Programmed cell death and redox metabolism protect Chlamydomonas reinhardtii populations from the galactic cosmic environment on the Artemis-1 mission

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Hammond, Sajanlal Panikkanvalappil, Patricia L. Allen, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5268750/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract We hypothesized that on the Artemis I mission, exposure to the galactic cosmic environment, specifically microgravity, lack of convection, and galactic cosmic radiation, would reduce survival of Chlamydomonas reinhardtii , a green unicellular flagellate alga, through the process of necrosis. An alternative hypothesis was that the radiation stimulus would induce a population survival response via a programmed cell death pathway. The Chlamydomonas strains were spotted on nutrient agar acetate plates and flown on Artemis I in the new Moonshot hardware that provided six hours of light daily to synchronize the algal cell cycle and tracked temperature, power use, and gravity over time. Synchronous ground controls were run in parallel. Analysis included spectrophotometry of chlorophylls and photosystems, flow cytometry of cell viability and lipid content, and Raman spectroscopy to identify DNA damage and cellular proteins. To test for radiation protection, select strains carried the tardigrade damage suppressor ( Dsup ) gene known to protect animal cells against gamma and ionizing radiation, dehydration, and temperature extremes in ground-based studies. A new flight hardware termed “Moonshot” was designed, built, and flown. “Moonshot” performed flawlessly, and is now available as flight-certified, flight-proven hardware for timed illumination and monitoring for flight and terrestrial applications. Within the limitations of the physical handling of specimens necessitated by the Artemis-1 mission flight around the Moon with exposure to the galactic cosmic environment: 1. Flown samples exposed to cosmic radiation were more viable than ground controls, with increased programmed cell death and decreased necrosis. 2. There was no difference in Chlamydomonas growth or content of chlorophylls and photosystems in flown versus ground controls, ruling out energy metabolism as the mediator of cell death. 3. Raman spectroscopy analysis showed that the redox-protective protein beta-carotene, a known cell death mediator, was increased during flight around the moon. 4. Compared to control inserts, insertion of the Dsup tardigrade gene was protective both on the ground and in flight. Biological sciences/Cell biology Biological sciences/Microbiology cosmic radiation Chlamydomonas reinhardtii protection β-carotene Dsup tardigrade spaceflight redox Artemis-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The study of the biological effects of the galactic cosmic environment has a dual purpose. First, understanding the biology of galactic cosmic environment should guide development of protective measures for astronauts flying beyond low Earth orbit [ 1 ], as well as leveraging biological systems to produce meaningful biologics in the space environment [ 2 , 3 ]. The second purpose is to begin to explore whether galactic cosmic environment mimics have utility for ground-based clinical applications [ 4 ]. Experiments flown on the Artemis 1 mission that circumnavigated the Moon, represent the first opportunity since the Apollo era to return to Earth biological samples exposed to the galactic cosmic environment ( https://www.nasa.gov/reference/artemis-i/ ). To understand the novelty of this opportunity, it is critical to remember that the International Space Station (ISS) is inside the radioprotective Van Allen Belts. Although experiments and personnel on the ISS are exposed to microgravity, reduced convection, and more radiation than on Earth [ 5 ], they are shielded from the vast majority of galactic cosmic radiation by the van Allen Belts [ 6 ]. The green alga Chlamydomonas reinhardtii was selected as the biological model system. It is a well-characterized, motile, single-celled green alga whose genome is fully sequenced and is relatively easy to engineer molecularly. During previous growth in space, a light-dependent increase in photosystem II has been observed [ 7 ]. The primary hypothesis was that exposure to galactic cosmic environment would reduce Chlamydomonas survival through unregulated cell death mechanisms called necrosis. An alternative balancing argument was that the radiation stimulus would induce a population survival response via a programmed cell death (PCD). PCD is sometimes a population survival mechanism in microalgae [ 8 – 10 ]. Measurements on the galactic cosmic environment exposed Chlamydomonas , and ground-based controls, included metabolic parameters of energy metabolism, mechanisms of cell death, and production of natural products. Raman spectroscopy was used for real-time chemical analysis of the Chlamydomonas cells [ 11 , 12 ]. The Curiosity rover on Mars uses fluorescence and Raman spectroscopy to search for organic molecules as a possible sign of life [ 13 ], but the potential of the biological application of these techniques is only starting to be realized [ 11 , 12 ]. Raman spectroscopy is a non-destructive and non-invasive technique, which offers several advantages in studying various stress-induced (including radiation) biomolecular modifications at the single-cell level without altering their integrity [ 14 , 15 ]. Moreover, the sensitivity of Raman spectroscopy enables the detection of subtle changes in cellular redox metabolism, which is fundamental in elucidating the dynamic alterations in the levels of redox-active species, as well as the cellular response to oxidative stress caused by cosmic radiation. Further, several lines of evidence show that multiple tardigrade genes, including Dsup , afford protection from a diverse array of stresses including various forms of radiation present in low Earth orbit [ 16 , 17 ]. An aliquot of the green algae strains flown in this experiment had the tardigrade gene Dsup inserted into the cell nucleus to test for protection from the galactic cosmic environment. The initiative began with the design and fabrication of affordable new flight hardware (named Moonshot) to support the growth of Chlamydomonas reinhardtii [ 18 ], while simultaneously meeting the constraints of the Artemis I mission profile ( https://www.nasa.gov/mission/artemis-i/ ). Discussion Artemis-1 The effects of the galactic cosmic environment on biological samples remains largely unknown, as the samples returned on Artemis-1 were the first samples returned following exposure to galactic cosmic environment beyond low Earth orbit since the Apollo era. Viability shift: protection from cosmic radiation The major finding of this study is that our hypothesis that exposure to the galactic cosmic environment would reduce Chlamydomonas survival through the process of necrosis was disproven. The alternative balancing argument that the radiation stimulus would induce a population survival response via a PCD pathway is supported. These findings are initially surprising, as the galactic cosmic environment contains high linear energy transfer (LET) particles that evoke complex DNA and other cellular damage [ 19 , 20 ], including significant damage to biological organisms, that include space radiation-induced carcinogenesis, cardiovascular disease, and central nervous system deficiencies [ 21 , 22 ]. Approximately 70% of galactic cosmic particles are high energy protons with similar relative biological effectiveness (RBE) slightly higher than low LET radiation such as gamma rays and X-rays. Protons are passing through living organisms with uniform distributions. However, heavier particles, which can cause devastating consequences on cells with direct hits, are distributed non-uniformly through the cell populations. Secondary effects are then caused by secondary particles or by signal transduced from the cells with direct hits [ 26 , 27 , 28 , 29 ]. The findings can be explained by understanding the properties of PCD [ 8 , 9 , 23 , 24 ]. PCD is a population stress response that is both adaptive and plastic [ 9 ]. Initial population decline due to PCD in response to stress helps the population rebound [ 9 ]. PCD can be a group-level stress response, where there is reconstitution of population density by expansion of survivors [ 23 ]. Microgravity simulation has been found to induce PCD in multiple cells, and tissues, both in vivo and in vitro [ 19 ]. In Chlamydomonas reinhardtii , PCD provides differential species-specific fitness effects that not only benefit others of the same species, but also have an inhibitory effect on the growth of other species [ 8 ]. The phenomenon of programmed cell death benefiting relatives is not limited to Chlamydomonas as it is also true in the green microalga Ankistrodesmus , (Sphaeropleales, Selenastraceae) [ 25 ]. There are many methods to assay PCD in algae and specifically in, Chlamydomonas reinhardtii [ 10 ]. PCD and necrosis assayed by flow cytometry analysis of Annexin V binding and propidium iodide uptake (described as a ‘hard sign’ of PCD), or cell size, give the best specificity and sensitivity of methods available within the parameters of the Artemis-1 mission profile [ 26 ]. Multiparameter flow cytometry can provide more robust data analysis [ 27 ], but no matter which combination of parameters we compared, there was always more overall cell survival, more PCD, and less necrosis in the spaceflight samples compared to the ground controls. Insertion of the tardigrade gene Dsup : Protection from cosmic radiation Tardigrades are microscopic animals renowned for their ability to survive a vast array of environmental extremes, including essentially complete desiccation for up to a decade as well as severe radiation damage [ 28 – 31 ]. Tardigrades survived ten days in the temperature, vacuum, and unshielded cosmic radiation of space aboard European Space Agency’s FOTON-M3 mission [ 31 ]. Several mechanisms contribute to the tardigrades' radio-resistance, and Tardigrade Disordered Proteins (TDPs) provide remarkable resistance to these stresses. RNA-seq and differential gene expression analysis revealed 11 TDP genes that are induced up to 20-fold during desiccation, along with a related class of TDPs that are expressed constitutively at extremely high levels. Amongst the tardigrade intrinsically disordered proteins ( Dsup ; Cytoplasmic, Secreted, and Mitochondrial Abundant Heat Soluble protein; and late embryogenesis-abundant proteins (LEA)) [ 32 ], Dsup was our choice to insert into the nucleus of Chlamydomonas based on evidence that Dsup suppresses the occurrence of DNA breaks by radiation in human-cultured cells[ 17 ]. As