Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

3D microenvironment attenuates simulated microgravity-mediated changes in T cell transcriptome.

Human space travel and exploration are of interest to both the industrial and scientific community. However, there are many adverse effects of spaceflight on human physiology. In particular, there is a lack of understanding of the extent to which microgravity affects the immune system. T cells, key players of the adaptive immune system and long-term immunity, are present not only in blood circulation but also reside within the tissue. As of yet, studies investigating the effects of microgravity on T cells are limited to peripheral blood or traditional 2D cell culture that recapitulates circulating blood. To better mimic interstitial tissue, 3D cell culture has been well established for physiologically and pathologically relevant models. In this work, we utilize 2D cell culture and 3D collagen matrices to gain an understanding of how simulated microgravity, using a random positioning machine, affects both circulating and tissue-resident T cells. T cells were studied in both resting and activated stages. We found that 3D cell culture attenuates the effects of simulated microgravity on the T cells transcriptome and nuclear irregularities compared to 2D cell culture. Interestingly, simulated microgravity appears to have less effect on activated T cells compared to those in the resting stage. Overall, our work provides novel insights into the effects of simulated microgravity on circulating and tissue-resident T cells which could provide benefits for the health of space travellers.

Lab-on-chip technologies for space research - current trends and prospects.

The in-depth analysis concerning application of microfluidic instruments for space biology research is presented. The article focuses on recently investigated key scientific fields, i.e., lab-on-chips applied to the biomedical studies performed in the (1) International Space Station and (2) CubeSat nanosatellites. The paper presents also the lab-on-chip devices that were fabricated with a view to future space biology research and to those that to date have been solely been tested under Earth laboratory conditions and/or simulated microgravity environments. NASA and ESA conceptual mission plans for future are also mentioned, concerning for instance "tissue chips" and the ESA-SPHEROIDS campaign. The paper ends with final conclusions and future perspectives regarding lab-on-chip application in the space biology sector and its impact on novel biomedical and pharmaceutical strategies.

Microfluidic-Assisted Human Cancer Cells Culturing Platform for Space Biology Applications.

In the paper, the lab-on-chip platform applicable for the long-term cultivation of human cancer cells, as a solution meeting the demands of the CubeSat biological missions, is presented. For the first time, the selected cancer cell lines-UM-UC-3 and RT 112 were cultured on-chip for up to 50 days. The investigation was carried out in stationary conditions (without medium microflow) in ambient temperature and utilizing the microflow perfusion system in the incubation chamber assuring typical cultivation atmosphere (37 °C). All the experiments were performed to imitate the conditions that are provided before the biological mission starts (waiting for the rocket launch) and when the actual experiment is initialized on a CubeSat board in space microgravity. The results of the tests showed appropriate performance of the lab-on-chip platform, especially in the context of material and technological biocompatibility. Cultured cells were characterized by adequate morphology-high attachment rate and visible signs of proliferation in each of the experimental stage. These results are a good basis for further tests of the lab-on-chip platform in both terrestrial and space conditions. At the end of the manuscript, the authors provide some considerations regarding a potential 3-Unit CubeSat biological mission launched with Virgin Orbit company. The lab-on-chip platform was modelled to fit a 2-Unit autonomous laboratory payload.

Interstellar space biology via Project Starlight.

Our ability to explore the cosmos by direct contact has been limited to a small number of lunar and interplanetary missions. However, the NASA Starlight program points a path forward to send small, relativistic spacecraft far outside our solar system via standoff directed-energy propulsion. These miniaturized spacecraft are capable of robotic exploration but can also transport seeds and organisms, marking a profound change in our ability to both characterize and expand the reach of known life. Here we explore the biological and technological challenges of interstellar space biology, focusing on radiation-tolerant microorganisms capable of cryptobiosis. Additionally, we discuss planetary protection concerns and other ethical considerations of sending life to the stars.

Plant responses to gravity.

