Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Gene”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42
All sourcesNASA NTRSDOE OSTI
NASA NTRS2018

Late Effects of Heavy-Ion Irradiation on Ex Vivo Osteoblastogenesis and Cancellous Bone Microarchitecture

Prolonged spaceflight causes degeneration of skeletal tissue with incomplete recovery even after return to Earth. We hypothesize that heavy-ion irradiation, a component of Galactic Cosmic Radiation, damages osteoblast progenitors and may contribute to bone loss during long duration space travel beyond the protection of the Earth's magnetosphere. Male, 16 week-old C57BL6/J mice were exposed to high-LET (56-Fe, 600MeV) radiation using either low (5 or 10cGy) or high (50 or 200cGy) doses at the NASA Space Radiation Lab and were euthanized 3-4, 7, or 35 days later. Bone structure was quantified by microcomputed tomography (6.8 μm pixel size) and marrow cell redox assessed using membrane permeable, free radical-sensitive fluorogenic dyes. To assess osteoblastogenesis, adherent marrow cells were cultured ex vivo, then mineralized nodule formation quantified by imaging and gene expression analyzed by RT-PCR. Interestingly, 3-4 days post-exposure, fluorogenic dyes that reflect cytoplasmic generation of reactive nitrogen/oxygen species (DAF-FM Diacetate or CM-H2DCFDA) revealed irradiation (50cGy) reduced free radical generation (20-45%) compared to sham-irradiated controls. Alternatively, use of a dye showing relative specificity for mitochondrial superoxide generation (MitoSOX) revealed an 88% increase compared to controls. One week after exposure, reactive oxygen/nitrogen levels remained lower (24%) relative to sham-irradiated controls. After one month, high dose irradiation (200 cGy) caused an 86% decrement in ex vivo nodule formation and a 16-31% decrement in bone volume to total volume and trabecular number (50, 200cGy) compared to controls. High dose irradiation (200cGy) up-regulated expression of a late osteoblast marker (BGLAP) and select genes related to oxidative metabolism (Catalase) and DNA damage repair (Gadd45). In contrast, lower doses (5, 10cGy) did not affect bone structure or ex vivo nodule formation, but did down-regulate iNOS by 0.54-0.58 fold. Thus, both low- and high-doses of heavy-ion irradiation cause time-dependent, adaptive changes in redox state within marrow cells but only high doses (50, 200cGy) inhibit osteoblastogenesis and cause cancellous bone loss. We conclude space radiation has the potential to cause persistent damage to bone marrow-derived stem and progenitor cells for osteoblasts despite adaptive changes in cellular redox state.

LATE EFFECTS OF HEAVY-ION IRRADIATION↗
NASA NTRS2018

Increasing the Statistical Rigor of Cross-Species Differential Expression Analysis

Microgravity inflicts substantial, but undercharacterized, pressure on organisms that induces metabolic responses such as increased microbial virulence and antibiotic resistance, altered organ weights in developing rats, and loss of bone tissue in astronauts. Numerous studies have analyzed the effects of microgravity on specific organisms, tissues, or test conditions, but these projects are necessarily limited by the small sample size of space research. Increasing the sample size of spaceflight studies is non-trivial; however, pooling data from numerous studies can greatly increase the statistical rigor of comparative analyses. The GeneLab houses datasets from 73 spaceflight studies that performed transcription profiling assays. These data encompass a diverse array of organisms ranging from Escherichia coli to Mus musculus to Homo sapiens and comprise studies analyzing ionizing radiation, mammalian pregnancy, etc. Collectively, the GeneLab database contains a large quantity of transcription assays and RNA sequence data analyzing Differential Gene Expression (DGE) between microand normogravity. Xspecies, a cross-species analysis method for DGE developed by Kristiansson, et al. in 2012, identifies homologous genes between species that are universally up- or downregulated in response to test conditions. Previous work by an intern at GeneLab applied Xspecies to 19 datasets containing seven different species and identified 14 homologous groups differentially expressed under spaceflight conditions including several heat shock proteins and cytoskeletal components. Unfortunately, these results may be biased by the disproportionate number of studies on Arabidopsis thaliana (5) and Mus musculus (6) and the results are not normalized by evolutionary distances. Here, we present modifications to the Xspecies algorithm that permits incorporation of multi-omic data and normalizes data for effect size, directionality, and evolutionary distances. We then apply this algorithm to all currently available GeneLab studies

Xspecies↗
NASA NTRS2018

Physical and Biological Properties of Cobalt- and Copper-Doped Calcium Phosphates as Bone Substitute Materials