tardigrades have high resistance to diverse stress factors associated with cosmic journeys, they are the focus of intense study by astrobiologists [ 33 , 34 ]. Inserting Dsup into the nucleus of Chlamydomonas did indeed improve survival, both in space and in the ground-based control samples. The substantial increase in survival of Chlamydomonas in space determined that the starting baseline survival of the two groups were very different. Certainly, Dsup induced a smaller increase in survival in space than on the ground, but it is unclear whether this is a ceiling phenomenon as the Dsup -induced final survival level is no different in the flight and ground groups. Raman spectroscopy and redox metabolism during spaceflight Redox signaling is activated in diverse tissues and cell systems by varied forms of stress, including radiation, spaceflight, and simulated microgravity [ 35 , 36 ]. Stress-induced redox activation can be alternatively adaptive or contribute to pathological outcomes [ 36 ]. Targeting of redox metabolism has been proposed for mitigation of radiation injury [ 37 ]. Carotenoids are a class of pigments critical for photoprotection [ 38 ]. It is likely that cosmic ray and/or microgravity induced stress in Chlamydomonas could potentially activate their defense mechanisms to prevent the radiation damage, which can result in enhanced carotenoid biosynthesis and production of carotenoids [ 39 ]. β-carotene administration is the subject of multiple clinical trials aiming to decrease cardiovascular disease or cancer risk [ 40 ]. The differential activation of unique survival strategies under space conditions, as observed through Raman spectroscopy in Chlamydomonas , highlights the complex nature of cellular responses to environmental stresses such as radiation and microgravity. The Raman spectroscopic analyses detected two types of cells, which we have designated as Type I and Type II. The enhanced production of carotenoids seen in Type II cells from the flown sample served as a protective mechanism, likely activated to scavenge reactive oxygen species generated by stress, thereby mitigating potential radiation damage. This aligns with various studies suggesting that carotenoid biosynthesis can be stimulated as an adaptive response to environmental challenges [ 41 , 42 ]. We also saw some algal cells without/weak β-carotene Raman bands (Type I) in the same flight samples, which is consistent with the flow cytometry and fluorescence microscopy data presented in this manuscript, that not all the cells can survive the cosmic ray stress. The lack of significant carotenoid vibrations in Type I cells (also in the azide-killed samples) suggests that these cells may not effectively induce protective responses, making them more susceptible to radiation-induced damage. This observation is critical as it implies that not all cells within a population will uniformly respond to stress, highlighting the importance of understanding individual cellular adaptations in space biology. In addition, our findings also underline the utility of Raman spectroscopy in detecting subtle changes in cellular composition and stress responses, such as shifts in carotenoid ratios and alterations in the electronic environment around chlorophyll molecules. These insights are pivotal for developing strategies to enhance the resilience of microorganisms in space. Chlorophylls and photosystems There was no detectible change in chlorophylls and photosystems during the flight, excluding energy metabolism as the cause of the changes in programmed cell death observed. Cost and practicality of Moonshot hardware Advances in materials, microelectronics, and molecular and cellular biology technologies provided a firm basis to produce cost-effective customized spaceflight hardware. Bionetics Corporation’s engineering team developed new hardware, called moonshot, within a cost that could be supported from the funded grant. Moonshot holds three 10 cm segmented Petri dishes, and provides timed daily blue and red-light sample exposure, while recording state, battery voltage, temperature, and acceleration in 3 dimensions at programmable times. The Moonshot hardware performed flawlessly, providing flight certified, flight proven hardware for future biological investigations [ 18 ]. Radiation dose The two sensors adjacent to the Moonshot hardware on Artemis I had different radiation spectrum sensitivities. Artemis I was initially planned in 2018, and at that time crew active dosimeters (CADs) were selected for radiation detection. When radiation area monitors (RAMs) became available in 2020, they were added to the radiation monitoring profile in flight [ 6 ]. The RAM is designed to record radiation exposure at a specific location, while a CAD is designed to move with a crew member. In our case both dosimeters were static on the hardware. The CAD was expected to have a slightly higher reading, due to differences in radiation spectrum collection by the two dosimeters. Solar energetic particles from solar flares or sunspots can moderate the observed radiation levels but no solar flares or sunspots occurred during the Artemis I mission. Other radiation detectors flown on the Orion capsule on the Artemis I mission included six CADS distributed around the capsule, and the Hybrid Electronic Radiation Assessor (HERA) system. HERA is a Timepix-based ionizing radiation detector built for NASA Exploration-class crewed missions [ 43 ]. Two ‘phantoms’ of the Matroshka AstroRad Radiation Experiment (MARE) flew in two of passenger seats (Seat #3 and Seat #4) in the Orion capsule [ 44 ]. The levels of radiation recorded in the RAM and CAD detectors during the Artemis-1 flight are similar to levels seen during the Curiosity rover flight to Mars, if the same period of time is extracted from the total radiation levels reported [ 45 ]. The radiation levels during the Artemis-1 mission flight were far greater than ambient terrestrial levels [ 46 ], about three times the levels on the ISS [ 5 ], and about the same total exposure as a medical computed tomography (CT) abdominal scan with and without contrast (Table 1 ) [ 47 ]. CAD and RAM measure quantity, but do not define the spectrum of the linear energy transfer for the galactic cosmic environment [ 6 ]. The radiation risk is directly influenced by this spectrum as the quality of the radiation characterized by its pattern of energy deposition at the micron/DNA scale determines damage [ 20 ]. The linear energy transfer profile as the Curiosity rover flew to Mars has been documented [ 45 ]. The linear energy transfer spectrum for the Artemis-1 flight has been defined [ 48 ], but a basis for biological interpretation is lacking. Parsing the elements of the galactic cosmic environment I deally, we would model and study the elements of microgravity, convection, and radiation of the galactic cosmic environment individually in ground-based studies. Unfortunately, the technical expertise to parse the elements of the galactic cosmic environment are lacking. Microgravity simulations balance gravity with an induced equal and opposite force, typically using liquid culture environment [ 49 ]. These model systems introduce multiple new stimuli, limiting comparison to an element of the galactic cosmic environment. There are scant, if any, mechanisms to model biological responses to low convection [ 49 ]. NASA’s ground-based Galactic Cosmic Ray Simulator at the NASA Space Radiation Lab [ 4 ] will be critical to understand the biology of galactic cosmic radiation but is beyond the scope and resources of the current study. Benefits and limitations The galactic cosmic environment poses many challenges but also provides opportunities. For life to survive in cosmic radiation, the first steps begin with research to understand the fundamental effect on biological processes; understand the fundamental biology of life during exposure to cosmic radiation; contribute knowledge to reduce human health risk beyond low Earth orbit; and contribute to improved system performance and reduced system risk. This data set is a first step to answer fundamental questions such as can we survive and thrive beyond Earth, and can we use knowledge gained from studying the biological effects of galactic space radiation for Earth-based benefits? Conclusions Within the limitations of the physical handling of specimens necessitated by the Artemis-1 mission, flight around the Moon with galactic cosmic environment exposure allows multiple conclusions. Flown samples exposed to the galactic cosmic environment were more viable than ground controls with increased programmed cell death and decreased necrosis. There was no difference in Chlamydomonas growth or content of chlorophylls and photosystems in flown versus ground controls, ruling out energy metabolism as the mediator of cell death. Compared to control inserts, insertion of the Dsup tardigrade gene was protective both on the ground and in flight, although the ground effect was far larger numerically. Raman spectroscopy analysis showed that the redox-protective protein beta-carotene, a known cell death mediator, was increased during flight around the Moon, defining this technology as an important new non-destructive tool for analysis of biological space flight samples. An inexpensive simple new flight hardware, termed Moonshot, can perform flawlessly, and is available as flight-certified, flight-proven hardware for timed illumination and monitoring of samples for flight and terrestrial applications. Methods and materials Chlamydomonas reinhardtii Chlamydomonas reinhardtii (CC-125 wild type mt+ [137c]), were purchased from the University of Minnesota Chlamydomonas collection (Minneapolis, MN). The Chlamydomonas were spotted on Tris-acetate-phosphate (TAP) agar plates, and aliquots reseeded onto fresh plates robotically every 2 weeks. TAP was 20 ml 1M Tris base, 1 ml phosphate K 2 HPO 4 /KH 2 PO 4 buffer, 1 ml Hunter’s trace metals, 10 ml of solution A [NH 4 Cl, MgSP 4 .7H 2 O, 1 ml CaCl 2 .2H 2 O], glacial acetic acid pH 7 made up to 1 liter with distilled water. Chemicals Life Technologies was the vendor for Annexin V, Alexa Fluor 488 conjugate, and Annexin-binding buffer – “5X concentrate” for flow cytometry. All other chemicals were purchased from Sigma/Aldrich, now Millipore-Sigma (Burlington, MA). Hutner’s trace elements were purchased from the University of Minnesota (Minneapolis, MN). Equipment Spectrophotometry was performed on a Molecular Dynamics (San Jose CA) M5e Spectrophotometer. Flow cytometry was performed on a Becton-Dickinson Accuri C + Plus flow cytometer (Franklin Lakes, NJ). Raman spectra were collected using a Renishaw inVia Raman Microscope coupled with Leica DM2500M microscope at the Dana-Farber Cancer Institute/Harvard