Tropisms are directed growth-mediated plant movements which allow plants to respond to their environment. Gravitropism is the ability of plants to perceive and respond to the gravity vector and orient themselves accordingly. The gravitropic pathway can be divided into three main components: perception, biochemical signaling, and differential growth. Perception of the gravity signal occurs through the movement/sedimentation of starch-filled plastids (termed statoliths) in gravity sensing cells. Once perceived, proteins interact with the settling statoliths to set a cascade of plant hormones to the elongation zones in the roots or shoots. Plant growth regulators that play a role in gravitropism include auxin, ethylene, gibberellic acid, jasmonic acid, among others. Differential growth on opposing sides of the root or shoot allow for the plant to grow relative to the direction of the perceived gravity vector. In this review, we detail how plants perceive gravity and respond biochemically in response to gravity as well as synthesize the recent literature on this important topic in plant biology. Keywords: auxin, gravitropism, gravity perception, plant growth regulators, space biology, statolith.

Engineered Microvessel for Cell Culture in Simulated Microgravity.

As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.

Tardigrades in Space Research - Past and Future.

To survive exposure to space conditions, organisms should have certain characteristics including a high tolerance for freezing, radiation and desiccation. The organisms with the best chance for survival under such conditions are extremophiles, like some species of Bacteria and Archea, Rotifera, several species of Nematoda, some of the arthropods and Tardigrada (water bears). There is no denying that tardigrades are one of the toughest animals on our planet and are the most unique in the extremophiles group. Tardigrada are very small animals (50 to 2,100 µm in length), and they inhabit great number of Earth environments. Ever since it was proven that tardigrades have high resistance to the different kinds of stress factors associated with cosmic journeys, combined with their relatively complex structure and their relative ease of observation, they have become a perfect model organism for space research. This taxon is now the focus of astrobiologists from around the world. Therefore, this paper presents a short review of the space research performed on tardigrades as well as some considerations for further studies.

Metabolic Pathways of the Warburg Effect in Health and Disease: Perspectives of Choice, Chain or Chance.

Focus on the Warburg effect, initially descriptive of increased glycolysis in cancer cells, has served to illuminate mitochondrial function in many other pathologies. This review explores our current understanding of the Warburg effect's role in cancer, diabetes and ageing. We highlight how it can be regulated through a chain of oncogenic events, as a chosen response to impaired glucose metabolism or by chance acquisition of genetic changes associated with ageing. Such chain, choice or chance perspectives can be extended to help understand neurodegeneration, such as Alzheimer's disease, providing clues with scope for therapeutic intervention. It is anticipated that exploration of Warburg effect pathways in extreme conditions, such as deep space, will provide further insights crucial for comprehending complex metabolic diseases, a frontier for medicine that remains equally significant for humanity in space and on earth.

Morphometric analyses of petioles of seedlings grown in a spaceflight experiment.

Gravity is a constant unidirectional stimulus on Earth, and gravitropism in plants involves three phases: perception, transduction, and response. In shoots, perception takes place within the endodermis. To investigate the cellular machinery of perception in microgravity, we conducted a spaceflight study with Arabidopsis thaliana seedlings, which were grown in microgravity in darkness using the Biological Research in Canisters (BRIC) hardware during space shuttle mission STS-131. In the 14-day-old etiolated plants, we studied seedling development and the morphological parameters of the endodermal cells in the petiole. Seedlings from the spaceflight experiment (FL) were compared to a ground control (GC), which both were in the BRIC flight hardware. In addition, to assay any potential effects from growth in spaceflight hardware, we performed another control by growing seedlings in Petri dishes in standard laboratory conditions (termed the hardware control, HC). Seed germination was significantly lower in samples grown in flight hardware (FL, GC) compared to the HC. In terms of cellular parameters of endodermal cells, the greatest differences also were between seedlings grown in spaceflight hardware (FL, GC) compared to those grown outside of this hardware (HC). Specifically, the endodermal cells were significantly smaller in seedlings grown in the BRIC system compared to those in the HC. However, a change in the shape of the cell, suggesting alterations in the cell wall, was one parameter that appears to be a true microgravity effect. Taken together, our results suggest that caution must be taken when interpreting results from the increasingly utilized BRIC spaceflight hardware system and that it is important to perform additional ground controls to aid in the analysis of spaceflight experiments.

High-altitude balloon platform for studying the biological response of living organisms exposed to near-space environments.

