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NASA NTRS2007

Multispectral Imaging Broadens Cellular Analysis

Amnis Corporation, a Seattle-based biotechnology company, developed ImageStream to produce sensitive fluorescence images of cells in flow. The company responded to an SBIR solicitation from Ames Research Center, and proposed to evaluate several methods of extending the depth of field for its ImageStream system and implement the best as an upgrade to its commercial products. This would allow users to view whole cells at the same time, rather than just one section of each cell. Through Phase I and II SBIR contracts, Ames provided Amnis the funding the company needed to develop this extended functionality. For NASA, the resulting high-speed image flow cytometry process made its way into Medusa, a life-detection instrument built to collect, store, and analyze sample organisms from erupting hydrothermal vents, and has the potential to benefit space flight health monitoring. On the commercial end, Amnis has implemented the process in ImageStream, combining high-resolution microscopy and flow cytometry in a single instrument, giving researchers the power to conduct quantitative analyses of individual cells and cell populations at the same time, in the same experiment. ImageStream is also built for many other applications, including cell signaling and pathway analysis; classification and characterization of peripheral blood mononuclear cell populations; quantitative morphology; apoptosis (cell death) assays; gene expression analysis; analysis of cell conjugates; molecular distribution; and receptor mapping and distribution.

Source record↗

NASA NTRS2016

Effects of Hindlimb Unloading and Ionizing Radiation on Murine Gene Expression in Skin and Bone

Long duration spaceflight causes a negative calcium balance and reduces bone density in astronauts. The underlying mechanisms of spaceflight-induced bone loss and the possible influences of both microgravity and radiation are not fully understood although emerging evidence suggests that these two factors may interact to result in increased bone loss. Previously, gene expression analysis of hair follicles from astronauts, as well as skin from space-flown mice, revealed changes in the expression of genes related to DNA damage and oxidative stress responses. These results resemble the responses of bone to spaceflight-like radiation and simulated weightlessness by hindlimb unloading (HU). Hence in this study, we initiated studies to determine whether skin can be used to predict the responses of bone to simulated microgravity and radiation. We examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR). To investigate the effects of irradiation andor HU on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated) (CT), hindlimb unloading (HU), 56Fe radiation (IR) and both HU+IR. Animals were euthanized 11 days post-IR, and results were analyzed by 1-way ANOVA. In skin samples, Cdkn1a was decreased to the same extent in HU and HU+IR (47 of CT). In addition, HU reduced FoxO3 expression (46 of CT) and IR increased Gadd45g expression 135 compared CT in skin. But in bone, HU increased FoxO3 expression 31 compared the level of CT. These results suggest that radiation and simulated weightlessness regulated simliar oxidative stress and cell cycle arrest genes in both skin and bone, although the time course and direction of changes may differ. This research may lead to the development of a relatively simple diagnostic tool for bone loss with the advantage that hair follicles and skin are relatively easy to acquire from subjects.

Terada, Masahiro↗

NASA NTRS2002

Genes encoding calmodulin-binding proteins in the Arabidopsis genome

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

NASA Discipline Plant Biology↗

NASA NTRS2018

Combined Effects of Simulated Microgravity and Radiation Exposure on Osteoclast Cell Fusion

The loss of bone mass and alteration in bone physiology during space flight are one of the major health risks for astronauts. Although the lack of weight bearing in microgravity is considered a risk factor for bone loss and possible osteoporosis, organisms living in space are also exposed to cosmic radiation and other environmental stress factors. As such, it is still unclear as to whether and by how much radiation exposure contributes to bone loss during space travel, and whether the effects of microgravity and radiation exposure are additive or synergistic. Bone is continuously renewed through the resorption of old bone by osteoclast cells and the formation of new bone by osteoblast cells. In this study, we investigated the combined effects of microgravity and radiation by evaluating the maturation of a hematopoietic cell line to mature osteoclasts. RAW 264.7 monocyte/macrophage cells were cultured in rotating wall vessels that simulate microgravity on the ground. Cells under static 1g or simulated microgravity were exposed to γ rays of varying doses, and then cultured in receptor activator of nuclear factor-κB ligand (RANKL) for the formation of osteoclast giant multinucleated cells (GMCs) and for gene expression analysis. Results of the study showed that radiation alone at doses as low as 0.1 Gy may stimulate osteoclast cell fusion as assessed by GMCs and the expression of signature genes such as tartrate resistant acid phosphatase (Trap) and dendritic cell-specific transmembrane protein (Dcstamp). However, osteoclast cell fusion decreased for doses greater than 0.5 Gy. In comparison to radiation exposure, simulated microgravity induced higher levels of cell fusion, and the effects of these two environmental factors appeared additive. Interestingly, the microgravity effect on osteoclast stimulatory transmembrane protein (Ocstamp) and Dcstamp expressions was significantly higher than the radiation effect, suggesting that radiation may not increase the synthesis of adhesion molecules as much as microgravity.

