Results for “RNA sequencing” — Page 7

NASA NTRSDate not supplied

A Gene Expression and Histologic Approach to Study Production and Outflow of Cerebrospinal Fluid in Hindlimb Suspended Rats

INTRODUCTION: The Spaceflight Associated Neuro-ocular Syndrome (SANS) is thought to be associated with weightlessness-induced cephalad fluid shift, possibly associated with a chronic elevation of intracranial pressure (ICP) during long-duration ISS missions. Changes in cerebrospinal fluid (CSF) dynamics and cranial compliance might be involved in the ICP increase. It is not known whether CSF production and/or outflow are altered in microgravity, but changes at the molecular and cellular level in the structures that produce and regulate the transcellular and paracellular secretion and reabsorption of CSF may be relevant. In this study, we used the rat hindlimb suspension (HS) model to examine the relationship between intracranial pressure (ICP) and the cellular responses to the prolonged change in body posture elicited specifically in the choroid plexus (CP). This was evaluated by transcriptomics, histopathology and ultrastructure of the CP and arachnoid villi (AV). METHODS: ICP was measured by telemetry. The morphology, ultrastructure, and gene expression profile of the CP was examined using male 9-month-old Long Evans rats subjected to HS for 14 and 90 days. A subset of animals completing 90-day HS returned to normal posture for 14 and 90 additional days of recovery. All HS rats had age-matched cage controls maintained in normal posture. A group of animals was maintained in conditions of 1% CO2 throughout the entire protocol. The rat brains were carefully removed and preserved for various analyses, including transmission electron microscopy (TEM), immunohistochemical analysis of specific targets involved in CSF regulation, and RNA sequencing analysis of laser capture micro-dissected CP tissue from the lateral ventricles. SUMMARY OF RESULTS: The ICP record was limited, which impacted any conclusions derived from the results. However, the ICP of rats in the CO2-enriched atmosphere appeared increased compared to those in a normal air composition. HS of rats resulted in an altered transcriptomics profile in the choroid plexus compared to animals maintained at normal posture. This was observed during both the HS period and during normal posture recovery period following 90 days of suspension. The greatest number of differentially expressed genes was observed at 90 days HS. Elevated CO2 also led to a different transcriptomics profile in the CP. The histology and ultrastructure results should be considered preliminary due to the limited number of samples. Examination of the CP by TEM showed blood vessel congestion and microvilli swelling in the CP, as well as some subpial and periventricular gliosis, but no associations were observed with treatment. There was no evidence of a reduced clearance of b-amyloid in the periventricular, perivascular and subpial regions of the brain in HS animals versus those in normal posture. Immunohistochemical staining of aquaporin 4, showed the localization to the subpial region and ependyma with increased immunoreactivity in the brains of CO2 exposed rats. This work was supported by awards NNX15AW48G to S.Z. Animal tissue and live data was provided under a tissue/data sharing agreement with Dr. Charles Fuller, UC Davis.

S B Zanello↗

NASA NTRSDate not supplied

Transcriptomic Changes in Peripheral Blood Mononuclear Cells of International Space Station Crewmembers

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 24 hrs (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the ground control subjects of all of the time points combined as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, including downregulations of SMAD7 and CDKN1A at MF and LF. Some of the genes such as SERPINE1 and VEGFA were downregulated at MF and LF, but upregulated at R0, while others such as NKG7 were down regulated at all of the 3 time points. Pathway analysis of these differentially expressed genes indicated that the NF-κB pathway was chronically activated in space. Analysis of the consequent diseases suggested potential associations with not only immune dysfunction, but also other health risks including osteoarthritis, cardiac hypertrophy and neuroinflammation.