The intangible desire to explore the mysteries of the universe has driven numerous advancements for humanity for centuries. Extraterrestrial journeys are becoming more realistic as a result of human curiosity and endeavors. Over the years, space biology research has played a significant role in understanding the hazardous effects of the space environment on human health during long-term space travel. The inevitable consequence of a space voyage is space ionizing radiation, which has deadly aftereffects on the human body. The paramount objective of this study is to provide a robust platform for performing biological experiments within the Earth's stratosphere by utilizing high-altitude balloons. This platform allows the use of a biological payload to simulate spaceflight missions within the unique properties of space that cannot be replicated in terrestrial facilities. This paper describes the feasibility and demonstration of a biological balloon mission suitable for students and scientists to perform space biology experiments within the boundary of the stratosphere. In this study, a high-altitude balloon was launched into the upper atmosphere (∼29 km altitude), where living microorganisms were exposed to a hazardous combination of UV irradiation, ultralow pressure and cold shock. The balloon carried the budding yeast Saccharomyces cerevisiae to investigate microbial survival potential under extreme conditions. The results indicated a notable reduction in biosample mortality two orders of magnitude (2-log) after exposure to 164.9 kJ m-2 UV. Postflight experiments have shown strong evidence that the effect of UV irradiation on living organisms is stronger than that of other extreme conditions.

Assessing Radish Health during Space Cultivation by Gene Transcription.

During the Advanced Plant Habitat experiment 2, radish plants were grown in two successive grow-outs on the International Space Station (ISS) for 27 days each. On days 10, 18, and 24, leaf punch (LP) samples were collected and frozen. At harvest, bulb tissue was sampled with oligo-dT functionalized Solid Phase Gene Extraction (SPGE) probes. The space samples were compared with samples from ground controls (GC) grown at the Kennedy Space Center (KSC) under the same conditions as on the ISS, with notably elevated CO2 (about 2500 ppm), and from lab plants grown under atmospheric CO2 but with light and temperature conditions similar to the KSC control. Genes corresponding to peroxidase (RPP), glucosinolate biosynthesis (GIS), protein binding (CBP), myrosinase (RMA), napin (RSN), and ubiquitin (UBQ) were measured by qPCR. LP from day 24 and bulb samples collected at harvest were compared with RNA-seq data from material that was harvested, frozen, and analyzed after return to Earth. The results showed stable transcription in LP samples in GC but decreasing values in ISS samples during both grow-outs, possibly indicative of stress. SPGE results were similar between GC and ISS samples. However, the RNA-seq analyses showed different transcription profiles than SPGE or LP results, possibly related to localized sampling. RNA-seq of leaf samples showed greater variety than LP data, possibly because of different sampling times. RSN and RPP showed the lowest transcription regardless of method. Temporal analyses showed relatively small changes during plant development in space and in ground controls. This is the first study that compares developmental changes in space-grown plants with ground controls based on a comparison between RNA-seq and qPCR analyses.

Designing a Novel Monitoring Approach for the Effects of Space Travel on Astronauts' Health.

Space exploration and extraterrestrial civilization have fascinated humankind since the earliest days of human history. It was only in the last century that humankind finally began taking significant steps towards these goals by sending astronauts into space, landing on the moon, and building the International Space Station. However, space voyage is very challenging and dangerous, and astronauts are under constant space radiation and microgravity. It has been shown that astronauts are at a high risk of developing a broad range of diseases/disorders. Thus, it is critical to develop a rapid and effective assay to monitor astronauts' health in space. In this study, gene expression and correlation patterns were analyzed for 10 astronauts (8 male and 2 female) using the publicly available microarray dataset E-GEOD-74708. We identified 218 differentially expressed genes between In-flight and Pre-flight and noticed that space travel decreased genome regulation and gene correlations across the entire genome, as well as individual signaling pathways. Furthermore, we systematically developed a shortlist of 32 genes that could be used to monitor astronauts' health during space travel. Further studies, including microgravity experiments, are warranted to optimize and validate the proposed assay.

Comparative genomic analysis of Cohnella hashimotonis sp. nov. isolated from the International Space Station.