Zhang, Ye↗

NASA NTRS2015

WetLab-2: Providing Quantitative PCR Capabilities on ISS

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a system capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project has developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage that it uses non-toxic chemicals, alcohols or other organics. The resulting RNA is transferred into a pipette and then dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. These reaction tubes are mounted on rotors to centrifuge the liquid to the reaction window of the tube using a cordless drill. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The resulting process takes less than 30 min to have tubes ready for loading into the qRT-PCR unit.The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, the Cepheid SmartCycler, that will fly in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid thermal ramp times and four-color detection. The ability to detect up to four fluorescent channels will enable multiplex assays that can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system will have the capability to downlink data from the ISS to the ground after a completed run and to uplink new programs. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time for subsequent trials, without the need for sample return and re-flight to sample multigenerational changes. The system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015. The WetLab-2 Project is supported by NASAs ISS Program at JSC, Code OZ.

ISS tools↗

NASA NTRSDate not supplied

RNASeq and Fluorescence Analysis of the Response of ERF2 and ERF104 in Arabidopsis thaliana under Simulated Altered Gravity

As NASA moves closer to long-term human space exploration, the need to understand how to sustain life in space is increasingly pressing. Plants are essential to human sustenance, making it important to understand how spaceflight affects plant health. We used differential gene expression analysis to examine GLDS-251 (RNAseq analysis of the response of Arabidopsis thaliana to fractional gravity under blue-light stimulation during spaceflight) from NASA’s GeneLab data repository and found downregulation of ERF2 and ERF104, transcription factors of the ethylene response factor families, that integrate hormonal pathways involved in abiotic stress responses. Downregulation of ERF2 and ERF104 during spaceflight may indicate a dysregulation of the ethylene signaling pathway. Our hypothesis is that altered gravity downregulates the expression of ERF2 and ERF104 in Arabidopsis thaliana, altering the ethylene signaling pathway and affecting the electron transport chain and light-dependent reactions in chloroplast thylakoids. To test this hypothesis, we propose to grow A. thaliana seedlings (wild-type and mutant/knockout of ERF2 and ERF104) in altered gravity conditions to determine the effects on the expression of ERF2, ERF104, and photosynthesis. We anticipate that ERF2 and ERF104 will be underexpressed in altered gravity conditions and result in decreased regulation of the ethylene signaling pathway.

Arabidopsis↗

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 NTRS2012

Mechanical Stress and Antioxidant Protection in the Retina of Hindlimb Suspended Rats

It has been postulated that hindlimb suspension (HS) causes a cephalad fluid shift in quadrupeds similar to that occurring to humans in microgravity. Therefore, HS may provide a suitable animal model in which to recapitulate the ocular changes observed in the human Visual Impairment and Intracranial Pressure (VIIP) syndrome. This work reports preliminary results from a tissue sharing project using 34 week-old Brown Norway rats. Two different experiments compared normal posture controls and HS rats for 2 weeks and rats exposed to HS for 2 weeks but allowed to recover in normal posture for 2 additional weeks. The effects of two nutritional countermeasures, green tea extract (GT) and plant polyphenol resveratrol (Rv), were also evaluated. Green tea contains the antioxidant epigallocatechin gallate (EGCG). qPCR gene expression analysis of selected targets was performed on RNA from isolated retinas, and histologic analysis was done on one fixed eye per rat. The transcription factor early growth response protein 1 (Egr1) was upregulated almost 2-fold in HS retinas relative to controls (P = 0.059), and its expression returned to control levels after 2 weeks of recovery in normal posture (P = 0.023). HS-induced upregulation of Egr1 was attenuated (but not significantly) in retinas from rats fed an antioxidant rich (GT extract) diet. In rats fed the GT-enriched diet, antioxidant enzymes were induced, evidenced by the upregulation of the gene heme oxygenase 1 (Hmox1) (P = 0.042) and the gene superoxide dismutase 2 (Sod2) (P = 0.0001). Egr1 is a stretch-activated transcription factor, and the Egr1 mechanosensitive response to HS may have been caused by a change in the translaminal pressure and/or mechanical deformation of the eye globe. The observed histologic measurements of the various retinal layers in the HS rats were lower in value than those of the control animal (n = 1), however insufficient data were available for statistical analysis. Aquaporin 4, a water-selective channel involved in interstitial fluid homeostasis, showed an upregulated trend in HS retinas; however, these results are preliminary. Total retinal thickness increased significantly (P = 0.049) in HS rats fed a resveratrol enriched diet compared to HS rats on a normal diet. This change appeared to be reversed during the 2 weeks of recovery post HS, but no differences in retina thickness were observed between HS animals and HS recovered animals when both groups consumed a normal diet. The reversibility of the increase in retinal thickness induced by resveratrol during HS may therefore reflect an interaction between the stress provoked by HS and the cytoprotective mechanisms elicited by resveratrol

