Results for “IN VITRO” — Page 16

NASA NTRS2010

Growing B Lymphocytes in a Three-Dimensional Culture System

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing within 3D cultures that have been modified to foster lymphopoiesis retain an immunophenotype that closely recapitulates cells in fresh bone marrow harvests. The 3D culture system has been found to be capable of supporting long-lived (8 weeks) populations of B and T lymphocytes from peripheral lymphoid organs, in the absence of activation signals, to an extent not achievable by conventional culture techniques. Interestingly, it has been found that 3D-culture B cells display a phenotype that has characteristics of both B1a and B2 cells. These promising preliminary observations suggest that the 3D culture system could be used with success in the study of peripheral-B-lymphocyte biology and in the development of biotechnological techniques and processes.

Wu, J. H. David↗

NASA NTRS2007

Effects of Bone Morphogenic Proteins on Engineered Cartilage

A report describes experiments on the effects of bone morphogenic proteins (BMPs) on engineered cartilage grown in vitro. In the experiments, bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured in, variously, a control medium or a medium supplemented with BMP-2, BMP-12, or BMP-13 in various concentrations. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks macroscopically and histologically resembled native cartilage. At a concentration of 100 ng/mL, BMP-2, BMP-12, or BMP-13 caused (1) total masses of the constructs to exceed those of the controls by 121, 80, or 62 percent, respectively; (2) weight percentages of glycosaminoglycans in the constructs to increase by 27, 18, or 15, respectively; and (3) total collagen contents of the constructs to decrease to 63, 89, or 83 percent of the control values, respectively. BMP-2, but not BMP-12 or BMP-13, promoted chondrocyte hypertrophy. These observations were interpreted as suggesting that the three BMPs increase the growth rates and modulate the compositions of engineered cartilage. It was also concluded that in vitro engineered cartilage is a suitable system for studying effects of BMPs on chondrogenesis in a well-defined environment.

Gooch, Keith, J.↗

NASA NTRS2011

Origins of Protein Functions in Cells

In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known. Recently it was found that, as in the previous case, the proteins have a structure unknown among modern enzymes. In this case, in vitro evolution started from a small, non-enzymatic protein. A similar selection process initiated from a library of random polypeptides is in progress. These results not only allow for estimating the occurrence of function in random protein assemblies but also provide evidence for the possibility of alternative protein worlds. Extant proteins might simply represent a frozen accident in the world of possible proteins. Alternative collections of proteins, even with similar functions, could originate alternative evolutionary paths.

Seelig, Burchard↗

NASA NTRS2010

Ocular Toxicity Testing of Lunar Dust

This slide presentation reviews the use of ocular testing to determine the toxicity of lunar dust. The OECD recommendations are reviewed. With these recommendations in mind the test methodology was to use EpiOcular, tissues derived from normal human epidermal keratinocytes, the cells of which have been differentiated on cell culture inserts to form a multi-layered structure, which closely parallels the corneal epithelium and to dose the tissue with 100 mg dust from various sources. The in-vitro study provides evidence that lunar dust is not severely corrosive or irritating, however, in vitro tests have limitations, and in vivo tests provides a more complete scenario, and information, it is recommended that in vivo tests be performed.

Meyers, Valerie E.↗

NASA NTRS2018

Coalescence of DNA Double Strand Breaks Induced by Galactic Cosmic Radiation is Modulated by Genetics in 15 Inbred Strains of Mice