Arthritis, osteoporosis, and other bone diseases and defects are common medical issues worldwide. By utilizing ceramic biomaterials as bone substitutes to treat these diseases, bone regeneration can be promoted and toxicity from the substitute material can be limited. Calcium phosphate (CaP) ceramics have become popular as bone substitutes due to their biocompatibility and similarity in composition to natural bone. The purpose of this research was to investigate the physical properties and biological responses of CaP-based bone substitutes, doped with metal ions. Some metal ions are present in natural bone in small amounts, and recent studies show they affect the biological responses of CaPs significantly. Dopants such as magnesium (Mg), strontium (Sr), silicon (Si) and iron (Fe) alter the phase composition, mechanical properties, osteogenesis and angiogenesis properties of CaPs, depending on their concentration and method of addition. In the present study, the effects of cobalt (Co) and copper (Cu) on the physical and biological properties of two main CaP materials, brushite cement (BrC) and tricalcium phosphate (TCP), have been investigated. Different concentrations of dopants in forms of oxide and/or chloride were selected and their effect on phase composition, sintering behavior, density, setting time, compressive strength and in vitro interaction with osteosarcoma and osteoblast cells were studied. Different techniques of sample preparation, challenges during cement preparation and compact sample pressing, sintering, and methods of dopant addition are discussed. The presence of Cu in tricalcium phosphate was found to increase thermal stability of the material and decrease the impact of sintering temperature on porosity. In addition, Cu caused a reduction in expression of inflammatory gene markers by human osteoblast cells and an increased expression at early time points due of osteoinductive markers.The addition of Cu and Co to brushite cement caused an increase in thermal stability of the material, and the addition of Cu caused significant increase in setting time. Small dopant amounts of Co caused decrease in setting time, but higher amounts caused an increase. In addition, Cu dopant in small amounts resulted in an increase of compressive strength. Both Cu and Co led to a decrease in expression of inflammatory gene markers by osteoblast and osteosarcoma cells respectively, and Cu also caused an increase in osteoinductive marker expression. Thus, these results indicate that Cu and Co have a positive impact on the physical, mechanical and biological properties of calcium phosphate biomaterials.

Cummings, Haley V.↗
NASA NTRS2017

Renin-Angiotensin System (RAS) in Hematopoietic Stem/Progenitor Cells (HS/PC) Predicts Vaso-Reparative Dysfunction and Progression of Diabetic Retinopathy (DR)

Purpose: We tested the hypothesis that loss of angiotensin converting enzyme 2 (ACE2) within diabetic HS/PCs (Hematopoietic Stem/Progenitor Cells) would be detrimental to HS/PC reparative function, and alter their ability to contribute to vascular remodeling in human subjects and rodent models of DR (Diabetic Retinopathy). Methods: Subjects (n52) were recruited as controls (n13) or diabetics (n39) with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ HS/PCs were isolated from peripheral blood. RAS (Renin-Angiotensin System) gene expression and migration was measured. Diabetic ACE2 knockout (KO)C57BL6-Ins2 (Akita) mice at 3, 6 and 9 months of diabetes were compared to age-matched controls. Bone marrow HS/PC populations were analyzed by flow cytometry and migration and proliferation studies performed. Results: ACE2 gene expression in human CD34+ cells from diabetics without DR was increased compared to controls (p0.0437). Mas receptor mRNA was also increased in diabetics without DR, but reduced with the onset of NPDR (p0.0002), suggesting a loss of compensation. DR was associated with CD34+ cell migratory dysfunction. By VESGEN analysis, vessel density measured by several confirming parameters in early NPDR (n3) was greater than in normal retina (n6) in both arteries and veins, which suggests active retinal remodeling. ACE2KO-Akita and Akita cohorts showed reduced retinal thickness by OCT (Optical Coherence Tomography) at 9 months of diabetes. Absence of ACE2 in 9-month Akita mice led to an accelerated increase in acellular capillaries compared to diabetic alone. Electroretinogram (ERG) in ACE2KO-Akita mice resulted in persistent deterioration of the neural retina. Reparative function studies showed that ACE2KO exacerbated diabetes-induced impairment of LK (Low Potassium) cell migration and proliferative functions as early as 3-month of diabetes (p0.0019). Conclusions: Retinopathy and adverse vascular remodeling in subjects with diabetes was associated with a loss of the protective arm of RAS in HS/PCs. Loss of ACE2 exacerbated vascular dysfunction in diabetic mice.

retina↗
NASA NTRS2017

The Role of CDKN1a/p21 in Cellular Senescence of Bone Marrow Stem Cells Under Spaceflight Stressors