Maria Moreno-Villanueva↗

NASA NTRSDate not supplied

A Gene Expression and Histologic Approach to Study Production and Outflow of Cerebrospinal Fluid in Hindlimb Suspended RATS

INTRODUCTION: The Spaceflight Associated Neuro-ocular Syndrome (SANS) is thought to be associated with weightlessness-induced cephalad fluid shift, possibly associated with a chronic elevation of intracranial pressure (ICP) during long-duration ISS missions. Changes in cerebrospinal fluid (CSF) dynamics and cranial compliance might be involved in the ICP increase. It is not known whether CSF production and/or outflow are altered in microgravity, but changes at the molecular and cellular level in the structures that produce and regulate the transcellular and paracellular secretion and reabsorption of CSF may be relevant. In this study, we used the rat hindlimb suspension (HS) model to examine the relationship between intracranial pressure (ICP) and the cellular responses to the prolonged change in body posture elicited specifically in the choroid plexus (CP). This was evaluated by transcriptomics, histopathology and ultrastructure of the CP and arachnoid villi (AV). METHODS: ICP was measured by telemetry. The morphology, ultrastructure, and gene expression profile of the CP was examined using male 9-month-old Long Evans rats subjected to HS for 14 and 90 days. A subset of animals completing 90-day HS returned to normal posture for 14 and 90 additional days of recovery. All HS rats had age-matched cage controls maintained in normal posture. A group of animals was maintained in conditions of 1% CO2 throughout the entire protocol. The rat brains were carefully removed and preserved for various analyses, including transmission electron microscopy (TEM), immunohistochemical analysis of specific targets involved in CSF regulation, and RNA sequencing analysis of laser capture micro-dissected CP tissue from the lateral ventricles. SUMMARY OF RESULTS: The ICP record was limited, which impacted any conclusions derived from the results. However, the ICP of rats in the CO2-enriched atmosphere appeared increased compared to those in a normal air composition. HS of rats resulted in an altered transcriptomics profile in the choroid plexus compared to animals maintained at normal posture. This was observed during both the HS period and during normal posture recovery period following 90 days of suspension. The greatest number of differentially expressed genes was observed at 90 days HS. Elevated CO2 also led to a different transcriptomics profile in the CP. The histology and ultrastructure results should be considered preliminary due to the limited number of samples. Examination of the CP by TEM showed blood vessel congestion and microvilli swelling in the CP, as well as some subpial and periventricular gliosis, but no associations were observed with treatment. There was no evidence of a reduced clearance of b-amyloid in the periventricular, perivascular and subpial regions of the brain in HS animals versus those in normal posture. Immunohistochemical staining of aquaporin 4, showed the localization to the subpial region and ependyma with increased immunoreactivity in the brains of CO2 exposed rats. This work was supported by awards NNX15AW48G to S.Z. Animal tissue and live data was provided under a tissue/data sharing agreement with Dr. Charles Fuller, UC Davis.

S B Zanello↗

NASA NTRSDate not supplied

Transcriptomic Changes in Peripheral Blood Mononuclear Cells of International Space Station Crewmembers

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) Blood was drawn at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 36 hours (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the ground control subjects of all time points combined as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, including downregulations of SMAD7 and CDKN1A at MF and LF. Some of the genes such as SERPINE1 and VEGFA were downregulated at MF and LF, but upregulated at R0, while others such as NKG7 were down regulated at all 3 time points. Pathway analysis of these differentially expressed genes indicated that the NF-κB pathway was chronically activated in space. Analysis of the consequent diseases suggested potential associations with not only immune dysfunction, but also other health risks including osteoarthritis, cardiac hypertrophy and neuroinflammation.

Maria Moreno-Villanueva↗

NASA NTRSDate not supplied

Differential Responses to Mechanostimulation in Embryonic Stem Cells Versus the Embryoid Body Model of Development Assessed at Single Cell RNA-Seq Resolution

Mechanicalforces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. In contrast disuse canreduce normal mechanical loading, resulting in altered cell and tissue function. Although loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how cells respondto this stimulusduring early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-minute pulse of 50xg hypergravity-generated 5kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the Embryoid Body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the hydrostatic pressurepulse. Single cell tSNE mapping shows limited transcriptome shifts in response to thispulse in either ESCs or EBs; this pulse,however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program.We assessed ESCs and EBs for differentially expressed (DE) genes with hydrostatic pressurepulse and found approximately 1/3 DE genes were shared. However, gene ontology (GO) pathway analysis show that EBs have choreographed responses associated with upregulation ofpathways formulticellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs showsmechanostimulationpromotes maintenance of transitory cell phenotypes in early development,including EB cluster co-expression of markers for progenitor, post-implant epiblast and primitive endoderm phenotypes versus expression exclusivity in the non-pulsed clusters. Pseudotime analysisidentified three branching cell types susceptible tohydrostatic pressureinduction of cell fate decisions. In summary, this study provides novel evidence that ESC maintenance and EB development can be regulated by mechanostimulation,and that stem cells committed to a differentiation program are more sensitive to force-induced changes to their transcriptome.