In this manuscript we address the challenges associated with the ability to predict radiation sensitivity associated with exposure to either cosmic radiation or X-rays in a population study, by monitoring DNA damage sensing protein 53BP1 forming small nuclear radiation-induced foci (RIF) as a surrogate biomarker of DNA double strand breaks (DSB). 76 primary skin fibroblasts were isolated from 10 collaborative cross strains and five reference inbred mice (C57Bl/6, BALB/CByJ, B6C3, C3H and CBA/CaJ) and exposed to three different charged nuclei of increasing LET (350 MeV/n Si, 350 MeV/n Ar and 600 MeV/n Fe) and X-ray. Our data brings strong evidence against the classic "contact-first" model where DSBs are assumed to be immobile and repaired at the lesion site. In contrast, our model suggests nearby DSBs move into single repair unit characterized by large RIF before the repair machinery kicks in. Such model has the advantage of being much more efficient molecularly but is poorly suited to deal with cosmic radiation, where energy is concentrated along the particle trajectory, inducing a large density of DSBs along each particle track. In accordance with this model, RIF quantification after X-ray exposition showed a saturated dose response for early time points post-irradiation for all strains. Similarly, the high-LET response showed that RIF number matched the number of track per cell, not the number of expected DSB per cell (1). At the temporal level, we noted that the percentage of unrepaired high-LET tracks over a 48 hour time-course increased with LET, confirming that the DNA repair process becomes more difficult as more DSB coalesce into single RIF. There was also good agreement between persistent RIF levels measured in-vitro in the primary skin cultures and survival levels of T-cells and B-cells collected in blood samples from 10 CC strains 24 hours after 0.1 Gy whole-body dose of X-ray. This suggests that persistent RIF 24 hour post-IR is a good surrogate in-vitro biomarker for in-vivo radiation toxicity. Finally, at the genomic level, large differences in repair rates between strains for high-LET allowed us to identify suggestive genetic loci associated with radiation sensitivity. Interestingly, the two highest LETs provided the most strain variation with a common locus on Chromosome 10 highly enriched for DNA repair associated genes we discussed in detail.

Radiation-Induced Foci↗

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

Systemic Alterations with Spaceflight Associated Health Risks Originating from Both Circulating miRNAs and Mitochondrial Biology

The many known health risks currently associated with space travel include increased risk of cardiovascular disease, cancer, central nervous system related diseases, muscle degeneration, and changes with host-gut microbiome interactions that can have profound impact with these and other health risks. The majority of the risk from space travel stem of the two components of the space environment which are microgravity and radiation. Two specific systemic effects have been uncovered by us to impact the body as a whole due to the space environment. One factor is related from our earlier work (Beheshti et al, PLOS One, 2018), we predicted that there is a systemic component of the host that causes general increased health risks due to spaceflight driven by a circulating microRNA (miRNA) signature consisting of 13 miRNAs that directly regulates both p53 and TGF1. MiRNAs are small non-coding RNA molecules with a negative and post-transcriptional regulation on gene expression) are increasingly recognized as major systemic regulators of responses to stressors, including microgravity, oxidative stress, and DNA damage. In addition, due to the size and stability of miRNAs, it is known that miRNAs can circulate throughout the body and have been found in the majority of the bodily fluids including blood, urine, saliva, and tears. Here, we start to dissect the actual impact of this miRNA signature on both the radiation and microgravity components and prove that this miRNA signature actually exists in the circulation of a host. The other systemic factor we uncovered was the impact the mitochondria on the whole body due to spaceflight. We hypothesize that spaceflight may promote a physiologic response driven by systemic mitochondria pathways leading to metabolic disorder stemming from the liver and directly impacting other organs and tissues. A systems biology method was implemented utilizing GeneLab datasets that involved in vitro experiments performed at the low Earth orbit, in vivo experiments involving mice flown to space, and finally human physiological data from astronauts. A comprehensive multi-omics approach was implemented which involved correlating transcriptomic analysis with proteomics, metabolomics, and methylation analysis. This approach led us to confirm our hypothesis that a systemic mitochondrial driven response is responsible for increasing potential health risk and is conserved from the in vitro studies, to the in vivo studies, and finally confirmed in astronauts.

