Microbiology of human spaceflight: microbial responses to mechanical forces that impact health and habitat sustainability.

SUMMARYUnderstanding the dynamic adaptive plasticity of microorganisms has been advanced by studying their responses to extreme environments. Spaceflight research platforms provide a unique opportunity to study microbial characteristics in new extreme adaptational modes, including sustained exposure to reduced forces of gravity and associated low fluid shear force conditions. Under these conditions, unexpected microbial responses occur, including alterations in virulence, antibiotic and stress resistance, biofilm formation, metabolism, motility, and gene expression, which are not observed using conventional experimental approaches. Here, we review biological and physical mechanisms that regulate microbial responses to spaceflight and spaceflight analog environments from both the microbe and host-microbe perspective that are relevant to human health and habitat sustainability. We highlight instrumentation and technology used in spaceflight microbiology experiments, their limitations, and advances necessary to enable next-generation research. As spaceflight experiments are relatively rare, we discuss ground-based analogs that mimic aspects of microbial responses to reduced gravity in spaceflight, including those that reduce mechanical forces of fluid flow over cell surfaces which also simulate conditions encountered by microorganisms during their terrestrial lifecycles. As spaceflight mission durations increase with traditional astronauts and commercial space programs send civilian crews with underlying health conditions, microorganisms will continue to play increasingly critical roles in health and habitat sustainability, thus defining a new dimension of occupational health. The ability of microorganisms to adapt, survive, and evolve in the spaceflight environment is important for future human space endeavors and provides opportunities for innovative biological and technological advances to benefit life on Earth.

Promoting Tech Transfer Between Space and Global Mental Health.

INTRODUCTION: Numerous issues in mental health benefit from technological innovation. An example involves the mental health challenges of long-duration spaceflight (such as a Mars mission), including prolonged confinement, microgravity, and different sunlight exposure lengths. Persisting on Earth are global mental health challenges stemming from disease burdens, limited interview-based diagnostic systems, trial-and-error treatment approaches, and suboptimal access. There is potential for cross-pollinating solutions between these seemingly disparate challenges using a range of emerging technologies such as sensors, omics, and big data. In this review, we highlight the bidirectional value of mental health technology transfer aimed to address issues both on Earth and in space.METHODS: We prepared a systematic review of studies pertaining to mental health technological innovation and space medicine.RESULTS: For Earth mental health technologies translatable to long-duration space missions, we cite several example technologies, including device-based psychotherapy and social support, conversational agents aka chatbots, and nutritional and physical activity focused mental health. Space technologies translatable to Earth mental health include remote sensing devices, global navigation satellite systems, satellite communications, chronotherapies, and nutritional advances.DISCUSSION: There is a rich history of space technologies informing Earth technological trends, including general health care on Earth, and vice versa. To avoid the traditional happenstance approach that results in delays, missed opportunities, and increased cost, and to improve outcomes for both Earth and space utilization of these technologies, we propose increased dialogue and training opportunities to enhance innovation and outcomes.Chang DD, Storch EA, Black L, Berk M, Pellis N, Lavretsky H, Sutton J, Ternes K, Shepanek M, Smith E, Abbott R, Eyre HA. Promoting tech transfer between space and global mental health. Aerosp Med Hum Perform. 2020; 91(9):737745.

A three-dimensional tissue culture model of bone formation utilizing rotational co-culture of human adult osteoblasts and osteoclasts.

Living bone is a complex, three-dimensional composite material consisting of numerous cell types spatially organized within a mineralized extracellular matrix. To date, mechanistic investigation of the complex cellular level cross-talk between the major bone-forming cells involved in the response of bone to mechanical and biochemical stimuli has been hindered by the lack of a suitable in vitro model that captures the "coupled" nature of this response. Using a novel rotational co-culture approach, we have generated large (>4mm diameter), three-dimensional mineralized tissue constructs from a mixture of normal human primary osteoblast and osteoclast precursor cells without the need for any exogenous osteoconductive scaffolding material that might interfere with such cell-cell interactions. Mature, differentiated bone constructs consist of an outer region inhabited by osteoclasts and osteoblasts and a central region containing osteocytes encased in a self-assembled, porous mineralized extracellular matrix. Bone constructs exhibit morphological, mineral and biochemical features similar to remodeling human trabecular bone, including the expression of mRNA for SOST, BGLAP, ACP5, BMP-2, BMP-4 and BMP-7 within the construct and the secretion of BMP-2 protein into the medium. This "coupled" model of bone formation will allow the future investigation of various stimuli on the process of normal bone formation/remodeling as it relates to the cellular function of osteoblasts, osteoclasts and osteocytes in the generation of human mineralized tissue.