Cassandra Juran↗

NASA NTRSDate not supplied

Evaluating Plant Suberin Mutants for Enhanced Water and Nutrient Uptake to Increase Biomass Production Under Elevated CO2 Concentrations

Spaceflight cabin environments such as the International Space Station (ISS) typically have elevated CO2(1500-7000 μmol mol-1), which can affect plant growth and development. On earth, findings from FACE (Free-Air CO2Enrichment) and climate change studies have shown that plants grown at elevated (~ 700 μmol mol-1) CO2contain reduced levels of essential elements such as nitrogen, zinc, and iron. We hypothesized that spacecraft environments with elevated CO2 might likewise result in less nutritious crops for human consumption. Literature showed that quantitative differences in root suberin content could determine the permeability of the internal plant tissues to both water and solutes. To evaluate if there was a correlation between growth under elevated CO2and root suberin content we grew Arabidopsis thaliana, wild type (WT)(col-0), esb1(Enhanced Suberin 1) and horst1(Hydroxylase of Root Suberized Tissue)under ambient (~ 420± 25μmol mol-1) and elevated (800 ± 25, 1600 ± 25, 4000 ± 25 μmol mol-1) CO2concentrations. Comparing the growth of esb1and horst1plants to WT under ambient and elevated CO2concentrations, esb1showed significantly (P-value < 0.05) lower total fresh biomass whereas the horst1exhibited no significant difference. RNA sequencing revealed genes related to water uptake or nutrient availability stress upregulated due to the exposure to 800 and 1600 μmol mol-1CO2.Comparing the elemental composition of leaf tissue between WT and horst1plants grown under elevated CO2,we found an overall decrease in elemental composition for WT, whereas horst1showed an average increase of 30-60 % in the elemental contentsat1600 μmol mol-1compared to plants grown at ambient CO2. In conclusion, our results showed that horst1like modification to edible crops could improve the nutritional (elemental) content to supplement astronaut’s diet.

Anirudha R Dixit↗

NASA NTRSDate not supplied

GL4U: Bioinformatics training for students and educators using space omics data

NASA’s GeneLab project provides researchers open access to space-relevant experiment multi-omics data that can be mined to understand the effects of spaceflight on biological systems. To maximize the number of scientists who understand and utilize GeneLab data and data processing pipelines, GeneLab has created GeneLab for Colleges and Universities (GL4U). GL4U provides space biology-relevant training in bioinformatics to the next generation of scientists through direct and indirect approaches. The GeneLab team plans to host two annual data processing bootcamps, one for college-level students (direct) and one for college educators (indirect – training of trainers), in which participants learn to analyze GeneLab’s space-relevant omics data. The GL4U direct training pilot program was conducted in June 2021. During the pilot, students participated in a week-long bootcamp consisting of space biology-specific lectures and hands-on instruction using Jupyter Notebooks to analyze RNA sequence data. This pilot demonstrated the capacity of GL4U for training young scientists and encouraging data re-use. During the educator pilot, scheduled for June 2022, educators will receive materials and training to enable them to run the bootcamp at their home institutions or alternatively to adapt the content to implement within existing courses, thereby extending the reach of this initiative.

Amanda Marie Saravia-butler↗

NASA NTRSDate not supplied

Gene expression changes in peripheral blood mononuclear cells of ISS crewmembers suggest impacts of spaceflight on cell death

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 24 hrs (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the data from all of the time points from the ground control subjects as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, including downregulations of several cell cycle related genes including CDKN1A and VEGFA at MF and LF. Pathway analysis of these differentially expressed genes indicated that, in space, pathways associated with autophagy and senescence were affected. Our analysis also indicated that the genes related to metabolisms were downregulated in the microgravity environment. Taken together, our data suggests that PBMC in the ISS crewmembers may be starved, resulting in autophagy and delayed senescence in space. Such findings are in agreement with delayed cell death in PBMC under simulated microgravity conditions on the ground and offer an explanation for telomere lengthening that has been reported among the ISS astronauts in flight.