Med. School, Boston, MA. A 785 nm diode laser was used for the surface enhanced Raman spectroscopy measurements. Moonshot hardware was designed and built by The Bionetics Corporation, (Kennedy Space Center, Brevard County, FL) as an ultralow-power growth system for use on Artemis-1 [ 18 ]. Each of the three growth modules holds one standard 100 mm diameter circular Petri dish. The plates are illuminated with blue (∼450 nm) and red (∼660 nm) light-emitting diode (LED) lights continuously for 6 h in every 24 h. The combination of red and blue light is optimum for Chlamydomonas [ 50 , 51 ]. The hardware monitors and records the temperature of the experiment, the power level, and draw from the batteries (as an indication that the lights were turned on and off), as well as the acceleration levels in three axes. Once activated and loaded with the biological samples, the flight hardware was placed in a custom lathed form-fitting Styrofoam support and encased in a Nitex bag. The assembly was termed the BioExpt-01 Science Bag. Crew Active Dosimeter (CAD) and Radiation Area Monitor (RAM) radiation detectors were placed adjacent to the biological flight hardware in the BioExpt-01 Science Bag. The detectors had been assembled and delivered to NASA/Kennedy Space Center prior to flight by the Space Radiation Analysis Group based at NASA Johnson Space Center (JSC) by Human Health and Performance Directorate/Leidos personnel. The detectors were collected post-flight and data was analyzed at NASA JSC [ 6 ]. Artemis-1 flight After an initial delay of 19 days on the launch pad, the Artemis − 1 mission was launched from Kennedy Space Center (KSC) on November 16, 2022 for a planned 25-day space mission. Orion completed one flyby of the Moon on November 21, followed by a distant retrograde orbit for six days and then a second flyby of the Moon on November 25, and subsequently returned to Earth. The Orion capsule was recovered from the Pacific Ocean, returned to California, and transported back to KSC by truck. However, the Space Biology experiments were first removed from Orion in California, flown on an accompanied commercial flight to Kennedy Space Center at ambient temperature (without security radiation scanning) and the Moonshot hardware containing the samples, released to the investigators 9 days after the landing. The sample arrived back in the investigator’s lab three days later, after decommissioning of the hardware at Bionetics facility adjacent to KSC. The samples for the payload described in the report were in the hardware for a total of 56.5 days (Fig. 1 ). The flight samples were exposed to microgravity in space, but also gravity transitions during launch and re-entry. Analysis Spectrophotometry Chlamydomonas colonies were scraped from the agar, washed three times in phosphate buffered saline, resuspended in TAP buffer and assayed by spectrophotometry for chlorophyll a (absorbance at 436 nm), chlorophyll b (absorbance at 472 nm), photosystem I and II (absorbance at 700 nm and 680nm respectively but the peaks are too broad to separate), cytochrome f (absorbance at 554 nm), and protein content (absorption at 800 nm). Net absorbance for each wavelength was calculated by subtracting the background absorbance of TAP buffer alone. The net absorbance values were then normalized to the protein content as measured by absorbance at 800 nm. Flow Cytometry Six colonies from flight and ground plates, plus four Dsup colonies from flight and ground plates, were harvested and resuspended in annexin-binding buffer, which is a phosphate buffer with calcium. The aggregates were disrupted by vortexing and then filtered through a 70µm nylon mesh to remove clumps that would plug the flow cytometer. The resuspended Chlamydomonas samples were aliquoted 100 µl per well into V-bottom microtiter plates. Individual aliquots were either stained with Alexa Fluor 488-annexin (5 µl/well) and propidium iodide (PI) (1 µl) to identify PCD and necrosis, or Nile Red (1:100 dilution from a stock solution of 100 µg ml − 1 in methanol) with 0.005% Triton-X (1:100 dilution from a stock solution of 0.5% in water) permeabilization to measure lipid content. We counted 2,000 Chlamydomonas cells from each sample. Background signal was estimated by appropriate no-dye controls. In every flow cytometry run, quality controls were performed on the instrument with three-color fluorescent beads, followed by assay of five control tubes: (1) no dyes, (2) Alexa fluor 488 annexin binding alone, (3) propidium iodide alone, (4) Alexa fluor 488 annexin binding with propidium iodide, and (5) Nile Red alone. Flow cytometric and spectrophotometric data were evaluated by two-tailed Student's t-test comparing heat- or cold-exposed Chlamydomonas to the relevant room temperature control. Single-cell confocal Raman spectroscopy of Chlamydomonas reinhardtii Multiple colonies of Chlamydomonas were harvested from a ground plate, and flight plate. The samples included colonies flown live and colonies pretreated with azide before flight. The colonies were harvested into 1.5 ml Eppendorf tubes with 500 µL TAP and resuspended by pipetting and vortexing. The resultant Chlamydomonas solution was filtered through a 70 µm mesh into 12 x 74 mm polypropylene tubes to remove multicellular aggregates. The remaining solution of cells was divided into two 500 µL Eppendorf tubes, spun at 3000g for 1 minute to pellet the cells, and the supernatant aspirated. One tube was left to air dry. The second tube had 100 µL of 10% electron microscopy paraformaldehyde added, incubated for 10 min, and then was spun to pellet the cells before the supernatant was aspirated. This paraformaldehyde-fixed sample was washed with 500 µL deionized water, spun, and the supernatant aspirated. A 2 µl aliquot of each sample rehydrated with distilled water was spotted on a quartz slide (25 mm x 25 mm x 1 mm) that was kept on a microscopic glass slide covered with aluminum foil. This substrate showed minimal background signal compared to various other substrates we tried such as glass, CaF 2 and Al 2 O 3 . A silicon wafer was not used as it has a strong Raman band at 521 cm − 1 and interfered with the current analysis. The samples were air dried at room temperature and were analyzed. A Horiba LabRAM Odyssey Raman microscope was used for the Raman spectral characterization of these samples. Laser power density was optimized in order to achieve better spectral intensity with characteristic Raman bands, which were not present at lower laser power densities. Exposure time and laser intensity were optimized by conducting a series of experiments to prevent charring of the samples to eliminate any unwanted signals due to laser-induced biomolecule denaturation. A 785 nm laser was used to acquire Raman spectra (acquisition time of 30 s with 300 lines/mm grating). First, white light focused on the individual algal cells at a magnification of 100X. Then, Raman spectra were collected in the range of 400–1800 cm − 1 . The spectral baselines were pre-processed by polynomial fitting. Statistics Flow cytometric and spectrophotometric data are presented as geometric mean ± standard error with six replicates (unless otherwise noted). Correlations were analyzed by Statistica 6.1 (StatSoft Inc. Tulsa OK) using correlation matrix product moment and partial correlations. For the Raman spectral analysis: Spectra were collected from five or more algal samples across three different colonies exposed to various conditions and subsequently averaged to better represent statistical variations in the intensities of vibrations corresponding to various biomolecular components within the spectra. The standard deviation of these intensities under different conditions was illustrated using error bars in the histogram to provide a clear visualization of the variability and reliability of our measurements. Methods and materials Chlamydomonas reinhardtii Chlamydomonas reinhardtii (CC-125 wild type mt+ [137c]), were purchased from the University of Minnesota Chlamydomonas collection (Minneapolis, MN). The Chlamydomonas were spotted on Tris-acetate-phosphate (TAP) agar plates, and aliquots reseeded onto fresh plates robotically every 2 weeks. TAP was 20 ml 1M Tris base, 1 ml phosphate K 2 HPO 4 /KH 2 PO 4 buffer, 1 ml Hunter’s trace metals, 10 ml of solution A [NH 4 Cl, MgSP 4 .7H 2 O, 1 ml CaCl 2 .2H 2 O], glacial acetic acid pH 7 made up to 1 liter with distilled water. Chemicals Life Technologies was the vendor for Annexin V, Alexa Fluor 488 conjugate, and Annexin-binding buffer – “5X concentrate” for flow cytometry. All other chemicals were purchased from Sigma/Aldrich, now Millipore-Sigma (Burlington, MA). Hutner’s trace elements were purchased from the University of Minnesota (Minneapolis, MN). Equipment Spectrophotometry was performed on a Molecular Dynamics (San Jose CA) M5e Spectrophotometer. Flow cytometry was performed on a Becton-Dickinson Accuri C + Plus flow cytometer (Franklin Lakes, NJ). Raman spectra were collected using a Renishaw inVia Raman Microscope coupled with Leica DM2500M microscope at the Dana-Farber Cancer Institute/Harvard Med. School, Boston, MA. A 785 nm diode laser was used for the surface enhanced Raman spectroscopy measurements. Moonshot hardware was designed and built by The Bionetics Corporation, (Kennedy Space Center, Brevard County, FL) as an ultralow-power growth system for use on Artemis-1 [ 18 ]. Each of the three growth modules holds one standard 100 mm diameter circular Petri dish. The plates are illuminated with blue (∼450 nm) and red (∼660 nm) light-emitting diode (LED) lights continuously for 6 h in every 24 h. The combination of red and blue light is optimum for Chlamydomonas [ 50 , 51 ]. The hardware monitors and records the temperature of the experiment, the power level, and draw from the batteries (as an indication that the lights were turned on and off), as well as the acceleration levels in three axes. Once activated and loaded with the biological samples, the flight hardware was placed in a custom lathed form-fitting Styrofoam support and encased in a Nitex bag. The assembly was termed the BioExpt-01 Science Bag. Crew Active Dosimeter (CAD) and Radiation Area Monitor (RAM) radiation detectors were placed adjacent to the biological flight hardware in the BioExpt-01 Science Bag. The detectors had been assembled and delivered to NASA/Kennedy Space Center prior to flight by the Space Radiation Analysis Group based at NASA Johnson Space Center (JSC) by Human Health and Performance Directorate/Leidos personnel. The detectors were collected post-flight and data was analyzed at NASA JSC [ 6 ]. Equipment Spectrophotometry was performed on a Molecular Dynamics (San Jose CA) M5e Spectrophotometer. Flow cytometry was performed on a Becton-Dickinson