Spaceflight environments and their associated conditions, such as microgravity and space radiation, cause many biological functions formerly considered to be standard to behave in nonstandard ways. Exposure to microgravity has shown to induce deleterious effects in stem cell-based tissue regeneration, leading to immune system and healing response impairments as well as muscle and bone density loss. Such risks must be mitigated in order for long-term human space exploration to proceed. Thus, our work seeks to explore mechanisms of stem cell-based tissue regeneration that experience changes in spaceflight environments. Cellular senescence is a process of inducing cell cycle arrest that can be initiated by various stimuli. This function is influenced by two major pathways, controlled by p53 and pRB tumor suppressor proteins. p53 activity targets the cyclin-dependent kinase inhibitor gene p21Cdkn1a in osteogenic cell cycle arrest. Under conditions of mechanical unloading, stem cell-based tissue regeneration has shown to be decreased in both proliferation and differentiation, as many cells are arrested in progenitor states. p21 has shown upregulation in expression under conditions of microgravity, suggesting its role in regenerative bone formation arrest in space. p21 levels are found to be elevated independent of p53, suggesting a decrease in proliferation and regeneration without apoptosis, but rather through cell cycle arrest alone. Thus, we hypothesize that p21 is a mediator of cellular senescence in bone marrow stem cells. Culturing of bone marrow stem cells from wild type and p21 knockout mice under osteoblastogenic conditions will be completed to explore the role of p21Cdkn1a in stem cell proliferation and maturation. We believe that decreases in somatic stem cell differentiation may occur after spaceflight due to signal pathway alterations that result in downstream inhibition of genes involved in differentiation, preventing tissue from repairing and regenerating normally.

Stem Cells↗
NASA NTRS2017

The Effects of CDKN1a/p21 on Oxidative Stress and Mitochondrial Function During Long Duration Spaceflight

Broad tissue degeneration and the failure of normal tissue regenerative processes in microgravity because of mechanical unloading are increasing concerns for sustaining life in space as the duration of future flight missions increases. Work in our laboratory has identified normal adult stem cell-based tissue regenerative processes, such as the formation of new bone, cartilage, and immune cells, as being particularly sensitive to the stresses of mechanical unloading in microgravity. Our studies have also identified the inhibition of differentiation of marrow mesenchymal stem cells and activation of CDKN1ap21-mediated cell cycle arrest in proliferative osteoprecursor cells on the bone surface as potential mechanisms for spaceflight-induced skeletal changes. This finding, in combination with the role of CDKN1ap21 as a suppressor of mammalian tissue regeneration, suggests that this gene could be responsible for suppressing stem cell-based tissue regeneration in response to disuse. In this work, we hypothesized that CDKN1ap21 regulates regenerative bone formation in response to alterations in mechanical load and tested this hypothesis by studying the skeletal phenotype and stem cell regenerative ability of juvenile (4-11 weeks old) and adult (18 weeks-12 months old) p21 (--) knockout (KO) mice. Additionally, we analyzed bone micro-architectural properties, bone formation rates and differentiation capacity of bone marrow stem cells (BMSCs) from male and female KO mice exposed to hindlimb unloading (HU) for 15-30 days. We found that juvenile KO mice exhibited increased femoral trabecular and cortical bone formation, whilst three-point bending of the tibias from KO mice showed decreased bone stiffness. Conversely, adult KO mice exhibited no significant differences in micro-architectural properties compared to WT (wild-type) but woven bone structure was indicative of rapid bone remodeling. Furthermore, cortical bone properties showed similar characteristics to aged bone, including increased cross-sectional area and perimeter, whilst three-point bending showed increased stiffness and toughness. Interestingly, in-vitro, KO mice exhibited increased differentiation and mineralized nodule formation in osteoblastogenesis assays compared to WT. Preliminary results from CDKN1ap21 KO mice subjected to HU suggest altered sensitivity to mechanical unloading resulting in decreased cortical thickness compared to WT mice. However, KO mice subjected to short and long-duration HU show increased in-vitro differentiation potential of BMSCs to from form mature, mineral-forming osteoblasts, indicating maintenance of regenerative potential. Analysis of bone formation rates, cell proliferation rates and key genes of interest are currently underway. These results indicate a novel role for CDKN1ap21 in load-dependent osteoprogenitor proliferation and differentiation and that deletion of CDKN1ap21 results in an age-dependent release of osteoblast proliferation inhibition and increased bone formation and turnover.