Radiation↗

NASA NTRS2019

NASA GeneLab Platform Utilized for Space Radiation Dosimetry Biological Response Compared to Radiation Ground Studies

Ionizing radiation from Galactic Cosmic Rays (GCR) is one of the major risk factors factor that will impact health of astronauts on extended missions outside the protective effects of the Earth's magnetic field. Currently there are gaps in our knowledge of the health risks associated with chronic low dose, low dose rate ionizing radiation, specifically ions associated with high (H) atomic number (Z) and energy (E). The NASA GeneLab project (genelab.nasa.gov) aims to provide a detailed library of Omics datasets associated with biological samples exposed to HZE. The GeneLab Data System (GLDS) includes datasets from both spaceflight and ground-based studies, a majority of which involved exposure to ionizing radiation. Recently GeneLab has also assessed radiation dosimetry data with omics datasets associated with samples flown to the International Space Station (ISS). The combination of the detailed information on radiation exposure for ground-based studies and curated dosimetry information for spaceflight experiments allows GeneLab to be the first comprehensive Omics database for space related research from which an investigator can generate hypotheses to direct future experiments utilizing both ground and space biological radiation data. We demonstrate the usefulness of these datasets by analyzing multiple GeneLab datasets associated with both radiation ground-based studies and spaceflight studies. The radiation ground based studies we analyzed includes both in vivo and in vitro work with a range ions from protons to iron particles with doses from 0.1Gy to 2Gy. These datasets were compared to both in vivo and in vitro datasets from samples flown to the ISS and on shorter shuttle missions with total doses of 0.1 mGy to 30 mGys. From this analysis we were able to associate distinct biological signatures associating specific ions to specific biological response to radiation exposure in space. For example, we discovered radiation biological response related to cardiovascular effects from proton ground studies are the dominating response for samples related to cardiovascular effects on the ISS. With this work we will provide a summary of how different ions will impact different biological response in space and how this can be used in future studies to assess optimal ground experiments to simulate space radiation.

Beheshti, Afshin↗

NASA NTRS2021

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high‐throughput sequencing

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell‐free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE‐based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)‐peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry‐over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti‐FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 10(exp 12) random sequences, a round‐by‐round high‐throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr(Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE‐based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

cDNA display, FLAG epitope, mRNA display, peptide ↗

NASA NTRSDate not supplied

Hazards of Lunar Surface Exploration: Determining the Immunogenicity/Allergenicity of Lunar Dust

There are multiple Apollo program reports of lunar dust (LD) exposure leading to significant upper respiratory symptoms in select crewmembers. Possible mechanisms include particulate irritation, oxidization and release of noxious gas, or legitimate adaptive immune-mediated response. Although sterile non-protein matter would not be expected to be an allergen, one Apollo flight surgeon reported increasing symptoms upon repeated exposure, with associated eosinophilia indicative of allergy (*Acta Astronautica. 2008 63 (7–10): 980–987). Many ISS crews display a pattern of persistent immune system dysregulation and latent virus reactivation (NPJ Microgravity. 2015 Sep 3; 1:15013; NPJ Microgravity. 2017 Apr 12; 3:11). Some ISS crews manifest atypical respiratory and/or dermatitis symptoms which could have an allergic pathogenesis (J Allergy Clin. Immunol. Pract. 2016 Jul-Aug; 4(4):759-762.e8). It is logical to anticipate crew immune dysregulation would worsen during prolonged deep space missions. Planetary surface hazards will only complicate crew health risks. This study hypothesizes that LD exposure can alter susceptible individuals’ immune responses such that repeated exposure will elicit an IgE mediated allergic response either to the LD itself or concomitant antigen exposure during spaceflight. This will adversely increase clinical and operational impacts for long-duration lunar astronauts and affect countermeasure requirements for surface vehicles. Specific aims for this study are (1) Does in vitro LD exposure result in increased histamine from human peripheral blood basophils? (2) Can LD impact the capacity of CD4+ helper and/or CD19+ B-cell mediated IgE production? To address these questions, a set of in-vitro cell culture experiments will be employed (short and long term) using human peripheral blood mononuclear cells (PBMC) and basophils from both atopic and non-atopic individuals, as well as established human basophil and mast cell lines. Cells will be co-cultured with cellular mitogens, common recall antigens (tetanus, Der p1), nickel (as a possible allergenic component of LD), with or without graded amounts of LD, to study whether LD exposure for varying time intervals will alter the generation of selective immune responses associated with clinical allergic reactions. Measured outputs include supernatant-derived IgE, tryptase, histamine and selected cytokine levels. Cellular activation will be monitored by assessing activation markers via flow cytometry. EM/x-ray analysis will be used to determine cellular interactions with dust particles. The minimal amount of LD (Apollo 14 dust) and controls/simulants have been requested. This study, originally planned as an FY20/21 activity, was delayed due to the COVID pandemic. It is now scheduled to be performed during FY22.