Maria Moreno-Villanueva↗

NASA NTRSDate not supplied

GL4U: Using Space Omics Data to Provide Bioinformatics Training for Students and Educators

NASA’s GeneLab project provides researchers open access to space-relevant experiment multi-omics data that can be mined to understand the effects of spaceflight on biological systems. To maximize the number of scientists who understand and utilize GeneLab data and data processing pipelines, GeneLab has created GeneLab for Colleges and Universities (GL4U). GL4U provides space biology-relevant training in bioinformatics to the next generation of scientists through direct and indirect approaches. The GeneLab team plans to host two annual data processing bootcamps, one for college-level students (direct) and one for college educators (indirect – training of trainers), in which participants learn to analyze GeneLab’s space-relevant omics data. The GL4U direct training pilot program was conducted in June 2021. During the pilot, students participated in a week-long bootcamp consisting of space biology-specific lectures and hands-on instruction using Jupyter Notebooks to analyze RNA sequence data. This pilot demonstrated the capacity of GL4U for training young scientists and encouraging data re-use. In June 2022, GL4U partnered with Jet Propulsion Laboratory’s (JPL) Planetary Protection Center of Excellence to conduct the indirect training pilot program by training educators at historically black colleges and universities (HBCUs) and minority serving institutions (MSIs). During the educator pilot, participants received materials, training, and will be provided the necessary compute resources to enable them to run the bootcamp at their home institutions or alternatively to adapt the content to implement within existing courses, thereby extending the reach of this initiative. The GL4U training program provides undergraduate students from underrepresented groups the opportunity to learn about NASA and Space Biology, and to enhance their career prospects by gaining hands-on experience analyzing omics data, a skillset that is highly applicable and marketable in the life sciences. Pre- and post-bootcamp surveys were completed by all participants and show the overwhelming success of the bootcamps.

Amanda M. Saravia-Butler↗

NASA NTRSDate not supplied

GL4U: Bioinformatics Training for Students and Educators Using Space Omics Data

NASA’s GeneLab project provides researchers open access to space-relevant experiment multi-omics data that can be mined to understand the effects of spaceflight on biological systems. To maximize the number of scientists who understand and utilize GeneLab data and data processing pipelines, GeneLab has created GeneLab for Colleges and Universities (GL4U). GL4U provides space biology-relevant training in bioinformatics to the next generation of scientists through direct and indirect approaches. The GeneLab team plans to host two annual data processing bootcamps, one for college-level students (direct) and one for college educators (indirect – training of trainers), in which participants learn to analyze GeneLab’s space-relevant omics data. The GL4U direct training pilot program was conducted in June 2021. During the pilot, students participated in a week-long bootcamp consisting of space biology-specific lectures and hands-on instruction using Jupyter Notebooks to analyze RNA sequence data. This pilot demonstrated the capacity of GL4U for training young scientists and encouraging data re-use. During the educator pilot, scheduled for June 2022, educators will receive materials and training to enable them to run the bootcamp at their home institutions or alternatively to adapt the content to implement within existing courses, thereby extending the reach of this initiative.

Amanda Marie Saravia-butler↗

NASA NTRSDate not supplied

Transcriptomic Changes in ISS Crewmembers Suggest Decreased Metabolic Activity in Peripheral Blood Mononuclear Cells

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) Blood was drawn at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 36 hours (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the ground control subjects of all time points combined as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, Pathway analysis of these differentially expressed genes indicated that several of the pathways related to metabolism, including LXR/RXR, NAD signaling and fatty acid betta-oxidation, were downregulated at MF and LF. We suggest that the decreased metabolic activity in space may contribute to the immune dysfunction and delayed cell cycle progression as observed in the astronauts.