Stem Cells↗
NASA NTRS2019

Deciphering the Effects of Microgravity Cell by Cell

Forces generated by gravity have a profound impact on the behavior of cells in tissues affecting the course of the cell cycle and differentiation fate of progenitors in mammalian tissues. These cells are contributing to normal tissue regenerative health and defence against disease. In Human space exploration context, it is extremely important to determine spaceflight provoked changes in tissue's regenerational capabilities. Microgravity experienced during spaceflight causes unloading and mechanical disuse on all orthostatic support tissues, therefore impacting stem cell fate and lineage commitment decisions. Investigating how ESCs respond to mechanical stimulation is a platform for fundamental developmental and regeneration research applicable for spaceflight. However, the gene expression programs associatiated with early committment stem cell pathways in response to physical stimulation are not readily known. Single-cell RNA-seq technologies have recently revolutionized the world of molecular biology by providing the capability to assess gene expression pattern within a single cell. Our method isolates and separately barcodes mRNAs from thousands of single cells and sequences their expressomes. Understanding regenerative processes on a molecular level would not only help reduce long-term spaceflight impact on health, but also may enable the development of novel tissue regenerative approaches to tissue degeneration on Earth.

Single cell sequencing↗
NASA NTRS2019

Evaluating Effects of Altered Gravity on the Nervous System Using D. melanogaster

A comprehensive understanding of the effects of spaceflight and altered gravity on human physiology is necessary for continued human space exploration and long-term space habitation. The oxidative stress response has been identified in astronauts exposed to short- and long-term space missions that are exposed to the multitude of stress factors of spaceflight, including altered gravity and radiation exposure. Reactive oxygen species (ROS) are byproducts of homeostatic cellular metabolism, yet when overproduced the oxidative stress response ensues, rendering molecules destructive causing cell death and inflammation. Controlling aberrant ROS production is necessary to prevent pathological consequences, in particular within the nervous system, since neurons are extremely sensitive overexpressed ROS insults. We hypothesize that exposure to altered gravity triggers the oxidative stress response, leading to impairments in the nervous system. In this study, we used a well-established spaceflight model organism, Drosophila melanogaster, to assess altered gravity associated changes in the nervous system using a ground-based hypergravity model. Acute hypergravity resulted in an induction of oxidative stress-related genes with an increase in reactive oxygen species (ROS) in fly brains (p<0.001). Also, qPCR analysis shows that parkin gene expression is significantly reduced in these fly brains(p<0.05). Additionally, chronic hypergravity resulted in depressed locomotor phenotype in these flies (p<0.05) in conjunction to decreased dopaminergic neuron counts (p<0.0001) and increased apoptosis in these fly brains (p<0.0001). Further, assessment of neurological changes, including the neuronal architecture, synaptic integrity and genetic regulation caused by hypergravity conditions were noted. Overall, our results validate chronic hypergravity simulation as a behavioral model to study spaceflight effects, and oxidative stress pathway as a potential avenue for countermeasure development for astronauts undergoing short- and long-term missions and for neurodegenerative research on Earth.

Mhatre, Siddhita↗
NASA NTRS2019

Cross Kingdom Analysis of Data Within the GeneLab Repository Identifies a Potential Conserved Response of Life to the Stress Associated with Spaceflight

It is important to determine the health risks and potential survival for astronauts associated with long-term space missions. This entails not only understanding the impact the space environment will have on humans, but also how it will affect other organisms needed for humans to survive in space such as plants. In addition, it has been reported in the literature that hundreds of genes seem to be conserved and/or transferred between different organisms from bacteria, archaea, fungi, microorganisms, and plants to animals. Since space travel involves humans in a closed environment over a long period of time, we hypothesize that potential conserved biological factors will occur between the different organisms in that environment possibly due to transfer of genes. Determining the conserved factors that are commonly being regulated in space can shed insight into possible universal master regulators and also determine the symbiotic relationship between the organisms in space. Utilizing NASA's GeneLab Data Repository (a rapidly expanding, curated clustering of spaceflight-related ‘omics-level datasets for all organisms), we were able to uncover a novel pathway and factors that were commonly shared between humans, mice, plants, C. Elegans, and drosophilas. Through ChIP-Seq enrichment analysis techniques utilizing various GeneLab datasets from each species that were flown in space, we found the following factors to be conserved across all species: oxidative stress, DNA damage (through GABPA/NRFs and NFY), SIX5, GTF2B and glutamine synthetase. Such commonalities would likely reflect the effects of factors such as microgravity and the increased radiation exposure inherent in spaceflight on basic physical processes shared by all biological systems at the cellular level. Differences between organismal responses revealed by GeneLab's data should also help understand the unique reactions to life in space that arise from the very different lifestyles of microbes, animals and plants.

