288 bacterial samples were prepared prior to the launch of the Northrop Grumman CRS-12 mission from NASA's Wallops Flight Facility. The samples were integrated into the spaceflight hardware, BioServe's Fluid Processing Apparatus (FPA) packed in sets of eight in Group Activation Packs (GAP). Half of these samples were activated and terminated on orbit by NASA astronauts Jessica Meir and Christina Koch, while the remaining half were processed equivalently on Earth. The spaceflight bacterial samples of Space Biofilms returned on board SpaceX' CRS-19 Dragon spacecraft, in early 2020. We here describe the test campaign implemented to verify the experiment design and confirm it would enable us to achieve the project's scientific goals. This campaign ended with the Experiment Verification Test (EVT), from which we here present example morphology and transcriptomic results. We describe in detail the sample preparation prior to flight, including cleaning and sterilization of the coupons of six materials (SS316, passivated-SS316, lubricant impregnated surface, catheter-grade silicone with and without a nanotopography, and cellulose membrane), loading and integration of growth media, bacterial inoculum, fixative and preservative to enable experiment termination on orbit. Additionally, we describe the performance of the experiment on board the ISS, including crew activities, use of assets, temperature profile, and experiment timeline; all leading to a successful spaceflight experiment.

Pamela Flores↗
NASA NTRS2006

Immune Response in Microgravity: Genetic Basis and Countermeasure Development Implications

Impairment of the immunity in astronauts and cosmonauts even in shortterm flights is a recognized risk. Longterm orbital space missions and anticipated interplanetary flights increase the concern for more pronounced effects on the immune system with potential clinical consequences. Studies in true and modeled microgravity (MG) have demonstrated that MG directly affects numerous lymphocyte functions. The purpose of this study was to screen for genes involved in lymphocytes response to modeled microgravity (MMG) that could explain the functional and structural changes observed earlier. The microgravity-induced changes in gene expression were analyzed by microarray DNA chip technology. CD3and IL2activated Tcells were cultured in 1g (static) and modeled microgravity (NASA Rotating Wall Vessel bioreactor) conditions for 24 hours. Total RNA was extracted using the RNeasy isolation kit (Qiagen, Valencia, CA). Microarray experiments were performed utilizing Affymetrix Gene Chips (U133A), allowing testing for 18,400 human genes. To decrease the biological variation and aid in detecting microgravity-associated changes, experiments were performed in triplicate using cells obtained from three different donors. Exposure to modeled microgravity resulted in alteration of 89 genes, 10 of which were upregulated and 79 down-regulated. Altered genes were categorized by their function, structural role and by association with metabolic and regulatory pathways. A large proportion was found to be involved in fundamental cellular processes: signal transduction, DNA repair, apoptosis, and multiple metabolic pathways. There was a group of genes directly related to immune and inflammatory responses (IL7R, granulysin, proteasome activator subunit 2, peroxiredoxin 4, HLADRA, lymphocyte antigen 75, IL18R and DOCK2 genes). Among these genes only one (IL7R) was upregulated, the rest were downregulated. The upregulation of the IL7 receptor gene was confirmed by RT PCR. Three genes with altered expression were identified in the apoptosis related group (Granzyme B, APO2 ligand and Beta3endonexin). All of them were downregulated. Gene expression changes in MG might appear pivotal in identifying potential molecular targets for countermeasure development. (Supported by NRA OLMSA02 and NSCORT NAG54072 grants).

Risin, Diana↗
NASA NTRS1997

Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

Jackson, Jacquelyn↗