Accuri C + Plus flow cytometer (Franklin Lakes, NJ). Raman spectra were collected using a Renishaw inVia Raman Microscope coupled with Leica DM2500M microscope at the Dana-Farber Cancer Institute/Harvard Med. School, Boston, MA. A 785 nm diode laser was used for the surface enhanced Raman spectroscopy measurements. Moonshot hardware was designed and built by The Bionetics Corporation, (Kennedy Space Center, Brevard County, FL) as an ultralow-power growth system for use on Artemis-1 [ 18 ]. Each of the three growth modules holds one standard 100 mm diameter circular Petri dish. The plates are illuminated with blue (∼450 nm) and red (∼660 nm) light-emitting diode (LED) lights continuously for 6 h in every 24 h. The combination of red and blue light is optimum for Chlamydomonas [ 50 , 51 ]. The hardware monitors and records the temperature of the experiment, the power level, and draw from the batteries (as an indication that the lights were turned on and off), as well as the acceleration levels in three axes. Once activated and loaded with the biological samples, the flight hardware was placed in a custom lathed form-fitting Styrofoam support and encased in a Nitex bag. The assembly was termed the BioExpt-01 Science Bag. Crew Active Dosimeter (CAD) and Radiation Area Monitor (RAM) radiation detectors were placed adjacent to the biological flight hardware in the BioExpt-01 Science Bag. The detectors had been assembled and delivered to NASA/Kennedy Space Center prior to flight by the Space Radiation Analysis Group based at NASA Johnson Space Center (JSC) by Human Health and Performance Directorate/Leidos personnel. The detectors were collected post-flight and data was analyzed at NASA JSC [ 6 ]. Artemis-1 flight After an initial delay of 19 days on the launch pad, the Artemis − 1 mission was launched from Kennedy Space Center (KSC) on November 16, 2022 for a planned 25-day space mission. Orion completed one flyby of the Moon on November 21, followed by a distant retrograde orbit for six days and then a second flyby of the Moon on November 25, and subsequently returned to Earth. The Orion capsule was recovered from the Pacific Ocean, returned to California, and transported back to KSC by truck. However, the Space Biology experiments were first removed from Orion in California, flown on an accompanied commercial flight to Kennedy Space Center at ambient temperature (without security radiation scanning) and the Moonshot hardware containing the samples, released to the investigators 9 days after the landing. The sample arrived back in the investigator’s lab three days later, after decommissioning of the hardware at Bionetics facility adjacent to KSC. The samples for the payload described in the report were in the hardware for a total of 56.5 days (Fig. 1 ). The flight samples were exposed to microgravity in space, but also gravity transitions during launch and re-entry. Analysis Spectrophotometry Chlamydomonas colonies were scraped from the agar, washed three times in phosphate buffered saline, resuspended in TAP buffer and assayed by spectrophotometry for chlorophyll a (absorbance at 436 nm), chlorophyll b (absorbance at 472 nm), photosystem I and II (absorbance at 700 nm and 680nm respectively but the peaks are too broad to separate), cytochrome f (absorbance at 554 nm), and protein content (absorption at 800 nm). Net absorbance for each wavelength was calculated by subtracting the background absorbance of TAP buffer alone. The net absorbance values were then normalized to the protein content as measured by absorbance at 800 nm. Flow Cytometry Six colonies from flight and ground plates, plus four Dsup colonies from flight and ground plates, were harvested and resuspended in annexin-binding buffer, which is a phosphate buffer with calcium. The aggregates were disrupted by vortexing and then filtered through a 70µm nylon mesh to remove clumps that would plug the flow cytometer. The resuspended Chlamydomonas samples were aliquoted 100 µl per well into V-bottom microtiter plates. Individual aliquots were either stained with Alexa Fluor 488-annexin (5 µl/well) and propidium iodide (PI) (1 µl) to identify PCD and necrosis, or Nile Red (1:100 dilution from a stock solution of 100 µg ml − 1 in methanol) with 0.005% Triton-X (1:100 dilution from a stock solution of 0.5% in water) permeabilization to measure lipid content. We counted 2,000 Chlamydomonas cells from each sample. Background signal was estimated by appropriate no-dye controls. In every flow cytometry run, quality controls were performed on the instrument with three-color fluorescent beads, followed by assay of five control tubes: (1) no dyes, (2) Alexa fluor 488 annexin binding alone, (3) propidium iodide alone, (4) Alexa fluor 488 annexin binding with propidium iodide, and (5) Nile Red alone. Flow cytometric and spectrophotometric data were evaluated by two-tailed Student's t-test comparing heat- or cold-exposed Chlamydomonas to the relevant room temperature control. Single-cell confocal Raman spectroscopy of Chlamydomonas reinhardtii Multiple colonies of Chlamydomonas were harvested from a ground plate, and flight plate. The samples included colonies flown live and colonies pretreated with azide before flight. The colonies were harvested into 1.5 ml Eppendorf tubes with 500 µL TAP and resuspended by pipetting and vortexing. The resultant Chlamydomonas solution was filtered through a 70 µm mesh into 12 x 74 mm polypropylene tubes to remove multicellular aggregates. The remaining solution of cells was divided into two 500 µL Eppendorf tubes, spun at 3000g for 1 minute to pellet the cells, and the supernatant aspirated. One tube was left to air dry. The second tube had 100 µL of 10% electron microscopy paraformaldehyde added, incubated for 10 min, and then was spun to pellet the cells before the supernatant was aspirated. This paraformaldehyde-fixed sample was washed with 500 µL deionized water, spun, and the supernatant aspirated. A 2 µl aliquot of each sample rehydrated with distilled water was spotted on a quartz slide (25 mm x 25 mm x 1 mm) that was kept on a microscopic glass slide covered with aluminum foil. This substrate showed minimal background signal compared to various other substrates we tried such as glass, CaF 2 and Al 2 O 3 . A silicon wafer was not used as it has a strong Raman band at 521 cm − 1 and interfered with the current analysis. The samples were air dried at room temperature and were analyzed. A Horiba LabRAM Odyssey Raman microscope was used for the Raman spectral characterization of these samples. Laser power density was optimized in order to achieve better spectral intensity with characteristic Raman bands, which were not present at lower laser power densities. Exposure time and laser intensity were optimized by conducting a series of experiments to prevent charring of the samples to eliminate any unwanted signals due to laser-induced biomolecule denaturation. A 785 nm laser was used to acquire Raman spectra (acquisition time of 30 s with 300 lines/mm grating). First, white light focused on the individual algal cells at a magnification of 100X. Then, Raman spectra were collected in the range of 400–1800 cm − 1 . The spectral baselines were pre-processed by polynomial fitting. Statistics Flow cytometric and spectrophotometric data are presented as geometric mean ± standard error with six replicates (unless otherwise noted). Correlations were analyzed by Statistica 6.1 (StatSoft Inc. Tulsa OK) using correlation matrix product moment and partial correlations. For the Raman spectral analysis: Spectra were collected from five or more algal samples across three different colonies exposed to various conditions and subsequently averaged to better represent statistical variations in the intensities of vibrations corresponding to various biomolecular components within the spectra. The standard deviation of these intensities under different conditions was illustrated using error bars in the histogram to provide a clear visualization of the variability and reliability of our measurements. Results Moonshot hardware The Moonshot hardware performed nominally throughout the flight and ground control experiments. The maximum temperature delta between flight and ground control hardware copies was 2.5 o F, well within the envelope of tolerable experimental conditions. At the end of the 25.5-day flight, the batteries still had more than 50% of power remaining. The power profile validated that the lights came on for 6 out of 24 h daily, as planned. Acceleration recordings show serial measurements of gravity in the z axis, but no acceleration in other directions as expected. The hardware met all the validation criteria for power use, power cycling, temperature control, g-force recording in three axes, data capture, and downloading for analysis, plus materials, and biological compatibility. The Moonshot hardware is now not only flight certified but flight proven. Spectrophotometry of chlorophyll a and b, cytochrome f, and photosystem levels Levels of the light absorbing pigments chlorophyll a and b, the mitochondrial heme protein cytochrome f, and the photon harvesting pigment photosystems were no different between ground and flight samples (Table 1 ). Radiation exposure in assorted environments in space and on Earth The total radiation exposure during the flight as measured in the RAM and CAD are reported in Table II. Comparison of the exposure levels during flight to other environmental systems (as shown in Table II) is examined in the discussion section. Flow cytometry assay of viability and PCD of Chlamydomonas in Flight vs Ground The percentages of PCD cells (Annexin V positive) and dead cells (propidium iodide positive) in the flight and ground control samples are shown in Fig. 2 . 45.1 ± 1.4% of the cells in flight had undergone PCD compared to 31.1 ± 2.0% of the cells in the ground control (mean ± standard error, n = 6, p < 0.001). 53.9 ± 1.3% of the cells in flight were had undergone PCD, compared to 67.6 ± 1.9% of the cells in the ground control (mean ± standard error, n = 6, p < 0.001). Flow cytometry assay of the effect of Dsup on the viability of Chlamydomonas in Flight vs Ground The percentages of live cells (PI negative) in flown and ground control Chlamydomonas are shown in Fig. 3 . 41.8 ± 1.7% of the flown cells with random gene inserts were living compared with 46.9 ± 2.3% of the flown cells with Dsup insertion (mean ± standard error, n = 6, p < 0.001). 