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NASA NTRSDate not supplied

LEIA: An Investigation of Radiation Risks to Biology at the Lunar South Pole

Radiation and reduced gravity pose biological risks to crewed deep space exploration. At the cellular level, radiation damage can be amplified by reduced gravity. Empirical evidence on cellular responses to beyond low Earth orbit (BLEO) environments is imperative to develop effective countermeasures for crew health and in-space biomanufacturing. The Lunar Explorer Instrument for Space Biology Applications (LEIA) project is developing an instrument suite to be delivered to the south polar region of the Moon by the Commercial Lunar Payload Services (CLPS) program. This presentation will provide an overview of the LEIA hardware, experiments, and mission timeline. The LEIA instruments include the BioSensor, the ARES charged particle detector, and the Mini-FND. The BioSensor is an autonomous light emitting diode (LED)-based spectrophotometer and microfluidic incubator. The BioSensor activates yeast cultures and can measure cell growth, metabolic activity, and carotenoid production. The ARES is a Timepix-based charged particle radiation detector that measures dose, dose rate, and linear energy transfer spectra. The Mini-FND is a fast neutron detector that measures albedo neutron flux and energy spectra. Combined, these instruments will be used for yeast genetics experiments to quantify growth, metabolism, and synthetic biology-enabled production of human nutrients, while taking real time measurements of biologically relevant radiation exposure on the lunar surface. These data will be used to test the importance of selected DNA damage repair and reactive oxygen species defense pathways in mitigating cellular damage from lunar surface radiation.

Yeast↗

NASA NTRSDate not supplied

LEIA: An Investigation of Radiation Risks to Biology at the Lunar South Pole

Yeast↗

DOE OSTI2023

Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet’s ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Bromodomain-containing protein 7 regulates matrix metabolism and apoptosis in human nucleus pulposus cells through the BRD7-PI3K-YAP1 signaling axis

Intervertebral disc degeneration (IDD) results from dysregulated metabolism of the extracellular matrix of the nucleus pulposus (NP) and involves the participation of inflammatory factors such as TNF-α. Bromodomain-containing protein 7 (BRD7) shows considerable potential for anti-inflammatory applications. Herein, we investigated the role of BRD7 in IDD. The immunohistochemistry results demonstrated decreased BRD7 expression in severely degenerated human NP tissues compared to those showing mild degeneration. Lentiviruses and adenoviruses were used to knock down or overexpress BRD7 and YAP1, respectively. Our results revealed that BRD7 knockdown promoted matrix degradation and suppressed PI3K and YAP1 expression, while BRD7 overexpression alleviated matrix degradation and promoted YAP1 and PI3K expression. In addition, PI3K inhibition augmented matrix degradation, enhanced apoptosis, and reduced YAP1 expression, whereas YAP1 overexpression promoted matrix synthesis, suppressed apoptosis and promoted PI3K expression. Besides, BRD7 overexpression reversed the reductions in sulfated glycosaminoglycan levels induced by TNF-α, but this effect was blocked by PI3K or YAP1 inhibitors. Moreover, YAP1 and PI3K were shown to interact through coimmunoprecipitation analysis. In summary, our results demonstrate that BRD7 can regulate matrix metabolism and apoptosis in human NP cells through the BRD7-PI3K-YAP1 signaling axis. This study might provide new insights into the prevention and treatment of IDD.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2020

BioSentinel ISS Control Experiment Final Investigation Summary Form

Each payload has 18 fluidic cards and each fluidic card has 16 microfluidic wells, for a total of 288 wells. Card activation occurs at different time points throughout the 6-month mission and following ground commands. At each timepoint, two fluidic cards are filled with growth medium containing nutrients and a metabolic indicator dye. In addition to card filling, the temperature of the cards increases from ~4°C (stasis mode) to 23°C to allow for cell growth. The metabolic dye changes color from blue to pink, then pink to clearin the presence of metabolically active yeast cells. The colorimetric changes and cell growth are monitored using a 3-light-emitting diode(LED)optical detection system. The optical data determines how fast the cells are able to recover after accumulating radiation damage over time.After ground command initiation, all the fluidics, thermal and optical detection steps in BioSentinel are fully automated.The biosensor response data is compared to physical dosimetry data collected onboard the same payload. This dosimeter or linear energy transfer (LET)spectrometer is based on TimePix chip technology. This chip measures the total ionizing radiation dose in addition to calculating the LET of each particle that traverses its sensor, thus allowing researchers to characterize the radiation environment around the payload.The biological response (and physical dosimetry) on ISS over time is compared to the deep space mission and to data obtained on the ground.This analysis provides insight on the biological effects of the space radiation exposure in preparation for future long-duration missions to space and inhabitation beyond low-Earth orbit (LEO).