Cellular and genetic adaptation in low-gravity environments.

Genetic response suites in human lymphocytes in response to microgravity are important to identify and study further to augment physiological adaptation to new milieus. Human peripheral blood from normal donors was used to isolate peripheral blood mononuclear cells. Blood traverses through most organs and hence is a suitable overall physiological predictor. The cells were cultured in 1g (T flask) and modeled microgravity for 24 and 72 h. Cell samples were collected and subjected to gene array analysis. Data were collected and subjected to a two-way analysis of variance. Different groups of genes related to the immune response, cardiovascular system, and stress response were then analyzed. These three groups focused on human adaptation to new environments. Many molecules related to T cell activation and second messengers, located both in the cell membrane and cytoplasm, were significantly altered (positive or negative regulation) in modeled microgravity. Cardiovascular biomarker expression and stress response gene expression also presented an aberrant response in analog microgravity. Previous findings in our laboratory showed lymphocyte activation and locomotion to be significantly suppressed in microgravity. Further analysis at the protein levels of genes involved in these responses could lead to development of prophylactic and countermeasure steps to augment human physiology for long-term space travel. Detailed results from the genetic analyses are presented in this study, including differential responses in stress response genes, cardiovascular and atherogenic genes, and T cell activation genes.

Proteomic analysis of mouse hypothalamus under simulated microgravity.

Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.

Melanoma growth and tumorigenicity in models of microgravity.

INTRODUCTION: Spaceflight involves numerous biological stressors that could affect long-term cancer incidence and tumor behavior. Ground-based models of microgravity can be used to investigate in vitro and in vivo tumor growth as a preparation for later work in space. The incidence of tumor growth and carcinogenesis in microgravity is as yet unknown. Hence, we investigated the effects of modeled microgravity on tumor growth and tumorigenicity using ground-based in vitro and in vivo models. METHODS: Murine B16-F10 melanoma cells were cultured in a tissue culture flask (FL) and in a rotating-wall vessel bioreactor (BIO) designed by NASA to simulate some aspects of microgravity. We then measured cell growth, melanin production, and apoptosis. After 48 h of cultures in FL and BIO, cells were inoculated subcutaneously in C57BL/6 mice, syngeneic hosts for B16-F10 tumor cells. Tumor sizes were then measured every other day. RESULTS: BIO cultures had 50% decreases in growth when compared with FL cultures while demonstrating an inversely proportional increase in doubling time. Melanin production (a marker of differentiation) increased at 24 and 48 h in BIO. Flow cytometry analysis demonstrated that there was an increase in the percentage of apoptotic cells in the BIO when compared with that in the FL. When BIO-cultured melanoma cells were inoculated subcutaneously in mice, there was a significant increase in tumorigenicity as compared with FL-cultured cells. CONCLUSION: Our results indicate that simulated microgravity may have altered the tumor cell characteristics and enhanced the invasive property. It is possible that the microgravity analogue culture environment may have selected highly tumorigenic cells for survival despite the decreased overall growth in the microgravity analogue.

Gene expression alterations in activated human T-cells induced by modeled microgravity.