Maria Moreno-Villanueva↗

NASA NTRSDate not supplied

Gene Expression Changes in Peripheral Blood Mononuclear Cells of ISS Crewmembers Suggest Impacts of Spaceflight on Cell Death

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 24 hrs (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the data from all of the time points from the ground control subjects as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, including downregulations of several cell cycle related genes including CDKN1A and VEGFA at MF and LF. Pathway analysis of these differentially expressed genes indicated that, in space, pathways associated with autophagy and senescence were affected. Our analysis also indicated that the genes related to metabolisms were downregulated in the microgravity environment. Taken together, we hypothesize that PBMC in the ISS crewmembers may be starved, resulting in autophagy and delayed senescence in space. Such findings are in agreement with delayed cell death in PBMC under simulated microgravity conditions on the ground and offer an explanation for telomere lengthening that has been reported among the ISS astronauts in flight

Maria Moreno-Villanueva↗

NASA NTRSDate not supplied

Genomics Study of Effect of Redox-Active Metalloporphyrin on Murine Retina During Spaceflight

Astronauts returning from spaceflight have experienced eye problems, which may decrease retinal performance and lead to long-term effects on visual acuity. This study leverages the collected data from spaceflown murine retinas that were treated with redox-active metalloporphyrin (BuOE) to mitigate spaceflight-induced changes. 10-week-old adult C57BL/6 male mice (n=5 in each of BuOE treated and saline control groups) were flown on Space-X 24 to the ISS national lab, kept in low earth orbit for 35 days and returned to Earth alive. Our analysis of RNA-sequencing data generated from subsequent murine retina tissues uncovered genes, pathways, and epigenetic modifications consistent with therapeutic potential of BuOE. For spaceflown murine samples, the treatment group show differentially expressed genes relative to saline controls that reached significance (adjusted p-value < 0.05) and included genes Gpx3 and Crhbp, which are related to protection against cell oxidative damage and cellular response to organonitrogen compounds. Ranked fold-changes from the same contrast were used for gene set enrichment analysis, which showed biological processes reaching significance (adjusted p-value < 0.05) including glutathione metabolic processes and cellular response to xenobiotic stimulus. The findings from this investigation have the potential to provide valuable insights into the molecular mechanisms underlying conditions like spaceflight associated neuro-ocular syndrome and assess the effectiveness of BuOE as a countermeasure for astronauts experiencing neuro-ophthalmic abnormalities, which can lead to long-term effects on visual acuity.

Machine Learning↗

NASA NTRSDate not supplied

GL4U: Using Space Biology Omics Data to Provide Bioinformatics Training for Students and Educators

NASA’s GeneLab project provides researchers open access to space-relevant multi-omics data via the Open Science Data Repository (OSDR) that can be mined to understand the effects of spaceflight on biological systems. To maximize the number of scientists who understand and utilize GeneLab data and data processing pipelines, GeneLab created GeneLab for Colleges and Universities (GL4U). GL4U provides space biology-relevant training in bioinformatics to the next generation of scientists through direct (training students) and indirect (training educators) approaches. The GL4U pilot programs were conducted in June 2021 (direct training) and 2022 (indirect training). During the pilots, students and educators at Historically Black Colleges and Universities (HBCUs) and Minority Serving Institutions (MSIs) participated in a week-long (direct training) or two-week-long (indirect training) bootcamp consisting of space biology-specific lectures and hands-on instruction using Jupyter Notebooks to analyze space biology RNA sequencing data from OSDR. During the educator pilot, participants received materials, training, and the necessary compute resources to enable them to run the bootcamp at their home institutions, thereby extending the reach of this initiative. In July 2023, GeneLab is partnering with JPL to expand GL4U to include amplicon sequencing (Amp-Seq) analysis training. During the GL4U Amp-Seq bootcamp, student and educator participants will receive training on how to analyze and interpret Amp-Seq data using the NASA GeneLab data processing pipeline. All bootcamp material, including instructions for requesting compute resources, will be made publicly available on GitHub for educators to teach the GL4U content in subsequent semesters. GL4U provides undergraduate students from underrepresented groups the opportunity to learn about NASA and Space Biology, and to enhance their career prospects by gaining hands-on experience analyzing omics data, a skillset that is highly applicable and marketable in the life sciences. We present results from pre- and post-training surveys completed by all participants of the Amp-Seq bootcamp.