Barker, Richard↗
NASA NTRS2019

Gravity as a Continuum: Effects of Altered Gravity on Drosophila Melanogaster Immunity

The impact of spaceflight on immune function is undoubtedly a critical focus in the area of space biology and human health research. Heat shock proteins (Hsp) are an evolutionarily conserved family of proteins that are expressed in response to cellular and physiological stressors, experienced during radiation exposure, confinement, circadian rhythm disruption, and altered gravity (hypergravity experienced at launch/landing and microgravity experienced in-flight). In particular, Hsp70 aids in the folding of proteins, facilitates the movement of proteins across the membranes during signal transductions and can stimulate innate immunity. Since Hsp70 is induced during cellular stress, and can act as a stimulator for innate immunity, we sought to address how a loss of Hsp70 affects immunity, under the stress-inducing model of acute and chronic hypergravity. Moreover, the effects of gravity as a continuum on the induction of Hsps and key immune genes were also assessed to determine if increased cellular stress, via increased gravity (g)-force, contributes to immune dysfunctions. For this, wildtype (W1118) and Hsp70 deficient (Hsp70null) Drosophila melanogaster were subjected to simulated hypergravity at increasing levels of g-force (1.2g, 3g, and 5g) for acute (1hr) and chronic (7-day) timepoints and were compared to 0g 'non-hypergravity' controls. Following simulation, whole bodies were sex-segregated, RNA was isolated and quantitative (q)PCR was performed to determine differential immune gene expression profiles. Further, functional output of hemocytes were assessed by a phagocytosis assay. Collectively, these studies evaluated the effects of Hsp70 in the context of immunity during acute and chronic hypergravity. Indeed, relevance for this work can directly translate to acute effects of launch/landing gravitational forces upon liftoff (~1.7g) and entry (~3.4g) that astronauts experience. In addition, the effects of chronic cellular stress is directly relevant to the immune health of astronauts on long duration missions, as well. Thus, as we approach the goal of returning to the Moon and landing the first humans on Mars, an evaluation of gravity as a continuum and the stress-inducing effects of altered gravity experienced during spaceflight on astronaut immunity and health are necessary.

Olivieri, Joe↗
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 NTRSDate not supplied

CDKN1A/P21’S Role in Osteoprogenitor Fate Regulation in Response to Mechanical Loading: A Single Cell Resolution Investigation

Forces generated by gravity in load-bearing tissues such as bone marrow promote stem cell-based tissue-regenerative processes by increasing proliferation and differentiation of tissue progenitors. Conversely, in microgravity, mouse bone marrow mesenchymal and hematopoietic precursors down-regulate differentiation markers and up-regulate stemness maintenance genes. During microgravity, these transcriptomic changes are associated with Cdkn1a overexpression in osteoprogenitors, and further the homozygous deletion of the CDKN1a gene in the mouse model results in elevated stem-cell based regeneration of severed digits and ear-hole punches. In this work we sought to test the hypothesis that gravity mechanotransduction regulates mesenchymal stem cell derived osteoprogenitor based osteogenesis by modulating proliferation and differentiation fates at specific cell cycle stages via a p21/CDKN1a-regulated mechanism.. To test our hypothesis, we isolated primary mouse bone marrow osteoprogenitors from Cdkn1a-/- (null) or wildtype mice, cultured the cells for 7-days unloaded in osteogenic differentiation media, to allow commitment of the adherent cell population, and subsequently, cultured an additional 48-hours under cyclic stretch or static control loading conditions. After 48-hours of dynamic mechanical loading, we used a 10X Genomics Chromium/Single Cell controller to generate bar-coded single cell Illumina libraries and sequenced expressomes for 6,000 static control and 6,000 cyclic stretch cells for each genotype. Stage-specific single cell analyses show Cdkn1a-/- osteogenic cultures are more differentiated with fewer progenitors, and that cyclic stretch further promotes the conversion of progenitors to osteoblasts in both wildtype and Cdkn1a-/- backgrounds. The proportion of late mineralizing osteoblasts doubled in Cdkn1a-/- cultures, but is not affected by load. Finally, single cell expression of Cdkn1a, in the wildtype cells, is most strongly suppressed by cyclic stretch both in early and late osteoblasts, and minimally in the progenitor population. Collectively, our results support the hypothesis that Cdkn1a constitutively plays a mechano-reversible anti-proliferative role during stem cell-based bone tissue regeneration, and suggests a new molecular target to counter regenerative deficits caused by disuse.