Brian Crucian↗

NASA NTRSDate not supplied

Hazards of Lunar Surface Exploration: Determining the Immunogenicity/Allergenicity of Lunar Dust

Although infrequent, there have been Apollo program reports of lunar dust (LD) exposure leading to notable upper respiratory symptoms in select crewmembers. Possible mechanisms include particulate irritation, oxidization and release of noxious gas, or legitimate adaptive immune-mediated response. Although sterile non-protein matter would not be expected to be an allergen, one Apollo flight surgeon reported increasing symptoms upon repeated exposure with associated eosinophilia, indicative of allergy (*Acta Astronautica. 2008 63 (7–10): 980–987). Many ISS crews display a pattern of persistent immune system dysregulation and latent virus reactivation (NPJ Microgravity. 2015 Sep 3; 1:15013; NPJ Microgravity. 2017 Apr 12; 3:11). Some ISS crews manifest atypical respiratory and/or dermatitis symptoms which could have an allergic pathogenesis (J Allergy Clin. Immunol. Pract. 2016 Jul-Aug; 4(4):759-762.e8). It is logical to anticipate crew immune dysregulation would worsen during prolonged deep space missions. Planetary surface hazards will only complicate crew health risks. This study with investigate if LD exposure will elicit an IgE mediated allergic response either to the LD itself or concomitant antigen exposure during spaceflight. Allergic reactivity could adversely increase clinical and operational impacts for long-duration lunar astronauts and affect countermeasure requirements for surface vehicles. Specific aims for this study are to answer two questions: (1) Does in vitro LD exposure result in increased histamine from human peripheral blood basophils? (2) Can LD impact the capacity of CD4+ T helper and/or CD19+ B-cell mediated IgE production? To address these questions, after the proposal and selection by NASA, our laboratory has separately requested and been approved for receipt of actual LD samples from the Apollo 16 mission. These samples will be used during the study to complete the proposed set of in vitro cell culture experiments (short and long term), using human peripheral blood mononuclear cells (PBMC) and basophils from both atopic and non-atopic individuals. Cells will be co-cultured with cellular mitogens, common recall antigens (Der p1), fine ground silica quartz (as a possible allergenic component of LD), or LD, to study whether LD exposure for varying time intervals will alter the generation of selective immune responses associated with clinical allergic reactions. Measured outputs include supernatant-derived IgE, tryptase, histamine, and selected cytokine levels. Cellular activation will be monitored by assessing activation markers via flow cytometry. EM/x-ray analysis will be used to determine cellular interactions with dust particles. A series of validation experiments was initiated in FY22 once the delivery of LD was received. Based on initial experimental findings, we are optimizing the culture conditions, LD concentrations, and refining our other protocol stimuli.