Glass, Aziza↗

NASA NTRS2016

Transgenic Mouse Model for Reducing Oxidative Damage in Bone

Bone loss can occur due to many challenges such age, radiation, microgravity, and Reactive Oxygen Species (ROS) play a critical role in bone resorption by osteoclasts (Bartell et al. 2014). We hypothesize that suppression of excess ROS in skeletal cells, both osteoblasts and osteoclasts, regulates skeletal growth and remodeling. To test our hypothesis, we used transgenic mCAT mice which overexpress the human anti-oxidant catalase gene targeted to the mitochondria, the main site for endogenous ROS production. mCAT mice have a longer life-span than wildtype controls and have been used to study various age-related disorders. To stimulate remodeling, 16 week old mCAT mice or wildtype mice were exposed to treatment (hindlimb-unloading and total body-irradiation) or sham treatment conditions (control). Tissues were harvested 2 weeks later for skeletal analysis (microcomputed tomography), biochemical analysis (gene expression and oxidative damage measurements), and ex vivo bone marrow derived cell culture (osteoblastogenesis and osteoclastogenesis). mCAT mice expressed the transgene and displayed elevated catalase activity in skeletal tissue and marrow-derived osteoblasts and osteoclasts grown ex vivo. In addition, when challenged with treatment, bone tissues from wildtype mice showed elevated levels of malondialdehyde (MDA), indicating oxidative damage) whereas mCAT mice did not. Correlation analysis revealed that increased catalase activity significantly correlated with decreased MDA levels and that increased oxidative damage correlated with decreased percent bone volume (BVTV). In addition, ex-vivo cultured osteoblast colony growth correlated with catalase activity in the osteoblasts. Thus, we showed that these transgenic mice can be used as a model to study the relationship between markers of oxidative damage and skeletal properties. mCAT mice displayed reduced BVTV and trabecular number relative to wildtype mice, as well as increased structural model index in the cancellous tibia. Treatment caused bone loss in wildtype mice, as expected. Treatment also caused deficits in microarchitecture of mCAT mice, although less severe than wildtype mice in some parameters (percent bone volume, structural model index and cortical area). In conclusion, our results indicate that endogenous ROS signaling in both osteoblast and osteoclast lineage cells contributes to skeletal growth and remodeling, and quenching oxidative damage could play a role in bone loss prevention.

transgenic mice↗

NASA NTRS2016

On-orbit RNA Purification and qRT-PCR Capabilities of the WetLab-2 System

NASA Ames Research Center’s WetLab-2 system brings new capabilities to the ISS for researchers. The system can lyse cells and extract RNA on-orbit from different sample types ranging from microbial cultures to animal tissues. Our purification method has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. qRT-PCR reactions are performed by dispensing the RNA into reaction tubes that contain all lyophilized reagents needed to perform the analysis. The system uses a Cepheid® SmartCycler that allows for multiplexing of assays, this can be used to normalize for RNA concentration and integrity and to study multiple genes of interest in each tube. There are a total of 16 independent PCR modules each capable of detecting up to four fluorescent channels. The WetLab- 2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to purify and stabilize RNA on-orbit can eliminate the confounding effects of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of some samples. It also has the benefit of minimizing the needed downmass. Conducting qRT-PCR and generating results on-orbit is also an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also provide benchmarking prior to sample return. The system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver is scheduled to launch on SpaceX-8 this spring. Pending operations, the preliminary results from the validation flight will be presented. To support the needs of future researchers, we are adapting our system to purify RNA from two additional sample types: fibrous tissue such as muscle and mammalian adherent cells grown on alginate beads. Progress of this work will also be presented. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