Amanda M Saravia-Butler↗

NASA NTRS2023

Uncovering Unique Molecular Adaptations in the Arabidopsis Thaliana Cvi-0 Ecotype

This research proposal aims to investigate the unique molecular adaptations exhibited by Arabidopsis Thaliana, specifically focusing on the Cape Verde Islands (Cvi-0) ecotype, in response to microgravity conditions. The study examines data from NASA’s Open Science Data Repository and applies a multifaceted RNAseq analysis pipeline using tools in the UseGalaxy.org open platform. Through transcriptomic analysis, differential gene expression patterns were identified in Cvi-0, revealing an absence of heat shock protein (HSP) upregulation and an upregulation of Rubisco Activase (RCA) and chloroplast-related pathways. To test the hypothesis that these adaptations may contribute to Cvi-0’s increased adaptability in microgravity, a three-fold experimental design is proposed. Four experimental groups will be cultivated under simulated microgravity and ground control conditions, including Cvi-0, Col-0, and genetically modified Col-0 with silenced HSP genes, and genetically modified Col-0 with upregulated RCA gene. Growth parameters will be measured to assess plant resilience, and RNA sequencing will provide transcriptomic data for pathway analysis. Anticipated outcomes include improved markers of plant health (mass, growth, etc.) of Cvi-0 in simulated microgravity and enhanced resilience in genetically altered Col-0 variants, providing insights into potential mechanisms of adaptation. This research would bear significance for space agriculture, nutrition for extended space missions, and sustainable terrestrial crop enhancement. Moreover, the insights gained could reshape crop engineering on Earth, enhancing robustness to climate induced stresses and bolstering global food security. The proposal’s trajectory blends scientific curiosity with practical applicability, forging a path towards sustainable food production and improving human exploration beyond our planet.

GL4HS↗

NASA NTRSDate not supplied

Cardiovascular Responses to Simulated Spaceflight: Molecular Signatures and Surrogate Outputs to Measure CVD Risk

During extended space missions beyond low Earth orbit, astronauts will encounter prolonged periods of weightlessness and low dose space radiation. Previous studies have shown that exposure to small doses of high LET radiation (< 50 cGy) can lead to both short-term and long-term alterations in heart function, structure and underlying molecular mechanisms. In this study, we aim to identify the molecular signature associated with the cardiovascular response to simulated galactic cosmic radiation (5-ion GCR) alone or in combination with simulated weightlessness at time intervals relevant to mission length and recovery. Additionally, we aim to determine whether sex impacts cardiovascular responses to these spaceflight factors. Our overarching goal is to enhance our understanding of the cardiovascular risks associated with extended space missions and the clinical endpoints they suggest. We hypothesize that exposure to simulated space radiation leads to enduring alterations in the transcriptome, redox signaling and cytokine environment of cardiovascular tissue, some which have known links with reduced cardiovascular performance, aging, and increased risk of cardiovascular disease (CVD). Furthermore, we posit that simulated space radiation exposure in combination with simulated microgravity exacerbates cardiovascular deficits compared to single factor exposure. Female and male C57BL/6J mice, aged 23-24 weeks, were exposed to a single dose of 5, 15, or 50 cGy of 5-ion GCR, or sham-treated (0 cGy). Euthanasia was performed at 14 days and ~4 months post-irradiation. Hearts, aorta and blood plasma were collected shortly thereafter. RNA-sequencing of left ventricles at ~4 months post-GCR exposure revealed sex differences in the heart transcriptome with a few genes showing radiation-dependent changes in expression levels. Notably, some of the differentially expressed genes in 15 and 50 cGy GCR groups are known to play roles in the development of CVD. Analysis of protein levels of a subset of inflammatory cytokines in the heart indicated sex differences but no differences between sham and 50 cGy groups. Results also showed correlations among differentially expressed genes and a subset of inflammatory cytokines, with some correlations altered by GCR exposure. These findings suggest that GCR exposure can modify protein and gene networks linked to inflammation and CVD progression. In the aorta, telomere lengths were comparable across treatment groups sexes. Mitochondrial copy number is a biomarker for mitochondrial function with decreased copy numbers associated with cardiometabolic disease traits. Mitochondrial copy numbers of aorta also showed no sex nor dose differences. In a second study, mice underwent one week of simulated microgravity by hindlimb unloading (HU) and then exposed to a single dose of 15 cGy of 5-ion GCR. HU was conducted for an additional two weeks following GCR exposure. Single factor exposure groups (HU or GCR only) also were included in the study. Euthanasia was then performed and the same tissues were collected. Protein levels of select inflammatory cytokines in the heart showed sex-dependent differences in expression. In the aorta, telomere lengths and mitochondrial copy number also showed sex differences. In summary, our results indicate differences between sexes in biomarkers related to cardiovascular health. Exposure to 5-ion GCR or HU, alone or in combination, did not result in changes in most of the cardiovascular biomarkers that were examined. However, in the heart, simulated space radiation at doses of 15 and 50 cGy led to long-term alterations in the expression levels of a small group of genes known to be associated with the progression of CVD. The long-term transcriptomic changes resulting from exposure to simulated space radiation should be carefully investigated to mitigate adverse cardiovascular events during and after deep space missions. Our results also highlight the importance of sex-specific strategies in monitoring and maintaining cardiovascular health during and after deep space missions.