Audrie A. Colorado↗

NASA NTRSDate not supplied

Hazards of Lunar Surface Exploration: Determining the Immunogenicity/Allergenicity of Lunar Dust

Although infrequent, there have been Apollo program reports of lunar dust (LD) exposure leading to notable upper respiratory symptoms in select crewmembers. Possible mechanisms include particulate irritation, oxidization and release of noxious gas, or legitimate adaptive immune-mediated response. Although sterile non-protein matter would not be expected to be an allergen, one Apollo flight surgeon reported increasing symptoms upon repeated exposure with associated eosinophilia, indicative of allergy (*Acta Astronautica. 2008 63 (7–10): 980–987). Many ISS crews display a pattern of persistent immune system dysregulation and latent virus reactivation (NPJ Microgravity. 2015 Sep 3; 1:15013; NPJ Microgravity. 2017 Apr 12; 3:11). Some ISS crews manifest atypical respiratory and/or dermatitis symptoms which could have an allergic pathogenesis (J Allergy Clin. Immunol. Pract. 2016 Jul-Aug; 4(4):759-762.e8). It is logical to anticipate crew immune dysregulation would worsen during prolonged deep space missions. Planetary surface hazards will only complicate crew health risks. This study with investigate if LD exposure will elicit an IgE mediated allergic response either to the LD itself or concomitant antigen exposure during spaceflight. Allergic reactivity could adversely increase clinical and operational impacts for long-duration lunar astronauts and affect countermeasure requirements for surface vehicles. Specific aims for this study are to answer two questions: (1) Does in vitro LD exposure result in increased histamine from human peripheral blood basophils? (2) Can LD impact the capacity of CD4+ T helper and/or CD19+ B-cell mediated IgE production? To address these questions, after the proposal and selection by NASA, our laboratory has separately requested and been approved for receipt of actual LD samples from the Apollo 16 mission. These samples will be used during the study to complete the proposed set of in vitro cell culture experiments (short and long term), using human peripheral blood mononuclear cells (PBMC) and basophils from both atopic and non-atopic individuals. Cells will be co-cultured with cellular mitogens, common recall antigens (Der p1), fine ground silica quartz (as a possible allergenic component of LD), or LD, to study whether LD exposure for varying time intervals will alter the generation of selective immune responses associated with clinical allergic reactions. Measured outputs include supernatant-derived IgE, tryptase, histamine, and selected cytokine levels. Cellular activation will be monitored by assessing activation markers via flow cytometry. EM/x-ray analysis will be used to determine cellular interactions with dust particles. A series of validation experiments was initiated in FY22 once the delivery of LD was received. Based on initial experimental findings, we are optimizing the culture conditions, LD concentrations, and refining our other protocol stimuli.

immunology↗

NASA NTRSDate not supplied

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

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

C M Ott↗

NASA NTRS2023

Peptide conjugates with polyaromatic hydrocarbons can benefit the activity of catalytic RNAs

Early stages of life likely employed catalytic RNAs (ribozymes) in many functions that are today filled by proteins. However, the earliest life forms must have emerged from heterogenous chemical mixtures, which included amino acids, short peptides, and many other compounds. Here we explored whether the presence of short peptides can help the emergence of catalytic RNAs. To do this, we conducted an in vitro selection for catalytic RNAs from randomized sequence in the presence of ten different peptides with a prebiotically plausible length of eight amino acids. This in vitro selection generated dozens of ribozymes, one of them with ∼900-fold higher activity in the presence of one specific peptide. Unexpectedly, the beneficial peptide had retained its N-terminal Fmoc protection group, and this group was required to benefit ribozyme activity. The same, or higher benefit resulted from peptide conjugates with prebiotically plausible polyaromatic hydrocarbons (PAHs) such as fluorene and naphthalene. This shows that PAH-peptide conjugates can act as potent cofactors to enhance ribozyme activity. The results are discussed in the context of the origin of life.