Parra, Macarena↗

NASA NTRS2014

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development; Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science. Mouse livers and spleens were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection operations including storage at -80 C for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RIN(is) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNAlater) and liver (fast-freezing) at various time points post-euthanasia (from 5 min up to 105 min). The RNA recovered was of high quality (spleen, RIN (is) greater than 8; liver, RIN (is) greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at the latest time (105 min). Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) Many additional tissues for gene expression analysis can be obtained by dissection following prolonged storage of the tissue in situ at -80 C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to all future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

tissues↗

NASA NTRS2003

Gene Expression Dynamics Inspector (GEDI): for integrative analysis of expression profiles

Genome-wide expression profiles contain global patterns that evade visual detection in current gene clustering analysis. Here, a Gene Expression Dynamics Inspector (GEDI) is described that uses self-organizing maps to translate high-dimensional expression profiles of time courses or sample classes into animated, coherent and robust mosaics images. GEDI facilitates identification of interesting patterns of molecular activity simultaneously across gene, time and sample space without prior assumption of any structure in the data, and then permits the user to retrieve genes of interest. Important changes in genome-wide activities may be quickly identified based on 'Gestalt' recognition and hence, GEDI may be especially useful for non-specialist end users, such as physicians. AVAILABILITY: GEDI v1.0 is written in Matlab, and binary Matlab.dll files which require Matlab to run can be downloaded for free by academic institutions at http://www.chip.org/~ge/gedihome.html Supplementary information: http://www.chip.org/~ge/gedihome.html.

Database Management Systems↗

NASA NTRS2001

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Non-NASA Center↗

NASA NTRSDate not supplied

Efficacy of Dried Plum to Countermeasure Simulated Spaceflight Exposure

Human exploration of the space environment has been a topic of interest for decades; however, the threat to physiological health posed by ionizing radiation and microgravity effects remains a concern. Previous research utilizing combined hindlimb unloading and irradiation to simulate spaceflight effects found that a dried plum diet can mitigate adverse effects of simulated spaceflight on skeletal health. Though the mechanism is not fully understood we believe this protective effect is due to its high antioxidant capacity and polyphenolic content. This study seeks to extend these findings to investigate if dried plum is an effective countermeasure in other tissues relating to aging and neurotoxicity. Male C57BL/6 J mice were assigned to one of eight groups (n = 10/group) and were fed either a controlled diet or dried plum diet, underwent normal loading or hindlimb unloading as well as exposure 2 Gy gamma radiation or sham exposure. At the end of the experiment, mice were euthanized, and RNA was extracted from brain and bone marrow tissue and PCR was employed to analyze significant changes in gene expression profiles. Analysis of five house-keeping genes revealed genes B2M (neurotoxicity) and GUSB (aging) to be the most stable genes across all treatments and were used to find the fold change within our genes of interest. Through this, and analysis via ANOVA, we were able to identify genes with significant altered expression levels within brain and bone marrow tissues related to neurotoxicity and aging. Considering previous research, we hypothesize that the dried plum diet’s antioxidative properties ameliorated the oxidative stress from irradiation and microgravity via altered levels of gene expression. Identified expression changes will provide insights into molecular mechanisms behind the observed protective effects. These findings present potential avenues for future interventions to alleviate negative impacts of spaceflight on astronaut health.

Faisal Ahmad↗

NASA NTRSDate not supplied

The Efficacy of Dried Plum to Counter Spaceflight Microgravity and Radiation

Faisal Ahmad↗

NASA NTRSDate not supplied

Combining RNA-SEQ Datasets from NASA GENELAB: An Evaluation of Correction Methods

Background: Conducting space biology experiments aboard the International Space Station, particularly those utilizing complex model organisms like mice, is expensive and difficult due to limited crew availability, hardware, and space. As a result, sample numbers from these studies are low, reducing the statistical power of any one experiment. Aggregating spaceflight datasets serves as a method to increase sample numbers, allowing for novel insights through bioinformatic analysis of ‘omics data from merged datasets. However, aggregating datasets can introduce unwanted variation including 1) differences in sample handling, processing, and sequencing platforms between datasets (technical variation) as well as 2) differences in experimental design between datasets. Methods: In the present study, NASA GeneLab-hosted RNAseq datasets from mouse liver tissues were used to evaluate several statistical methods to correct for this unwanted variation through two approaches, reference-based and standard. The following correction algorithms were applied with (reference-based) and/or without (standard) considering Universal Mouse RNA Reference samples: ComBat and ComBat_seq from the SVA package, the median polish, empirical Bayes, and ANOVA-based algorithms from the MBatch package, and negative binomial regression normalization in the DESeq2 package. For each approach, after the correction algorithm was applied, differential gene expression (DGE) analysis of flight and ground control samples was performed with the combined data. The robustness of each tool was evaluated using BatchQC to determine statistical differences between datasets before and after correction, Principal Component Analysis to evaluate global gene expression in samples before and after correction, and by comparing DGE analysis of individual datasets and combined datasets before and after correction. Results: The results showed that the reference-based approach introduced several additional (and likely artificial) differentially expressed genes when compared with the respective standard approach. Conclusions: Of the methods tested, standard ComBat_seq and DESeq2 were identified as the most robust correction methods for combining spaceflight mouse liver RNAseq datasets hosted on GeneLab.