BioSentinel↗

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↗

DOE OSTI2024

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

3D spheroids↗

DOE OSTI2023

Validation of a metabolite–GWAS network for Populus trichocarpa family 1 UDP-glycosyltransferases

Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as Populus trichocarpa , a biofuel feedstock and model woody plant species. Despite their growing importance in plant genetics and metabolomics, few mGWASs are experimentally validated. Here, we present a functional genomics workflow for validating mGWAS-predicted enzyme–substrate relationships. We focus on uridine diphosphate–glycosyltransferases (UGTs), a large family of enzymes that catalyze sugar transfer to a variety of plant secondary metabolites involved in defense, signaling, and lignification. Glycosylation influences physiological roles, localization within cells and tissues, and metabolic fates of these metabolites. UGTs have substantially expanded in P. trichocarpa , presenting a challenge for large-scale characterization. Using a high-throughput assay, we produced substrate acceptance profiles for 40 previously uncharacterized candidate enzymes. Assays confirmed 10 of 13 leaf mGWAS associations, and a focused metabolite screen demonstrated varying levels of substrate specificity among UGTs. A substrate binding model case study of UGT-23 rationalized observed enzyme activities and mGWAS associations, including glycosylation of trichocarpinene to produce trichocarpin, a major higher-order salicylate in P. trichocarpa. We identified UGTs putatively involved in lignan, flavonoid, salicylate, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress response that determine sustainable biomass crop production. Our results provide new support for in silico analyses and evidence-based guidance for in vivo functional characterization.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal reorganization. However the underlying mechanism for these cellular changes have not been clearly defined. We examined alterations in endothelial migration, and cytoskeleton architecture (microfilamentous f-actin and vimentin-rich- intermediate filaments) following wounding under simulated microgravity. We also examined the possibility that altered signal transduction pathways, involving protooncogenes, may play a crucial role in microgravity-induced retardation of cell migration and alterations in cytoskeletal organization. We hypothesize that, based on the tensegrity theory, cytoskeletal organization respond to gravitational unloading and through this response, cell behavior, function and gene expression are modified.

Melhado, C. D.↗

DOE OSTI2021

Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent states

Highlights: • PKM1/2 protein abundance is greater in formative mEpiLCs compared to naïve mESCs or primed mEpiSCs. The ratio of PKM1/2 is maintained. • PKM1/2 are localized in both nuclear and cytoplasmic regions relative to GAPDH and OCT4 across the pluripotent continuum. • PKM1 localization is strongly correlated to OCT4 localization, and moderately to GAPDH in formative mEpiLCs. • PKM1/2 localization is moderately correlated to OCT4 and GAPDH localization in naïve mESCs. • PKM1/2 localization are moderately correlated to GAPDH localization in primed mEpiSCs. Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways, including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate, however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg Effect. Both isoforms catalyze the final, rate limiting step of glycolysis, generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm, but also accumulate in differential subnuclear regions of mESC, mEpiLCs, and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 changes during the transition from mESCs, mEpiLCs, and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson's correlation coefficient and Manders's overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N 2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A ( Candidatus Atelocyanobacterium thalassa ). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N 2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Transcriptomic Analysis of ISS Crewmembers’ Peripheral Blood Mononuclear Cells Reveals Homeostatic Regulations in Space