Kevin J. Sweeney↗

NASA NTRS2024

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

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

C.M. Ott↗

NASA NTRS2003

Effects of hindlimb unweighting on the mechanical and structure properties of the rat abdominal aorta

Previous studies have shown that hindlimb unweighting of rats, a model of microgravity, reduces evoked contractile tension of peripheral conduit arteries. It has been hypothesized that this diminished contractile tension is the result of alterations in the mechanical properties of these arteries (e.g., active and passive mechanics). Therefore, the purpose of this study was to determine whether the reduced contractile force of the abdominal aorta from 2-wk hindlimb-unweighted (HU) rats results from a mechanical function deficit resulting from structural vascular alterations or material property changes. Aortas were isolated from control (C) and HU rats, and vasoconstrictor responses to norepinephrine (10(-9)-10(-4) M) and AVP (10(-9)-10(-5) M) were tested in vitro. In a second series of tests, the active and passive Cauchy stress-stretch relations were determined by incrementally increasing the uniaxial displacement of the aortic rings. Maximal Cauchy stress in response to norepinephrine and AVP were less in aortic rings from HU rats. The active Cauchy stress-stretch response indicated that, although maximum stress was lower in aortas from HU rats (C, 8.1 +/- 0.2 kPa; HU, 7.0 +/- 0.4 kPa), it was achieved at a similar hoop stretch. There were also no differences in the passive Cauchy stress-stretch response or the gross vascular morphology (e.g., medial cross-sectional area: C, 0.30 +/- 0.02 mm(2); HU, 0.32 +/- 0.01 mm(2)) between groups and no differences in resting or basal vascular tone at the displacement that elicits peak developed tension between groups (resting tension: C, 1.71 +/- 0.06 g; HU, 1.78 +/- 0.14 g). These results indicate that HU does not alter the functional mechanical properties of conduit arteries. However, the significantly lower active Cauchy stress of aortas from HU rats demonstrates a true contractile deficit in these arteries.

Non-NASA Center↗

NASA NTRS2000

Endogenous circulating sympatholytic factor in orthostatic intolerance

Sympathotonic orthostatic hypotension (SOH) is an idiopathic syndrome characterized by tachycardia, hypotension, elevated plasma norepinephrine, and symptoms of orthostatic intolerance provoked by assumption of an upright posture. We studied a woman with severe progressive SOH with blood pressure unresponsive to the pressor effects of alpha(1)-adrenergic receptor (AR) agonists. We tested the hypothesis that a circulating factor in this patient interferes with vascular adrenergic neurotransmission. Preincubation of porcine pulmonary artery vessel rings with patient plasma produced a dose-dependent inhibition of vasoconstriction to phenylephrine in vitro, abolished vasoconstriction to direct electrical stimulation, and had no effect on nonadrenergic vasoconstrictive stimuli (endothelin-1), PGF-2alpha (or KCl). Preincubation of vessels with control plasma was devoid of these effects. SOH plasma inhibited the binding of an alpha(1)-selective antagonist radioligand ([(125)I]HEAT) to membrane fractions derived from porcine pulmonary artery vessel rings, rat liver, and cell lines selectively overexpressing human ARs of the alpha(1B) subtype but not other AR subtypes (alpha(1A) and alpha(1D)). We conclude that a factor in SOH plasma can selectively and irreversibly inhibit adrenergic ligand binding to alpha(1B) ARs. We propose that this factor contributes to a novel pathogenesis for SOH in this patient. This patient's syndrome represents a new disease entity, and her plasma may provide a unique tool for probing the selective functions of alpha(1)-ARs.

Case Reports↗

NASA NTRS1997

Measurement of shear-wave velocity by ultrasound critical-angle reflectometry (UCR)

There exists a growing body of research that relates the measurement of pressure-wave velocity in bone to different physiological conditions and treatment modalities. The shear-wave velocity has been less studied, although it is necessary for a more complete understanding of the mechanical properties of bone. Ultrasound critical-angle reflectometry (UCR) is a noninvasive and nondestructive technique previously used to measure pressure-wave velocities both in vitro and in vivo. This note describes its application to the measurement of shear-wave velocity in bone, whether directly accessible or covered by soft tissue.

NASA Discipline Cardiopulmonary↗