Eduardo Almeida↗
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↗
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

A METHOD TO REDUCE BIOBURDEN IN ASTROMATERIALS CURATION FACILITIES WITHOUT INTRODUCING UNWANTED CONTAMINATION

Introduction: NASA curates its Astromaterials collections in cleanrooms that are carefully monitored for particulate, inorganic and trace metal contamination. Current sample collections are not particularly susceptible to organic contamination or biological alteration. However, new collections like those from the OSIRIS-REx and Hayabusa2 missions will have organic contamination requirements and are susceptible to biodegradation. It will be necessary sterilize or at least disinfect curation labs, as well as tools and equipment in a manner that does not introduce additional contamination and does not affect the samples 1. Current curation cleaning procedures utilize isopropyl alcohol which offers some bioburden reduction, but is not effective against spore-forming bacteria or fungal spores 2. We present a modified disinfection method that uses ultrapure hydrogen peroxide to reduce bioburden inside curation labs and glove boxes without introducing contamination or damaging curation equipment. We tested this method in the meteorite processing lab as well as on a glovebox being cleaned for use in processing ANGSA (Apollo Next Generation Sample Analysis) samples and present the results of those tests. We discuss the limitations of this method and describe potential situations in which it will not be applicable. The CDC guidelines for disinfection andsterilization in healthcare facilities discusses over 15different methods for reducing bioburden in hospitalsettings 3. The most common method, steamsterilization, is well suited to sterilizing curationprocessing tools but cannot easily be used to sterilizecleanroom surfaces or large equipment likegloveboxes. Chemical sterilization with bleach(NaOCl) is also a common strategy in healthcare andpharmaceutical settings that presents materialcompatibility issues as well as serious inorganiccontamination concerns for curation facilities.Introducing a new source of Na and Cl into curationlabs is not acceptable. Other chemical methods likeethylene oxide, formaldehyde, iodophors andquaternary ammonium compounds could introduceorganic and inorganic contamination. We chose tofocus on hydrogen peroxide because it is generallycompatible with commonly used curation materialslike stainless steel, aluminum and Teflon and becauseit decomposes to oxygen and water. The CDCguidelines for hydrogen peroxide specify using a 7.5wt% solution at 25 ̊C with a contact time of 30 minutesfor high level disinfection and 6 hours for sterilization.High level disinfection is defined as a technique thatwill kill all microorganisms except large numbers ofbacterial spores 3. Methods: We prepared a solution of 7.5 wt%hydrogen peroxide from a stock solution of ultrapure30 wt% peroxide (JT Baker) and curation gradeultrapure water. This ultrapure water is already used incuration cleaning procedures and thus is not consideredand additional source of contamination. We conducteda materials compatibility test by exposing unanodizedand anodized 6061 T6 Al alloy to the peroxide solutionfor up to six hours and periodically inspecting thesurfaces for visible defects. We used this peroxide todisinfect the floor of the meteorite processing lab andthe interior of a curation glovebox by exposing thesesurfaces to the peroxide solution for 30 min. Thesurfaces were swabbed with a dry macrofoam swabbefore (Puritan Brand 2518051PFRNDFD) and afterperoxide treatment to collect microbes present on thesurfaces. Microbes were extracted by sonication fromthe swab into 15 ml of PBS (phosphate buffered saline)and inoculated onto the following media: TSA (trypticsoy agar) BA (blood agar), R2A (Reasoners 2 agar),Potato Dextrose Agar, Saboraud Dextrose Agar andSaboraud Dextrose Agar with 0.1 mg/ mlchloramphenicol. Four TSA plates and two BA plateswere inoculated with 0.1 ml of PBS each andincubated at 35 and 37 for 48 hours. Two R2A°C°Cplates (0.1 ml of PBS each) were incubated at 25 .°CThe remaining plates were inoculated with 0.2ml ofPBS and incubated at 30 ̊C for seven days. Afterincubation bacterial and fungal isolates were countedand transferred to new plates for identification usingthe VITEK24 automated system or by sequencing aportion of the barcode gene (16S rRNA for bacteria,small subunit gene for fungi) on an ABI 3500 Sangersequencer. Negative controls consisted of swabs thatwere opened in the sampling environment andanalyzed alongside the experimental samples.Results: A 6 hour exposure to hydrogen peroxideresulted in visible pitting on un-anodized 6061 Al, butnot on anodized surfaces. No visible pitting occurredafter a 30 minute exposure. Therefore, we decided tolimit our experimental tests to 30 min. exposures. 17bacterial CFU (colony forming units) representing 4distinct organisms were isolated from the meteorite processing lab floor prior to hydrogen peroxidetreatment. We were unable culture any organisms afterperoxide treatment. In the glovebox we were able toculture three bacterial CFU representing three distinctspecies, including a spore forming bacterium prior todisinfection with peroxide. After the peroxidetreatment we were unable to culture any organisms.Routine monitoring of the meteorite processing lab andthe glovebox did not indicate any increase in unwantedinorganic contamination after these peroxidetreatments. Discussion: A 30 minute treatment with 7.5 wt%peroxide appears to be an effective method forreducing bioburden on typical cleanroom surfaces. Themethod does not introduce unwanted organic orinorganic contamination and is compatible withcommonly used curation materials like stainless steel,Teflon and anodized aluminum alloys. Special careshould be taken with un-anodized aluminum.Prolonged exposure to hydrogen peroxide can causepitting on this material. We recommend using thismethod to disinfect curation labs and equipment whenbiological alteration is a concern. This method iseffective at room temperature and cannot be used todisinfect labs and equipment where the ambienttemperature is < 0 ̊C. Astromaterials samples shouldbe removed from the area where disinfection is tooccur. Hydrogen peroxide is a powerful oxidizingagent and will react with any organic carbon present inthe sample. References: [1.] Mccubbin, F. M. et al.Sp. Sci Rev(2019) doi:10.1007/s11214-019-0615-9. [2.] Mogul, R.et al.Astrobiology 18, ast.2017.1814 (2018). [3.]Rutala, W. A. & Weber, D. J. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. [4.] Pincus, D. H. in Encyclopedia of Rapid Microbiological Methods (2005).