16.4 ± 0.9% of the ground control cells with random gene inserts were living compared with 53.7 ± 2.6% of the ground control cells with Dsup insertion (mean ± standard error, n = 6, p < 0.001). Single-cell confocal Raman spectroscopy of Chlamydomonas reinhardtii in Flight vs Ground In this pioneering use of Raman spectroscopy for the analysis of space biology samples, we noticed a differential survival strategy among Chlamydomonas exposed to cosmic rays and/or microgravity. Specifically, we found two different cell types (Type I and Type II) with distinct Raman spectral features in flight samples compared to the ground samples. The intensity of Raman vibration corresponding to carotenoids in Type I cells (FL-Type I) was much weaker. Whereas, in Type II cells (FL-Type II), characteristic carotenoid vibrations were enhanced significantly (Fig. 4 A). In Raman spectra of Chlamydomonas , the presence of prominent vibrations at 1001, 1156, and 1523 cm − 1 indicate the presence of carotenoids [ 52 – 55 ], which are attributed to C–CH 3 deformation, C-C stretching, and C = C stretching vibrations of polyene, respectively [ 54 , 56 ]. The significant variation in these carotenoid Raman vibrations found in the spectral profiles between Type I and Type II algal cells offers a compelling insight into the heterogeneity of stress responses within a single-cell population in two-cell populations. The lack of significant carotenoid vibrations in the Type I flight sample implies that these cells may not have been able to induce the same protective response and might have yielded to radiation-induced damage. Moreover, the azide-killed flight samples exhibited nearly identical Raman spectra with weak carotenoid signal as in Type I cells, corroborating our proposed link between the absence of carotenoid and cell death pathways. The weaker carotenoid signals in these samples suggest that the living cells in the flight environment actively maintain or increase their carotenoid content. In contrast, the azide treatment or cell death ceases metabolic activity, thereby reducing the carotenoid levels. For better understanding the relative increase in the carotenoid content at various conditions, the intensity ratio (I Caro/Chlo ) of carotenoid-chlorophyll vibration (1523 cm − 1 and 1359 cm − 1 , respectively) was used (Fig. 4 C). We also used Raman spectroscopy to identify any significant changes in chlorophyll composition by analyzing prominent and characteristic Raman bands of chlorophylls found at 1359 (vibrations of C-C and C-N bonds within the porphyrin ring) and 1549 cm − 1 (primarily involves the C = C stretching vibrations within the porphyrin ring) [ 57 ]. We noticed that the intensity ratio (Fig. 4 B) of these bands (I 1549/1359 ) in flight samples (FL-Type I and FL-Type II) as well as in azide-killed samples, showed no significant differences compared to the ground sample, suggesting a comparable level chlorophyll levels across these conditions. This was in concordance with the data obtained from the spectrophotometry. A slight variation in the I 1549/1359 found in FL-Type II could potentially be due to the variation in chlorophyll a to chlorophyll b ratio as vibration corresponds to the stretching vibrations of the carbonyl (C = O) group in the aldehyde group present in Chlorophyll b also contributes to the intensity of Raman vibration at 1549 cm − 1 (this ratio has slightly increased in FL-Type II samples). In addition, a minor shift in the 1549 cm − 1 band to 1546 cm − 1 in FL-Type II samples was also found, which could possibly be attributed to the change in the electronic environment around the chlorophyll molecules as an adaptive response to radiation stress and subsequent variation in chlorophyll b content. Declarations Author contributions Author contributions were: Hardware design and build HWW, JMR, and TGH; Hands on biology experiments TGH, PLA, and HHB; Strain cloning HK, CN, and GG; Data management and statistics TGH and HHB; Administration services TGH, HLG, YZ, and HHB; Radiation dosimetry selection and management DD, and RG; Raman spectroscopy SP. Prose drafting TGH, SP, PD and HHB. All authors reviewed, edited, and approved multiple manuscript versions. Competing interests The authors declare no competing interests. Author Contribution Author contributions were: Hardware design and build HWW, JMR, and TGH; Hands on biology experiments TGH, PLA, and HHB; Strain cloning HK, CN, and GG; Data management and statistics TGH and HHB; Administration services TGH, HLG, YZ, and HHB; Radiation dosimetry selection and management DD, and RG; Raman spectroscopy SP. Prose drafting TGH, SP, PD and HHB. All authors reviewed, edited, and approved multiple manuscript versions. 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Mars' surface radiation environment measured with the Mars Science Laboratory's Curiosity rover. Science . 343 , 1244797. 10.1126/science.1244797 (2014). Tables Table 1 Relative quantities of protein in flown vs. ground control Chlamydomonas Protein (OD 800) Flight Avg Flight SEM Ground Avg Ground SEM T-test Chlorophyll a (OD 436) 1.244 0.049 1.257 0.038 NS Chlorophyll b (OD 472) 1.273 0.044 1.266 0.030 NS Cytochrome f (OD 554) 1.111 0.027 1.119 0.014 NS Photosystem I and II (OD 700) 1.153 0.017 1.141 0.014 NS Table 1. Aliquots of flown and ground control Chlamydomonas were suspended in TAP and the optical densities (ODs) for chlorophyll a (OD 436), chlorophyll b (OD 472), cytochrome f (OD 554) and photosystem I and II (OD 700) were measured by spectrophotometry. Values shown are the net OD above background absorbance for TAP media alone, normalized to the total quantity of protein measured as OD 800. Values are the mean and SEM of six replicates. Significance was estimated by unpaired t-test, with non-significant differences (NS) defined as p>0.05. Table II Radiation exposure in assorted environments in space and on Earth Exposure Radiation (mGy) Ref. Radiation Area Monitor Artemis I Mission Dose 11.4 ± 0.4 [ 6 ] Crew Active Dosimeter Artemis I Mission Dose 12.6 ± 0.5 [ 6 ] Naturally occurring "background" for 25.5 days (Earth) 0.17 [ 46 ] CAT-Scan of Abdomen & Pelvis, ± contrast 15.4 [ 47 ] Exposure on Mars for 25.5 days (Curiosity rover) 5.4 [ 45 , 58 ] Mars transit for 25.5 days (Curiosity rover) 11.8 [ 45 , 58 ] International Space Station for 25.5 days 5.7 [ 5 ] Table II. Comparison of total radiation doses for 25.5 days during the Artemis I mission, plus 25.5 days of exposure to background radiation on Earth, the Curiosity rover in transit to Mars, and on Mars, and 25.5 days on the ISS. CAT-scan exposure is a one-time dose. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Dec, 2024 Reviews received at journal 07 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers invited by journal 04 Nov, 2024 Editor assigned by journal 04 Nov, 2024 Editor invited by journal 04 Nov, 2024 Submission checks completed at journal 30 Oct, 2024 First submitted to journal 15 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5268750","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":375388108,"identity":"6b31efec-e1c6-4d1c-ba9e-e1f3eab75c2a","order_by":0,"name":"Timothy G. 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Levine","email":"","orcid":"","institution":"NASA-Kennedy Space Center, Utilization \u0026 Life Sciences Office","correspondingAuthor":false,"prefix":"","firstName":"Howard","middleName":"G.","lastName":"Levine","suffix":""},{"id":375388116,"identity":"db783ce6-07bf-48a6-bea8-3cfdf168d3b6","order_by":8,"name":"Ramona Gaza","email":"","orcid":"","institution":"Leidos","correspondingAuthor":false,"prefix":"","firstName":"Ramona","middleName":"","lastName":"Gaza","suffix":""},{"id":375388117,"identity":"ce29b55d-6379-4dbe-93ae-af360eff47f4","order_by":9,"name":"Dinah Dimapilis","email":"","orcid":"","institution":"NASA Johnson Space Center","correspondingAuthor":false,"prefix":"","firstName":"Dinah","middleName":"","lastName":"Dimapilis","suffix":""},{"id":375388118,"identity":"9bf5abd3-ca27-456a-a369-d497e63c6e97","order_by":10,"name":"Howard W. Wells","email":"","orcid":"","institution":"The Bionetics Corporation","correspondingAuthor":false,"prefix":"","firstName":"Howard","middleName":"W.","lastName":"Wells","suffix":""},{"id":375388119,"identity":"b431fe36-8437-4725-a31c-5ecb97143f32","order_by":11,"name":"James M. Russick","email":"","orcid":"","institution":"The Bionetics Corporation","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"M.","lastName":"Russick","suffix":""},{"id":375388120,"identity":"7ce8eae9-f9cb-4741-8b1b-9198a8707ae5","order_by":12,"name":"Pierre M. Durand","email":"","orcid":"","institution":"University of the Witwatersrand","correspondingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"M.","lastName":"Durand","suffix":""},{"id":375388121,"identity":"f6ec930c-7d01-4957-8617-d7eb7cd6954a","order_by":13,"name":"Holly H. Birdsall","email":"","orcid":"","institution":"Baylor College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Holly","middleName":"H.","lastName":"Birdsall","suffix":""}],"badges":[],"createdAt":"2024-10-15 12:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5268750/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5268750/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05419-w","type":"published","date":"2025-07-02T15:58:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68689617,"identity":"37c741b0-bb63-4718-865b-a0bc2a673f4a","added_by":"auto","created_at":"2024-11-11 05:36:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of experiments. \u003c/strong\u003eA summary diagram of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eChlamydomonas\u003c/em\u003e preparation, flight details, and analysis modalities.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5268750/v1/5c42c48409d437559505ba29.png"},{"id":68689616,"identity":"6cb8933b-703e-433f-8831-9348fdb73736","added_by":"auto","created_at":"2024-11-11 05:36:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability and programmed cell death (PCD) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlamydomonas \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein Flight vs Ground.