Finsam Samson↗

NASA NTRSDate not supplied

Evaluation of Correction Methods for NASA GeneLab Transcriptomic Datasets

Conducting space biology experiments aboard the International Space Station, particularly those utilizing complex model organisms like mice, is expensive and difficult due to limited crew availability, hardware, and space. As a result, sample numbers from these studies are low, reducing the statistical power of any one experiment. Aggregating spaceflight datasets serves as a method to increase sample numbers, allowing for novel insights through bioinformatic analysis of ‘omics data from merged datasets. However, aggregating datasets can introduce unwanted variation including 1) differences in sample handling, processing, and sequencing platforms between datasets (technical variation) as well as 2) differences in experimental design between datasets. In the present study, NASA GeneLab-hosted RNAseq datasets from mouse liver tissues were used to evaluate several statistical methods to correct for this unwanted variation through two approaches, reference-based and standard. The following correction algorithms were applied with (reference-based) and/or without (standard) considering Universal Mouse RNA Reference samples: ComBat and ComBat_seq from the SVA package, median polish, empirical Bayes, and ANOVA-based algorithms from the MBatch package, and negative binomial regression normalization in the DESeq2 package. For each approach, after the correction algorithm was applied, differential gene expression (DGE) analysis of flight and ground control samples was performed with the combined data. The robustness of each tool was evaluated using BatchQC to determine statistical differences between datasets before and after correction, Principal Component Analysis to evaluate global gene expression in samples before and after correction, and by comparing DGE analysis of individual datasets and combined datasets before and after correction. The results showed that the reference-based approach introduced several additional (and likely artificial) DEGs when compared with the respective standard approach. Of the methods tested, standard ComBat and DESeq2 were identified as the most robust correction methods for combining spaceflight mouse liver RNAseq datasets hosted on GeneLab.

GeneLab↗

NASA NTRSDate not supplied

Evaluation of Correction Methods for NASA GeneLab Transcriptomic Datasets

Conducting space biology experiments aboard the International Space Station, particularly those utilizing complex model organisms like mice, is expensive and difficult due to limited crew availability, hardware, and space. As a result, sample numbers from these studies are low, reducing the statistical power of any one experiment. Aggregating spaceflight datasets serves as a method to increase sample numbers, allowing for novel insights through bioinformatic analysis of ‘omics data from merged datasets. However, aggregating datasets can introduce unwanted variation including 1) differences in sample handling, processing, and sequencing platforms between datasets (technical variation) as well as 2) differences in experimental design between datasets such as sex or age of the model organism used. In the present study, NASA GeneLab-hosted RNAseq datasets from rodent liver tissues were used to evaluate several statistical methods to correct for this unwanted variation through two approaches, reference-based and standard. The following correction algorithms were applied with (reference-based) and/or without (standard) considering Universal Mouse RNA Reference samples: ComBat and ComBat_seq from the SVA package, median polish, empirical Bayes, and ANOVA-based algorithms from the MBatch package, and negative binomial regression normalization in the DESeq2 package. For each approach, after the correction algorithm was applied, differential gene expression (DGE) analysis of flight and ground control samples was performed with the combined data. The robustness of each tool was evaluated using BatchQC, to determine statistical differences between datasets before and after correction, Principal Component Analysis, to evaluate global gene expression in samples before and after correction, and by comparing DGE analysis of individual datasets and combined datasets before and after correction. The results showed that the reference-based approach introduced several additional (and likely artificial) DEGs when compared with the standard approach. Thus, the most robust standard correction will be implemented in the GeneLab Visualization 2.0 platform when datasets are combined.

GeneLab, RNA-seq, Batch Correction↗