A. B. Regberg↗
NASA NTRSDate not supplied

BLOOD-BASED MULTI-SCALE MODEL FOR CANCER RISK FROM GCR IN GENETICALLY DIVERSE POPULATIONS

OBJECTIVES AND METHODS This project addresses the challenge of understanding and predicting individual radiation sensitivity by integrating genetics, demographics and biomarker characteristics across species (mice and humans). We hypothesize that ex vivo DNA repair response to GCR components is a central determinant of cancer risk from space radiation and can serve as a biomarker of radiation risk in combination with genetics. Automated image quantification of 53BP1+ radiation-induced foci (RIF) during the first 4-48 h post-irradiation was performed as a function of dose and LET in non-immortalized primary skin fibroblasts derived from 76 mice across 15 strains (5 inbred reference strains and 10 collaborative-cross strains) exposed to X rays (0.1, 1 and 4 Gy), 350 MeV/n 40Ar and 600 MeV/n 56Fe (1.1 and 3 particles/100μm2), as well as in peripheral blood mononuclear cells (PBMCs) from 768 healthy donors (matched ethnicity, 50/50 male/female, 18-70 years old) exposed to gamma rays (0.1 and 1 Gy), 350 MeV/n 28Si, 350 MeV/n 40Ar and 600 MeV/n 56Fe (1.1 and 3 particles/100μm2). QUANTIFICATION OF 53BP1+ FOCI IN VITRO AND ASSOCIATIONS TO IN VIVO RADIATION SUSCEPTIBILITY IN 15 MOUSE STRAINS We reported in vitro repair kinetic and repairable fractions of RIF for the 15 mouse strains and introduced a mathematical model for RIF as a function of time, dose and LET. We noted that the metabolic activity of cells modulates the RIF response, and we introduced the open access tool terRIFic (Tool for Enhanced Results of RIF In Cells, https://radbiolab.shinyapps.io/terrific/) to correct for such bias using confluence level. Notably, at 4h post-irradiation, RIF/Gy decreased with dose or LET: as the dose or LET increases, so does the proximity of DNA double-strand-breaks (DSB) and our data suggest that proximal DSBs are brought together inside isolated RIF for repair. The RIF/Gy trend was inverted at 24h, suggesting RIF with high DSB content are more difficult to repair. We showed that in vitro metrics correlate with in vivo measurements in the same 15 mouse strains, such as survival levels of immune cells or spontaneous cancer incidence, suggesting a relationship between the efficiency of DSB repair and cancer risk or radiation toxicity. In addition to the efficiency of repair and persistent RIF, the amount of spontaneous foci before irradiation was also found to be strain dependent. Finally, we performed genome-wide association study in the same 15 mouse strains using all RIF phenotypes measured in vitro, identifying genes of interest and validating RIF as an ideal biomarker for individual radiation sensitivity. BASELINE 53BP1+ FOCI PREDICTS INDIVIDUAL HUMAN RESPONSE TO GCR COMPONENTS Based on the analysis of radiation responses of 576 donor PBMCs (using quantification of 53BP1+ foci, oxidative stress and cell death), we observed a wide variability of subject- and LET-dependent radiation responses, with radiation-induced DNA repair foci increasing with LET, though oxidative stress being notably reduced by high-LET irradiation, potentially due to a switch between hydrogen peroxide and oxygen radical-based mechanisms. We identified a relationship between few spontaneous DNA foci at baseline and increased DNA repair after irradiation, accompanied by an alteration in immunoregulatory cytokine secretion, which might be adapted as biomarkers to predict ionizing radiation sensitivity. Among demographic variables, only latent cytomegalovirus infection and age were predictive of high baseline foci formation. Finally, we have performed low-throughput whole genome sequencing of all samples and are currently in the process of identifying the genes and pathways associated with low and high-LET ionizing radiation sensitivity in humans.