\u003c/strong\u003e Aliquots of flown and ground control \u003cem\u003eChlamydomonas\u003c/em\u003e were stained with Annexin V, to identify PCD, and propidium iodide, to identify dead cells, and analyzed by flow cytometry. Values shown are the % positively staining cells and are the mean ± SEM of six replicates. The p values for significance were evaluated by unpaired two-tailed t-test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5268750/v1/63e3c1de1dbfa6ca2a904119.png"},{"id":68689615,"identity":"bc4c21c1-f7a0-4073-aa01-e70241a80062","added_by":"auto","created_at":"2024-11-11 05:36:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDsup\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on the viability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlamydomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in Flight vs Ground\u003c/strong\u003e. \u003cem\u003eChlamydomonas \u003c/em\u003ewith insertion of the \u003cem\u003eDsup\u003c/em\u003e gene were compared to wild type (WT) strains in flown versus ground control samples. Aliquots of each were stained with propidium iodide and analyzed by flow cytometry. Values shown are the percent of viable cells, defined as excluding propidium iodide, and are the mean ± SEM of six replicates. The p values for significance were evaluated by unpaired two-tailed t-test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5268750/v1/de758d10f0c04b73a021f11f.png"},{"id":68689612,"identity":"be08d214-e9be-4b63-91ec-70aa16c50448","added_by":"auto","created_at":"2024-11-11 05:36:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":316585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRaman spectra from ground control \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlamydomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Type I, Type II, and azide-killed cells from flown \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlamydomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaman spectra of \u003cem\u003eChlamydomonas\u003c/em\u003e under different conditions are shown in \u003cstrong\u003eFig 4A\u003c/strong\u003e.\u0026nbsp; Prominent Raman shifts at 1001, 1156, 1359, 1523, and 1549 cm\u003csup\u003e-1\u003c/sup\u003e, associated with various molecular vibrations, are highlighted with dashed boxes. Histograms showing the ratio of the intensities of the Raman bands at 1549 cm\u003csup\u003e-1\u003c/sup\u003e and 1359 cm\u003csup\u003e-1\u003c/sup\u003e (I\u003csub\u003e1549/1359\u003c/sub\u003e) for various samples are shown in \u003cstrong\u003eFig 4B\u003c/strong\u003e. Flown \u003cem\u003eChlamydomonas\u003c/em\u003e contained two different cell types (Type I and Type II) with distinct Raman spectral features. The intensity of Raman vibration corresponding to carotenoids in Type I cells (FL-Type I) was much weaker. Whereas, in Type II cells (FL-Type II), characteristic carotenoid vibrations enhanced significantly.\u0026nbsp; This ratio provides the relative chlorophyll content across different conditions. Histogram illustrating the carotenoid to chlorophyll ratio (I\u003csub\u003eCaro/Chlo\u003c/sub\u003e) across the various sample types are shown in \u003cstrong\u003eFig 4C\u003c/strong\u003e. The data points represent the mean value and error bars indicate the standard deviation, reflecting the variability of the response in each sample type. These spectra and ratios provide insights into the biochemical adaptations of \u003cem\u003eChlamydomonas\u003c/em\u003e to galactic radiation and microgravity, with significant variations noted between the two cell types we found in live flight samples, suggesting differential stress responses and survival strategies.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5268750/v1/abd6b8fe1655d0c368198fd0.png"},{"id":86180957,"identity":"de316f87-2d9c-4e5d-a112-71e598227614","added_by":"auto","created_at":"2025-07-07 16:23:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1874800,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5268750/v1/9fbfdf8f-c449-441b-aafd-6546792d3f45.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Programmed cell death and redox metabolism protect Chlamydomonas reinhardtii populations from the galactic cosmic environment on the Artemis-1 mission","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe study of the biological effects of the galactic cosmic environment has a dual purpose. First, understanding the biology of galactic cosmic environment should guide development of protective measures for astronauts flying beyond low Earth orbit [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], as well as leveraging biological systems to produce meaningful biologics in the space environment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The second purpose is to begin to explore whether galactic cosmic environment mimics have utility for ground-based clinical applications [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExperiments flown on the Artemis 1 mission that circumnavigated the Moon, represent the first opportunity since the Apollo era to return to Earth biological samples exposed to the galactic cosmic environment (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nasa.gov/reference/artemis-i/\u003c/span\u003e\u003cspan address=\"https://www.nasa.gov/reference/artemis-i/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To understand the novelty of this opportunity, it is critical to remember that the International Space Station (ISS) is inside the radioprotective Van Allen Belts. Although experiments and personnel on the ISS are exposed to microgravity, reduced convection, and more radiation than on Earth [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], they are shielded from the vast majority of galactic cosmic radiation by the van Allen Belts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe green alga \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e was selected as the biological model system. It is a well-characterized, motile, single-celled green alga whose genome is fully sequenced and is relatively easy to engineer molecularly. During previous growth in space, a light-dependent increase in photosystem II has been observed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe primary hypothesis was that exposure to galactic cosmic environment would reduce \u003cem\u003eChlamydomonas\u003c/em\u003e survival through unregulated cell death mechanisms called necrosis. An alternative balancing argument was that the radiation stimulus would induce a population survival response via a programmed cell death (PCD). PCD is sometimes a population survival mechanism in microalgae [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMeasurements on the galactic cosmic environment exposed \u003cem\u003eChlamydomonas\u003c/em\u003e, and ground-based controls, included metabolic parameters of energy metabolism, mechanisms of cell death, and production of natural products.\u003c/p\u003e \u003cp\u003eRaman spectroscopy was used for real-time chemical analysis of the \u003cem\u003eChlamydomonas\u003c/em\u003e cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The Curiosity rover on Mars uses fluorescence and Raman spectroscopy to search for organic molecules as a possible sign of life [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], but the potential of the biological application of these techniques is only starting to be realized [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Raman spectroscopy is a non-destructive and non-invasive technique, which offers several advantages in studying various stress-induced (including radiation) biomolecular modifications at the single-cell level without altering their integrity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, the sensitivity of Raman spectroscopy enables the detection of subtle changes in cellular redox metabolism, which is fundamental in elucidating the dynamic alterations in the levels of redox-active species, as well as the cellular response to oxidative stress caused by cosmic radiation.\u003c/p\u003e \u003cp\u003eFurther, several lines of evidence show that multiple tardigrade genes, including \u003cem\u003eDsup\u003c/em\u003e, afford protection from a diverse array of stresses including various forms of radiation present in low Earth orbit [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. An aliquot of the green algae strains flown in this experiment had the tardigrade gene \u003cem\u003eDsup\u003c/em\u003e inserted into the cell nucleus to test for protection from the galactic cosmic environment.\u003c/p\u003e \u003cp\u003eThe initiative began with the design and fabrication of affordable new flight hardware (named Moonshot) to support the growth of \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while simultaneously meeting the constraints of the Artemis I mission profile (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nasa.gov/mission/artemis-i/\u003c/span\u003e\u003cspan address=\"https://www.nasa.gov/mission/artemis-i/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eArtemis-1\u003c/h2\u003e \u003cp\u003eThe effects of the galactic cosmic environment on biological samples remains largely unknown, as the samples returned on Artemis-1 were the first samples returned following exposure to galactic cosmic environment beyond low Earth orbit since the Apollo era.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eViability shift: protection from cosmic radiation\u003c/h3\u003e\n\u003cp\u003eThe major finding of this study is that our hypothesis that exposure to the galactic cosmic environment would reduce \u003cem\u003eChlamydomonas\u003c/em\u003e survival through the process of necrosis was disproven. The alternative balancing argument that the radiation stimulus would induce a population survival response via a PCD pathway is supported. These findings are initially surprising, as the galactic cosmic environment contains high linear energy transfer (LET) particles that evoke complex DNA and other cellular damage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], including significant damage to biological organisms, that include space radiation-induced carcinogenesis, cardiovascular disease, and central nervous system deficiencies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Approximately 70% of galactic cosmic particles are high energy protons with similar relative biological effectiveness (RBE) slightly higher than low LET radiation such as gamma rays and X-rays. Protons are passing through living organisms with uniform distributions. However, heavier particles, which can cause devastating consequences on cells with direct hits, are distributed non-uniformly through the cell populations. Secondary effects are then caused by secondary particles or by signal transduced from the cells with direct hits [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings can be explained by understanding the properties of PCD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. PCD is a population stress response that is both adaptive and plastic [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Initial population decline due to PCD in response to stress helps the population rebound [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PCD can be a group-level stress response, where there is reconstitution of population density by expansion of survivors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Microgravity simulation has been found to induce PCD in multiple cells, and tissues, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e, PCD provides differential species-specific fitness effects that not only benefit others of the same species, but also have an inhibitory effect on the growth of other species [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The phenomenon of programmed cell death benefiting relatives is not limited to \u003cem\u003eChlamydomonas\u003c/em\u003e as it is also true in the green microalga \u003cem\u003eAnkistrodesmus\u003c/em\u003e, (Sphaeropleales, Selenastraceae) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are many methods to assay PCD in algae and specifically in, \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. PCD and necrosis assayed by flow cytometry analysis of Annexin V binding and propidium iodide uptake (described as a \u0026lsquo;hard sign\u0026rsquo; of PCD), or cell size, give the best specificity and sensitivity of methods available within the parameters of the Artemis-1 mission profile [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Multiparameter flow cytometry can provide more robust data analysis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], but no matter which combination of parameters we compared, there was always more overall cell survival, more PCD, and less necrosis in the spaceflight samples compared to the ground controls.