A single strain from the family Paenibacillaceae was isolated from the wall behind the Waste Hygiene Compartment aboard the International Space Station (ISS) in April 2018, as part of the Microbial Tracking mission series. This strain was identified as a gram-positive, rod-shaped, oxidase-positive, catalase-negative motile bacterium in the genus Cohnella, designated as F6_2S_P_1T. The 16S sequence of the F6_2S_P_1T strain places it in a clade with C. rhizosphaerae and C. ginsengisoli, which were originally isolated from plant tissue or rhizosphere environments. The closest 16S and gyrB matches to strain F6_2S_P_1T are to C. rhizosphaerae with 98.84 and 93.99% sequence similarity, while a core single-copy gene phylogeny from all publicly available Cohnella genomes places it as more closely related to C. ginsengisoli. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values to any described Cohnella species are <89 and <22%, respectively. The major fatty acids for strain F6_2S_P_1T are anteiso-C15:0 (51.7%), iso-C16:0 (23.1%), and iso-C15:0 (10.5%), and it is able to metabolize a wide range of carbon compounds. Given the results of the ANI and dDDH analyses, this ISS strain is a novel species within the genus Cohnella for which we propose the name Cohnella hashimotonis, with the type strain F6_2S_P_1T (=NRRL B-65657T and DSMZ 115098T). Because no closely related Cohnella genomes were available, this study generated the whole-genome sequences (WGSs) of the type strains for C. rhizosphaerae and C. ginsengisoli. Phylogenetic and pangenomic analysis reveals that F6_2S_P_1T, C. rhizosphaerae, and C. ginsengisoli, along with two uncharacterized Cohnella strains, possess a shared set of 332 gene clusters which are not shared with any other WGS of Cohnella species, and form a distinct clade branching off from C. nanjingensis. Functional traits were predicted for the genomes of strain F6_2S_P_1T and other members of this clade.

Phylogenetic affiliations and genomic characterization of novel bacterial species and their abundance in the International Space Station.

Background: With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habits, and how microbes survive, proliferate and spread in space conditions, is coming more and more important. The Microbial Tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the International Space Station (ISS). Results: The analysis of their 16S rRNA gene sequences revealed <99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing (WGS) was undertaken. For all strains, the gyrB gene exhibited <93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average ucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, when compared to any known bacterial species, were less than <94% and 50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including ß-lactone and type III polyketide synthase (T3PKS) clusters. Conclusions: Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10T (=NRRL B-65660T), Leifsonia virtsii, F6_8S_P_1AT (=NRRL B-65661T), Leifsonia williamsii, F6_8S_P_1BT (=NRRL B- 65662T and DSMZ 115932T), Paenibacillus vandeheii, F6_3S_P_1CT(=NRRL B-65663T and DSMZ 115940T), and Sporosarcina highlanderae F6_3S_P_2 T(=NRRL B-65664T and DSMZ 115943T). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions.

Gravity's effect on biology.

Gravity is a fundamental interaction that permeates throughout our Universe. On Earth, gravity gives weight to physical objects, and has been a constant presence throughout terrestrial biological evolution. Thus, gravity has shaped all biological functions, some examples include the growth of plants (e.g., gravitropism), the structure and morphology of biological parts in multicellular organisms, to its effects on our physiological function when humans travel into space. Moreover, from an evolutionary perspective, gravity has been a constant force on biology, and life, to our understanding, should have no reason to not experience the effects of gravity. Interestingly, there appear to be specific biological mechanisms that activate in the absence of gravity, with the space environment the only location to study the effects of a lack of gravity on biological systems. Thus, in this perspective piece, biological adaptations from the cellular to the whole organism levels to the presence and absence of gravity will be organized and described, as well as outlining future areas of research for gravitational biological investigations to address. Up to now, we have observed and shown how gravity effects biology at different levels, with a few examples including genetic (e.g., cell cycle, metabolism, signal transduction associated pathways, etc.), biochemically (e.g., cytoskeleton, NADPH oxidase, Yes-associated protein, etc.), and functionally (e.g., astronauts experiencing musculoskeletal and cardiovascular deconditioning, immune dysfunction, etc., when traveling into space). Based from these observations, there appear to be gravity-sensitive and specific pathways across biological organisms, though knowledge gaps of the effects of gravity on biology remain, such as similarities and differences across species, reproduction, development, and evolutionary adaptations, sex-differences, etc. Thus, here an overview of the literature is provided for context of gravitational biology research to-date and consideration for future studies, as we prepare for long-term occupation of low-Earth Orbit and cis-Lunar space, and missions to the Moon and Mars, experiencing the effects of Lunar and Martian gravity on biology, respectively, through our Artemis program.

Understanding the Complexities and Changes of the Astronaut Microbiome for Successful Long-Duration Space Missions.