cardiovascular↗

NASA NTRSDate not supplied

Genomics Study of Effect of Redox-Active Metalloporphyrin on Murine Retina During Spaceflight

Astronauts returning from spaceflight have experienced eye problems, which may decrease retinal performance and lead to long-term effects on visual acuity. This study leverages the collected data from spaceflown murine retinas that were treated with redox-active metalloporphyrin (BuOE) to mitigate spaceflight-induced changes. 10-week-old adult C57BL/6 male mice (n=5 in each of BuOE treated and saline control groups) were flown on Space-X 24 to the ISS national lab, kept in low earth orbit for 35 days and returned to Earth alive. Our analysis of RNA-sequencing data generated from subsequent murine retina tissues uncovered genes, pathways, and epigenetic modifications consistent with therapeutic potential of BuOE. For spaceflown murine samples, the treatment group show differentially expressed genes relative to saline controls that reached significance (adjusted p-value < 0.05) and included genes Gpx3 and Crhbp, which are related to protection against cell oxidative damage and cellular response to organonitrogen compounds. Ranked fold-changes from the same contrast were used for gene set enrichment analysis, which showed biological processes reaching significance (adjusted p-value < 0.05) including glutathione metabolic processes and cellular response to xenobiotic stimulus. The findings from this investigation have the potential to provide valuable insights into the molecular mechanisms underlying conditions like spaceflight associated neuro-ocular syndrome and assess the effectiveness of BuOE as a countermeasure for astronauts experiencing neuro-ophthalmic abnormalities, which can lead to long-term effects on visual acuity.

Machine Learning↗

NASA NTRS2021

Water/Oil Emulsions with Controlled Droplet Sizes for In Vitro Selection Experiments

In the early history of life, RNA might have had many catalytic functions as ribozymes that do not exist today. To explore this possibility, catalytically active RNAs can be identified by in vitro selection experiments. Some of these experiments are best performed in nanodroplets to prevent diffusion between individual RNA sequences. In order to explore the suitability for the large-scale in emulsio selection of water-in-oil emulsions made by passing a mixture of mineral oil, the emulsifier ABIL-EM90, and a few percent of an aqueous phase through a microfluidizer, we used dynamic light scattering to characterize the size of aqueous droplets dispersed throughout the oil. We found that seven or more passes through the microfluidizer at 8000 psi with close to half molar inorganic salts and 10% polyethylene glycol produced droplets with sizes below 100 nm that were ideal for our purposes. We also identified conditions that would produce larger or smaller droplets, and we demonstrate that the emulsions are stable over weeks and months, which is desirable for different types of in vitro selection experiments.

Douglas Magde↗