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsertion of the tardigrade gene\u003c/b\u003e \u003cb\u003eDsup\u003c/b\u003e: \u003cb\u003eProtection from cosmic radiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTardigrades are microscopic animals renowned for their ability to survive a vast array of environmental extremes, including essentially complete desiccation for up to a decade as well as severe radiation damage [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Tardigrades survived ten days in the temperature, vacuum, and unshielded cosmic radiation of space aboard European Space Agency\u0026rsquo;s FOTON-M3 mission [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Several mechanisms contribute to the tardigrades' radio-resistance, and Tardigrade Disordered Proteins (TDPs) provide remarkable resistance to these stresses. RNA-seq and differential gene expression analysis revealed 11 TDP genes that are induced up to 20-fold during desiccation, along with a related class of TDPs that are expressed constitutively at extremely high levels. Amongst the tardigrade intrinsically disordered proteins (\u003cem\u003eDsup\u003c/em\u003e; Cytoplasmic, Secreted, and Mitochondrial Abundant Heat Soluble protein; and late embryogenesis-abundant proteins (LEA)) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eDsup\u003c/em\u003e was our choice to insert into the nucleus of \u003cem\u003eChlamydomonas\u003c/em\u003e based on evidence that \u003cem\u003eDsup\u003c/em\u003e suppresses the occurrence of DNA breaks by radiation in human-cultured cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As tardigrades have high resistance to diverse stress factors associated with cosmic journeys, they are the focus of intense study by astrobiologists [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInserting \u003cem\u003eDsup\u003c/em\u003e into the nucleus of \u003cem\u003eChlamydomonas\u003c/em\u003e did indeed improve survival, both in space and in the ground-based control samples. The substantial increase in survival of \u003cem\u003eChlamydomonas\u003c/em\u003e in space determined that the starting baseline survival of the two groups were very different. Certainly, \u003cem\u003eDsup\u003c/em\u003e induced a smaller increase in survival in space than on the ground, but it is unclear whether this is a ceiling phenomenon as the \u003cem\u003eDsup\u003c/em\u003e-induced final survival level is no different in the flight and ground groups.\u003c/p\u003e\n\u003ch3\u003eRaman spectroscopy and redox metabolism during spaceflight\u003c/h3\u003e\n\u003cp\u003eRedox signaling is activated in diverse tissues and cell systems by varied forms of stress, including radiation, spaceflight, and simulated microgravity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Stress-induced redox activation can be alternatively adaptive or contribute to pathological outcomes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Targeting of redox metabolism has been proposed for mitigation of radiation injury [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Carotenoids are a class of pigments critical for photoprotection [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. It is likely that cosmic ray and/or microgravity induced stress in \u003cem\u003eChlamydomonas\u003c/em\u003e could potentially activate their defense mechanisms to prevent the radiation damage, which can result in enhanced carotenoid biosynthesis and production of carotenoids [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. β-carotene administration is the subject of multiple clinical trials aiming to decrease cardiovascular disease or cancer risk [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe differential activation of unique survival strategies under space conditions, as observed through Raman spectroscopy in \u003cem\u003eChlamydomonas\u003c/em\u003e, highlights the complex nature of cellular responses to environmental stresses such as radiation and microgravity. The Raman spectroscopic analyses detected two types of cells, which we have designated as Type I and Type II. The enhanced production of carotenoids seen in Type II cells from the flown sample served as a protective mechanism, likely activated to scavenge reactive oxygen species generated by stress, thereby mitigating potential radiation damage. This aligns with various studies suggesting that carotenoid biosynthesis can be stimulated as an adaptive response to environmental challenges [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We also saw some algal cells without/weak β-carotene Raman bands (Type I) in the same flight samples, which is consistent with the flow cytometry and fluorescence microscopy data presented in this manuscript, that not all the cells can survive the cosmic ray stress.\u003c/p\u003e \u003cp\u003eThe lack of significant carotenoid vibrations in Type I cells (also in the azide-killed samples) suggests that these cells may not effectively induce protective responses, making them more susceptible to radiation-induced damage. This observation is critical as it implies that not all cells within a population will uniformly respond to stress, highlighting the importance of understanding individual cellular adaptations in space biology. In addition, our findings also underline the utility of Raman spectroscopy in detecting subtle changes in cellular composition and stress responses, such as shifts in carotenoid ratios and alterations in the electronic environment around chlorophyll molecules. These insights are pivotal for developing strategies to enhance the resilience of microorganisms in space.\u003c/p\u003e\n\u003ch3\u003eChlorophylls and photosystems\u003c/h3\u003e\n\u003cp\u003eThere was no detectible change in chlorophylls and photosystems during the flight, excluding energy metabolism as the cause of the changes in programmed cell death observed.\u003c/p\u003e\n\u003ch3\u003eCost and practicality of Moonshot hardware\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAdvances in materials, microelectronics, and molecular and cellular biology technologies provided a firm basis to produce cost-effective customized spaceflight hardware. Bionetics Corporation\u0026rsquo;s engineering team developed new hardware, called moonshot, within a cost that could be supported from the funded grant. Moonshot holds three 10 cm segmented Petri dishes, and provides timed daily blue and red-light sample exposure, while recording state, battery voltage, temperature, and acceleration in 3 dimensions at programmable times. The Moonshot hardware performed flawlessly, providing flight certified, flight proven hardware for future biological investigations [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRadiation dose\u003c/h2\u003e \u003cp\u003eThe two sensors adjacent to the Moonshot hardware on Artemis I had different radiation spectrum sensitivities. Artemis I was initially planned in 2018, and at that time crew active dosimeters (CADs) were selected for radiation detection. When radiation area monitors (RAMs) became available in 2020, they were added to the radiation monitoring profile in flight [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The RAM is designed to record radiation exposure at a specific location, while a CAD is designed to move with a crew member. In our case both dosimeters were static on the hardware. The CAD was expected to have a slightly higher reading, due to differences in radiation spectrum collection by the two dosimeters. Solar energetic particles from solar flares or sunspots can moderate the observed radiation levels but no solar flares or sunspots occurred during the Artemis I mission. Other radiation detectors flown on the Orion capsule on the Artemis I mission included six CADS distributed around the capsule, and the Hybrid Electronic Radiation Assessor (HERA) system. HERA is a Timepix-based ionizing radiation detector built for NASA Exploration-class crewed missions [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Two \u0026lsquo;phantoms\u0026rsquo; of the Matroshka AstroRad Radiation Experiment (MARE) flew in two of passenger seats (Seat #3 and Seat #4) in the Orion capsule [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe levels of radiation recorded in the RAM and CAD detectors during the Artemis-1 flight are similar to levels seen during the Curiosity rover flight to Mars, if the same period of time is extracted from the total radiation levels reported [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The radiation levels during the Artemis-1 mission flight were far greater than ambient terrestrial levels [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], about three times the levels on the ISS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and about the same total exposure as a medical computed tomography (CT) abdominal scan with and without contrast (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. CAD and RAM measure quantity, but do not define the spectrum of the linear energy transfer for the galactic cosmic environment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The radiation risk is directly influenced by this spectrum as the quality of the radiation characterized by its pattern of energy deposition at the micron/DNA scale determines damage [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The linear energy transfer profile as the Curiosity rover flew to Mars has been documented [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The linear energy transfer spectrum for the Artemis-1 flight has been defined [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], but a basis for biological interpretation is lacking.