During space missions, astronauts are faced with a variety of challenges that are unique to spaceflight and that have been known to cause physiological changes in humans over a period of time. Several of these changes occur at the microbiome level, a complex ensemble of microbial communities residing in various anatomic sites of the human body, with a pivotal role in regulating the health and behavior of the host. The microbiome is essential for day-to-day physiological activities, and alterations in microbiome composition and function have been linked to various human diseases. For these reasons, understanding the impact of spaceflight and space conditions on the microbiome of astronauts is important to assess significant health risks that can emerge during long-term missions and to develop countermeasures. Here, we review various conditions that are caused by long-term space exploration and discuss the role of the microbiome in promoting or ameliorating these conditions, as well as space-related factors that impact microbiome composition. The topics explored pertain to microgravity, radiation, immunity, bone health, cognitive function, gender differences and pharmacomicrobiomics. Connections are made between the trifecta of spaceflight, the host and the microbiome, and the significance of these interactions for successful long-term space missions.

NASA's Ground-Based Microgravity Simulation Facility.

Since opportunities to conduct experiments in space are scarce, various microgravity simulators and analogs have been widely used in space biology ground studies. Even though microgravity simulators do not produce all of the biological effects observed in the true microgravity environment, they provide alternative test platforms that are effective, affordable, and readily available to facilitate microgravity research. The Microgravity Simulation Support Facility (MSSF) at the National Aeronautics and Space Administration (NASA) John F. Kennedy Space Center (KSC) has been established for conducting short duration experiments, typically less than 1 month, utilizing a variety of microgravity simulation devices for research at different gravity levels. The simulators include, but are not limited to, 2D Clinostats, 3D Clinostats, Random Positioning Machines, and Rotating Wall Vessels. In this chapter, we will discuss current MSSF capabilities, development concepts, and the physical characteristics of these microgravity simulators.

Conducting Plant Experiments in Space and on the Moon.

The growth and development of plants during spaceflight have important implications for both basic and applied research supported by NASA and other international space agencies. While there have been many reviews of plant space biology, this chapter attempts to fill a gap in the literature on the actual process and methods of performing plant research in the spaceflight environment. One of the authors (JZK) has been a principal investigator on eight spaceflight projects. These experiences include using the U.S. Space Shuttle, the former Russian Space Station Mir, and the International Space Station, utilizing the Space Shuttle and Space X as launch vehicles. While there are several ways to fly an experiment into space and to obtain a spaceflight opportunity, this review focuses on using the NASA peer-reviewed sciences approach to get an experiment manifested for flight. Three narratives for the implementation of plant space biology experiments are considered from rapid turn around of a few months to a project with new hardware development that lasted 6 years. The many challenges of spaceflight research include logistical and resource constraints such as crew time, power, cold stowage, data downlinks, among others. Additional issues considered are working at NASA centers, hardware development, safety concerns, and the engineering versus science culture in space agencies. The difficulties of publishing the results from spaceflight research based on such factors as the lack of controls, limited sample size, and the indirect effects of the spaceflight environment also are summarized. Lessons learned from these spaceflight experiences are discussed in the context of improvements for future space-based research projects with plants. We also will consider new opportunities for Moon-based research via NASA's Artemis lunar exploration program.

Nitric oxide, gravity response, and a unified schematic of plant signaling.

Plant signaling components are often involved in numerous processes. Calcium, reactive oxygen species, and other signaling molecules are essential to normal biotic and abiotic responses. Yet, the summation of these components is integrated to produce a specific response despite their involvement in a myriad of response cascades. In the response to gravity, the role of many of these individual components has been studied, but a specific sequence of signals has not yet been assembled into a cohesive schematic of gravity response signaling. Herein, we provide a review of existing knowledge of gravity response and differential protein and gene regulation induced by the absence of gravity stimulus aboard the International Space Station and propose an integrated theoretical schematic of gravity response incorporating that information. Recent developments in the role of nitric oxide in gravity signaling provided some of the final contextual pillars for the assembly of the model, where nitric oxide and the role of cysteine S-nitrosation may be central to the gravity response. The proposed schematic accounts for the known responses to reorientation with respect to gravity in roots-the most well studied gravitropic plant tissue-and is supported by the extensive evolutionary conservation of regulatory amino acids within protein components of the signaling schematic. The identification of a role of nitric oxide in regulating the TIR1 auxin receptor is indicative of the broader relevance of the schematic in studying a multitude of environmental and stress responses. Finally, there are several experimental approaches that are highlighted as essential to the further study and validation of this schematic.