53BP1↗
NASA NTRSDate not supplied

3D Construction of Biologically Derived Materials

System for the 3D Construction of Biologically Derived Materials, Structures, and Parts NASA has developed a novel approach for macroscale biomaterial production by combining synthetic biology with 3D printing. Cells are biologically engineered to deposit desired materials, such as proteins or metals, derived from locally available resources. The bioengineered cells build different materials in a specified 3D pattern to produce novel microstructures with precise molecular composition, thickness, print pattern, and shape. Scaffolds and reagents can be used for further control over material product. This innovation provides modern design and fabrication techniques for custom-designed organic or organic-inorganic composite biomaterials produced from limited resources. Benefits Conserves resources. Few raw or bulk starting materials needed Enables custom design of diverse materials Fast, portable, macroscale, on-demand manufacturing High-fidelity microstructures Uses commercially available parts Applications Biomaterials, biotechnology Organic-inorganic composite materials On-demand manufacturing In situ resource utilization Space stations Military Infrastructure materials The Technology Once genes for a desired material type, delivery mode, control method and affinity have been chosen, assembling the genetic components and creating the cell lines can be done with well-established synthetic biology techniques. A 3D microdeposition system is used to make a 3D array of these cells in a precise, microstructure pattern and shape. The engineered cells are suspended in a printable 'ink'. The 3D microdeposition system deposits minute droplets of the cells onto a substrates surface in a designed print pattern. Additional printer passes thicken the material. The cell array is fed nutrients and reagents to activate the engineered genes within the cells to create and deposit the desired molecules. These molecules form the designed new material. If desired, the cells may be removed by flushing. The end product is thus a 3D composite microstructure comprising the novel material. This innovation provides a fast, controlled production of natural, synthetic, and novel biomaterials with minimum resource overhead and reduced pre- and post-processing requirements.

3D↗
NASA NTRSDate not supplied

Single-Cell mRNA Sequencing of Bone Marrow Mesenchymal Stem Cell Lineages Shows that Gravity Mechanical Loading Regulates Regenerative Osteogenesis in a CDKN1A-Dependent Manner

Weight-bearing at 1g is increasingly recognized as an important stimulus driving stem cell-based tissue regenerative homeostasis and health. In the bone marrow compartment, mechanical stimuli from gravity can drive osteogenesis from mesenchymal progenitors and hematopoiesis from hematopoietic progenitors. Conversely, hematopoietic monocyte/macrophage progenitors can also be driven into osteoclastogenesis by mechanical unloading in microgravity. Although the cell cycle inhibitor gene Cdkn1a is known to be upregulated in microgravity, the great diversity of stem cell lineages and stages of differentiation in bone marrow, make it difficult to attribute gene expression changes to specific cell types. To overcome this limitation, we used single-cell mRNAseq analyses with both in-vitro and in-vivo strategies, including in-vitro stretch of marrow osteogenic cultures, and in-vivo hindlimb unloading, running wheels, treadmills, and exposure to real microgravity. For in-vitro studies marrow osteoprogenitors from WT and Cdkn1a-null mice were subjected to substrate stretch during osteogenesis, with static cultures simulating unloading. From those experiments we determined Cdkn1a-null cells transition more readily from progenitors to early osteoblasts and further to mineralizing osteoblasts. Additionally, single-cell mRNAseq resolution identified Cdkn1a suppression in response to mechanical loading predominantly in early proliferative osteoblasts rather than progenitor or mineralizing osteoblasts. These findings support the hypothesis that CDKN1A plays a mechano-reversible, anti-proliferative role during bone regenerative osteogenesis. To investigate in-vivo mechanoregulation of osteogenesis, mice were flown in microgravity during the Rodent Research-10 experiment comparing wildtype and Cdkn1a-null mice kept on ISS for 30 days. The mice were euthanized and dissected on-orbit in microgravity and femur bone marrow was collected using a custom sample isolation and live-cell cryo-preservation technique allowing for single cell transcriptomics post-flight processing. Ground control experiments with hindlimb-unloading, running wheels, and treadmills, were also conducted for a comprehensive analysis of mechanical loading effects on the regenerative health of bone tissue and its marrow stem cell progenitor lineages.

single cell sequencing↗