Studies conducted in real Space and in ground-based microgravity analog systems (MAS) have demonstrated changes in numerous lymphocyte functions. In this investigation we explored whether the observed functional changes in lymphocytes in MAS are associated with changes in gene expression. NASA-developed Rotating Wall Vessel (RWV) bioreactor was utilized as a MAS. Activated T lymphocytes were obtained by adding 100 ng/ml of anti-CD3 and 100 U/ml of IL-2 in RPMI medium to blood donor mononuclear cells for 4 days. After that the cells were washed and additionally cultured for up to 2 weeks with media (RPMI, 10% FBS and 100 U/ml IL-2) replacement every 3-4 days. Flow cytometry analysis had proven that activated T lymphocytes were the only cells remaining in culture by that time. They were split into two portions, cultured for additional 24 h in either static or simulated microgravity conditions, and used for RNA extraction. The gene expression was assessed by Affymetrix GeneChip Human U133A array allowing screening for expression of 18,400 genes. About 4-8% of tested genes responded to MG by more than a 1.5-fold change in expression; however, reproducible changes were observed only in 89 genes. Ten of these genes were upregulated and 79 were downregulated. These genes were categorized by associated pathways and viewed graphically through histogram analysis. Separate histograms of each pathway were then constructed representing individual gene expression fold changes. Possible functional consequences of the identified reproducible gene expression changes are discussed.

Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis.

In vitro cell culture models used to study how Salmonella initiates disease at the intestinal epithelium would benefit from the recognition that organs and tissues function in a three-dimensional (3-D) environment and that this spatial context is necessary for development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3-D models of human colonic epithelium and apply them to study the early stages of enteric salmonellosis. The human colonic cell line HT-29 was cultured in 3-D and characterized by immunohistochemistry, histology, and scanning electron microscopy. Wild-type Salmonella typhimurium and an isogenic SPI-1 type three secretion system (TTSS) mutant derivative (invA) were used to compare the interactions with 3-D cells and monolayers in adherence/invasion, tissue pathology, and cytokine expression studies. The results showed that 3-D culture enhanced many characteristics normally associated with fully differentiated, functional intestinal epithelia in vivo, including better organization of junctional, extracellular matrix, and brush-border proteins, and highly localized mucin production. Wild-type Salmonella demonstrated increased adherence, but significantly lower invasion for 3-D cells. Interestingly, the SPI-I TTSS mutant showed wild-type ability to invade into the 3-D cells but did not cause significant structural changes to these cells. Moreover, 3-D cells produced less interleukin-8 before and after Salmonella infection. These results suggest that 3-D cultures of human colonic epithelium provide valuable alternative models to study human enteric salmonellosis with potential for novel insight into Salmonella pathogenesis.

Proteomic analysis of mice hippocampus in simulated microgravity environment.

Space travel induces many deleterious effects on the flight crew due to the '0' g environment. The brain experiences a tremendous fluid shift, which is responsible for many of the detrimental changes in physical behavior seen in astronauts. It therefore indicates that the brain may undergo major changes in its protein levels in a '0' g environment to counteract the stress. Analysis of these global changes in proteins may explain to better understand the functioning of brain in a '0' g condition. Toward such an effort, we have screened proteins in the hippocampus of mice kept in simulated microgravity environment for 7 days and have observed a few changes in major proteins as compared to control mice. Essentially, the results show a major loss of proteins in the hippocampus of mice subjected to simulated microgravity. These changes occur in structural proteins such as tubulin, coupled with the loss of proteins involved in metabolism. This preliminary investigation leads to an understanding of the alteration of proteins in the hippocampus in response to the microgravity environment.

Recent NASA research accomplishments aboard the ISS.

The activation of the US Laboratory Module "Destiny" on the International Space Station (ISS) in February 2001 launched a new era in microgravity research. Destiny provides the environment to conduct long-term microgravity research utilizing human intervention to assess, report, and modify experiments real time. As the only available pressurized space platform, ISS maximizes today's scientific resources and substantially increases the opportunity to obtain much longed-for answers on the effects of microgravity and long-term exposure to space. In addition, it evokes unexpected questions and results while experiments are still being conducted, affording time for changes and further investigation. While building and outfitting the ISS is the main priority during the current ISS assembly phase, seven different space station crews have already spent more than 2000 crew hours on approximately 80 scientific investigations, technology development activities, and educational demonstrations.

Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog.

Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.

Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity.

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.