The impact of spaceflight on the immune system has been investigated for decades. Studies conducted in cell models, animals and humans suggest that the spaceflight environment affects the innate and acquired immune systems, as the ability to recognize antigens, defend against foreign invaders, and orchestrate repair is significantly hindered. However, the molecular mechanisms behind spaceflight-induced immune dysregulations are still unclear. In this study, blood from eleven (11) International Space Station (ISS) crewmembers was collected before, during and after long duration space missions, as well as from 11 matched ground control subjects. Transcriptomic analysis was performed in isolated peripheral blood mononuclear cells (PBMCs) using the RNA-sequencing technique. In comparison to the blood samples collected from the crewmembers pre-flight, a total of ~1000 genes were found to be upregulated and ~1000 genes downregulated in PBMC collected between 4 and 6 months after they were in space. The most significantly DEGs (differentially expressed genes) include activation of RUBCNL which is an autophagy enhancer and inhibition of GRASP which regulates cell trafficking. Genes involved in cell adhesion, cell cycle progression and other functions were also dysregulated. Pathway analysis of the DEGs indicates mitochondria dysfunction, particularly reduced ATP production in the electron transport chain. Other pathways impacted by spaceflight include glycolysis, autophagy and inflammatory response. Our results suggest that, in space, blood cells may have also experienced energy depletion and reduced metabolism. Consequently, the cells may become autophagic, which is a known homeostatic mechanism for blood cells to become quiescent, but to stay alive. Further analysis of the data shows recovery of the crewmembers after mission and potential differential responses between genders to the space environment. Our data potentially explains some of the physiological changes that have been observed in space such as mitochondria dysfunction, inhibition of T cell activation and telomere lengthening. Comparison of our results with other transcriptomics studies of ISS crewmembers’ blood cells will also be presented.

Maria Moreno Villanueva↗

NASA NTRS2016

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

yeast↗

DOE OSTI2021

CPT2 downregulation triggers stemness and oxaliplatin resistance in colorectal cancer via activating the ROS/Wnt/β-catenin-induced glycolytic metabolism

Highlights: • CPT2 was downregulated in CRC. • CPT2 suppressed the proliferation and glycolytic metabolism in CRC cells. • CPT2 inhibited cancer stem cell properties and oxaliplatin resistance in CRC. • CPT2 attenuated CRC tumor growth and enhanced oxaliplatin sensitivity in vivo. • CPT2 functioned via suppressing ROS/Wnt/β-catenin pathway in CRC. Carnitine palmitoyltransferase 2 (CPT2) has been demonstrated to act as a tumor promotor or suppressor in different types of cancers. However, little is known about the effect of CPT2 on colorectal cancer (CRC). In the present study, we analyzed CPT2 expression in CRC tissues and cells. CPT2 was overexpressed in CRC cell lines (SW480 and RKO), and its effects and molecular mechanism on the proliferation, glycolysis, stemness, and oxaliplatin sensitivity were investigated. The xenograft experiment was used to confirm the influence of CPT2 on CRC tumorigenesis in vivo. We found that CPT2 expression was significantly downregulated in CRC patients, and its lower expression was associated with the poor prognosis, large tumor size, advanced TNM stage, and poor histological grade differentiation of patients. Upregulation of CPT2 significantly inhibited the proliferation, glycolytic metabolism, cancer stem cell properties, and oxaliplatin resistance in CRC cells. Also, the increase of CPT2 inhibited tumorigenesis, stemness and glycolysis, while enhanced oxaliplatin sensitivity in mouse models. Mechanistically, CPT2 functioned via suppressing the activation of Wnt/β-catenin pathway through repressing ROS production. In conclusion, our results demonstrated that CPT2 was decreased in CRC, and CPT2 downregulation could trigger stemness and oxaliplatin resistance in CRC via activating the ROS/Wnt/β-catenin-induced glycolytic metabolism. This study indicates that CPT2 is a potential therapeutic target for CRC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Spatiotemporal metabolic responses to water deficit stress in distinct leaf cell-types of poplar

The impact of water-deficit (WD) stress on plant metabolism has been predominantly studied at the whole tissue level. However, plant tissues are made of several distinct cell types with unique and differentiated functions, which limits whole tissue ‘omics’-based studies to determine only an averaged molecular signature arising from multiple cell types. Advancements in spatial omics technologies provide an opportunity to understand the molecular mechanisms underlying plant responses to WD stress at distinct cell-type levels. Here, we studied the spatiotemporal metabolic responses of two poplar ( Populus tremula× P. alba ) leaf cell types -palisade and vascular cells- to WD stress using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). We identified unique WD stress-mediated metabolic shifts in each leaf cell type when exposed to early and prolonged WD stresses and recovery from stress. During water-limited conditions, flavonoids and phenolic metabolites were exclusively accumulated in leaf palisade cells. However, vascular cells mainly accumulated sugars and fatty acids during stress and recovery conditions, respectively, highlighting the functional divergence of leaf cell types in response to WD stress. By comparing our MALDI-MSI metabolic data with whole leaf tissue gas chromatography-mass spectrometry (GC-MS)-based metabolic profile, we identified only a few metabolites including monosaccharides, hexose phosphates, and palmitic acid that showed a similar accumulation trend at both cell-type and whole leaf tissue levels. Overall, this work highlights the potential of the MSI approach to complement the whole tissue-based metabolomics techniques and provides a novel spatiotemporal understanding of plant metabolic responses to WD stress. This will help engineer specific metabolic pathways at a cellular level in strategic perennial trees like poplars to help withstand future aberrations in environmental conditions and to increase bioenergy sustainability.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1997

Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

Many of the physiological changes of the cardiovascular system during space flight may originate from the dysfunction of basic biological mechanisms caused by microgravity. The weightlessness affects the system when blood and other fluids move to the upper body causing the heart to enlarge to handle the increased blood flow to the upper extremities and decrease circulating volume. Increase arterial pressure triggers baroreceptors which signal the brain to adjust heart rate. Hemodynarnic studies indicate that the microgravity-induced headward fluid redistribution results in various cardiovascular changes such as; alteration of vascular permeability resulting in lipid accumulation in the lumen of the vasculature and degeneration of the the vascular wall, capillary alteration with extensive endothelial invagination. Achieving a true microgravity environment in ground based studies for prolonged periods is virtually impossible. The application of vector-averaged gravity to mammalian cells using horizontal clinostat produces alterations of cellular behavior similar to those observed in microgravity. Similarly, the low shear, horizontally rotating bioreactor (originally designed by NASA) also duplicates several properties of microgravity. Additionally, increasing gravity, i.e., hypcrgravity is easily achieved. Hypergravity has been found to increase the proliferation of several different cell lines (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. The effect of altered gravity on cells maybe similar to those of other physical forces, i.e. shear stress. Previous studies examining laminar flow and shear stress on endothelial cells found that the cells elongate, orient with the direction of flow, and reorganize their F-actin structure, with concomitant increase in cell stiffness. These studies suggest that alterations in the gravity environment will change the behavior of most cells, including vascular cells. However, few studies have been directed at assessing the effect of altered gravitational field on vascular cell fiction and metabolism, Using image analysis we examined how bovine aortic endothelial cells altered their morphological characteristics and their response to a denudation injury when cells were subjected to simulated microgravity and hypergravity.

Melhado, Caroline↗

DOE OSTI2021

TMEM180 contributes to SW480 human colorectal cancer cell proliferation through intra-cellular metabolic pathways

Not Available

Anzai, Takahiro↗

DOE OSTI2022

Colorectal Cancer Metastases in the Liver Establish Immunosuppressive Spatial Networking between Tumor-Associated SPP1 + Macrophages and Fibroblasts

Abstract Purpose: The liver is the most frequent metastatic site for colorectal cancer. Its microenvironment is modified to provide a niche that is conducive for colorectal cancer cell growth. This study focused on characterizing the cellular changes in the metastatic colorectal cancer (mCRC) liver tumor microenvironment (TME). Experimental Design: We analyzed a series of microsatellite stable (MSS) mCRCs to the liver, paired normal liver tissue, and peripheral blood mononuclear cells using single-cell RNA sequencing (scRNA-seq). We validated our findings using multiplexed spatial imaging and bulk gene expression with cell deconvolution. Results: We identified TME-specific SPP1-expressing macrophages with altered metabolism features, foam cell characteristics, and increased activity in extracellular matrix (ECM) organization. SPP1+ macrophages and fibroblasts expressed complementary ligand–receptor pairs with the potential to mutually influence their gene-expression programs. TME lacked dysfunctional CD8 T cells and contained regulatory T cells, indicative of immunosuppression. Spatial imaging validated these cell states in the TME. Moreover, TME macrophages and fibroblasts had close spatial proximity, which is a requirement for intercellular communication and networking. In an independent cohort of mCRCs in the liver, we confirmed the presence of SPP1+ macrophages and fibroblasts using gene-expression data. An increased proportion of TME fibroblasts was associated with the worst prognosis in these patients. Conclusions: We demonstrated that mCRC in the liver is characterized by transcriptional alterations of macrophages in the TME. Intercellular networking between macrophages and fibroblasts supports colorectal cancer growth in the immunosuppressed metastatic niche in the liver. These features can be used to target immune-checkpoint–resistant MSS tumors.

60 APPLIED LIFE SCIENCES↗