\u003c/p\u003e \u003ch3\u003eParsing the elements of the galactic cosmic environment\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003edeally, we would model and study the elements of microgravity, convection, and radiation of the galactic cosmic environment individually in ground-based studies. Unfortunately, the technical expertise to parse the elements of the galactic cosmic environment are lacking. Microgravity simulations balance gravity with an induced equal and opposite force, typically using liquid culture environment [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. These model systems introduce multiple new stimuli, limiting comparison to an element of the galactic cosmic environment. There are scant, if any, mechanisms to model biological responses to low convection [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. NASA\u0026rsquo;s ground-based Galactic Cosmic Ray Simulator at the NASA Space Radiation Lab [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e] will be critical to understand the biology of galactic cosmic radiation but is beyond the scope and resources of the current study.\u003c/p\u003e\n\u003ch3\u003eBenefits and limitations\u003c/h3\u003e\n\u003cp\u003eThe galactic cosmic environment poses many challenges but also provides opportunities. For life to survive in cosmic radiation, the first steps begin with research to understand the fundamental effect on biological processes; understand the fundamental biology of life during exposure to cosmic radiation; contribute knowledge to reduce human health risk beyond low Earth orbit; and contribute to improved system performance and reduced system risk. This data set is a first step to answer fundamental questions such as can we survive and thrive beyond Earth, and can we use knowledge gained from studying the biological effects of galactic space radiation for Earth-based benefits?\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWithin the limitations of the physical handling of specimens necessitated by the Artemis-1 mission, flight around the Moon with galactic cosmic environment exposure allows multiple conclusions. Flown samples exposed to the galactic cosmic environment were more viable than ground controls with increased programmed cell death and decreased necrosis. There was no difference in \u003cem\u003eChlamydomonas\u003c/em\u003e growth or content of chlorophylls and photosystems in flown versus ground controls, ruling out energy metabolism as the mediator of cell death. Compared to control inserts, insertion of the \u003cem\u003eDsup\u003c/em\u003e tardigrade gene was protective both on the ground and in flight, although the ground effect was far larger numerically. Raman spectroscopy analysis showed that the redox-protective protein beta-carotene, a known cell death mediator, was increased during flight around the Moon, defining this technology as an important new non-destructive tool for analysis of biological space flight samples. An inexpensive simple new flight hardware, termed Moonshot, can perform flawlessly, and is available as flight-certified, flight-proven hardware for timed illumination and monitoring of samples for flight and terrestrial applications.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2 name=\"removable\"\u003eMethods and materials\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2 name=\"removable\"\u003eChlamydomonas reinhardtii\u003c/h2\u003e \u003cp name=\"removable\"\u003e \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e (CC-125 wild type mt+ [137c]), were purchased from the University of Minnesota \u003cem\u003eChlamydomonas\u003c/em\u003e collection (Minneapolis, MN).\u003c/p\u003e \u003cp name=\"removable\"\u003eThe \u003cem\u003eChlamydomonas\u003c/em\u003e were spotted on Tris-acetate-phosphate (TAP) agar plates, and aliquots reseeded onto fresh plates robotically every 2 weeks. TAP was 20 ml 1M Tris base, 1 ml phosphate K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e/KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e buffer, 1 ml Hunter’s trace metals, 10 ml of solution A [NH\u003csub\u003e4\u003c/sub\u003eCl, MgSP\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 1 ml CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO], glacial acetic acid pH 7 made up to 1 liter with distilled water.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2 name=\"removable\"\u003eChemicals\u003c/h2\u003e \u003cp name=\"removable\"\u003eLife Technologies was the vendor for Annexin V, Alexa Fluor 488 conjugate, and Annexin-binding buffer – “5X concentrate” for flow cytometry. All other chemicals were purchased from Sigma/Aldrich, now Millipore-Sigma (Burlington, MA). Hutner’s trace elements were purchased from the University of Minnesota (Minneapolis, MN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\" name=\"removable\"\u003e \u003ch2 name=\"removable\"\u003eEquipment\u003c/h2\u003e \u003cp name=\"removable\"\u003eSpectrophotometry was performed on a Molecular Dynamics (San Jose CA) M5e Spectrophotometer. Flow cytometry was performed on a Becton-Dickinson Accuri C + Plus flow cytometer (Franklin Lakes, NJ). Raman spectra were collected using a Renishaw inVia Raman Microscope coupled with Leica DM2500M microscope at the Dana-Farber Cancer Institute/Harvard Med. School, Boston, MA. A 785 nm diode laser was used for the surface enhanced Raman spectroscopy measurements.\u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp name=\"removable\"\u003eMoonshot hardware was designed and built by The Bionetics Corporation, (Kennedy Space Center, Brevard County, FL) as an ultralow-power growth system for use on Artemis-1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Each of the three growth modules holds one standard 100 mm diameter circular Petri dish. The plates are illuminated with blue (∼450 nm) and red (∼660 nm) light-emitting diode (LED) lights continuously for 6 h in every 24 h. The combination of red and blue light is optimum for \u003cem\u003eChlamydomonas\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The hardware monitors and records the temperature of the experiment, the power level, and draw from the batteries (as an indication that the lights were turned on and off), as well as the acceleration levels in three axes.\u003c/p\u003e\u003cp name=\"removable\"\u003eOnce activated and loaded with the biological samples, the flight hardware was placed in a custom lathed form-fitting Styrofoam support and encased in a Nitex bag. The assembly was termed the BioExpt-01 Science Bag.\u003c/p\u003e\u003c/div\u003e\u003cp name=\"removable\"\u003e\u003c/p\u003e \u003cp name=\"removable\"\u003eCrew Active Dosimeter (CAD) and Radiation Area Monitor (RAM) radiation detectors were placed adjacent to the biological flight hardware in the BioExpt-01 Science Bag. The detectors had been assembled and delivered to NASA/Kennedy Space Center prior to flight by the Space Radiation Analysis Group based at NASA Johnson Space Center (JSC) by Human Health and Performance Directorate/Leidos personnel. The detectors were collected post-flight and data was analyzed at NASA JSC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2 name=\"removable\"\u003eArtemis-1 flight\u003c/h2\u003e \u003cp name=\"removable\"\u003eAfter an initial delay of 19 days on the launch pad, the Artemis − 1 mission was launched from Kennedy Space Center (KSC) on November 16, 2022 for a planned 25-day space mission. Orion completed one flyby of the Moon on November 21, followed by a distant retrograde orbit for six days and then a second flyby of the Moon on November 25, and subsequently returned to Earth. The Orion capsule was recovered from the Pacific Ocean, returned to California, and transported back to KSC by truck. However, the Space Biology experiments were first removed from Orion in California, flown on an accompanied commercial flight to Kennedy Space Center at ambient temperature (without security radiation scanning) and the Moonshot hardware containing the samples, released to the investigators 9 days after the landing. The sample arrived back in the investigator’s lab three days later, after decommissioning of the hardware at Bionetics facility adjacent to KSC. The samples for the payload described in the report were in the hardware for a total of 56.5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The flight samples were exposed to microgravity in space, but also gravity transitions during launch and re-entry.\u003c/p\u003e \u003cp name=\"removable\"\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2 name=\"removable\"\u003eAnalysis\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2 name=\"removable\"\u003eSpectrophotometry\u003c/h2\u003e \u003cp name=\"removable\"\u003e \u003cem\u003eChlamydomonas\u003c/em\u003e colonies were scraped from the agar, washed three times in phosphate buffered saline, resuspended in TAP buffer and assayed by spectrophotometry for chlorophyll a (absorbance at 436 nm), chlorophyll b (absorbance at 472 nm), photosystem I and II (absorbance at 700 nm and 680nm respectively but the peaks are too broad to separate), cytochrome f (absorbance at 554 nm), and protein content (absorption at 800 nm). Net absorbance for each wavelength was calculated by subtracting the background absorbance of TAP buffer alone. The net absorbance values were then normalized to the protein content as measured by absorbance at 800 nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2 name=\"removable\"\u003eFlow Cytometry\u003c/h2\u003e \u003cp name=\"removable\"\u003eSix colonies from flight and ground plates, plus four \u003cem\u003eDsup\u003c/em\u003e colonies from flight and ground plates, were harvested and resuspended in annexin-binding buffer, which is a phosphate buffer with calcium. The aggregates were disrupted by vortexing and then filtered through a 70µm nylon mesh to remove clumps that would plug the flow cytometer. The resuspended \u003cem\u003eChlamydomonas\u003c/em\u003e samples were aliquoted 100 µl per well into V-bottom microtiter plates. Individual aliquots were either stained with Alexa Fluor 488-annexin (5 µl/well) and propidium iodide (PI) (1 µl) to identify PCD and necrosis, or Nile Red (1:100 dilution from a stock solution of 100 µg ml\u003csup\u003e− 1\u003c/sup\u003e in methanol) with 0.005% Triton-X (1:100 dilution from a stock solution of 0.5% in water) permeabilization to measure lipid content.\u003c/p\u003e \u003cp name=\"removable\"\u003eWe counted 2,000 \u003cem\u003eChlamydomonas\u003c/em\u003e cells from each sample. Background signal was estimated by appropriate no-dye controls. In every flow cytometry run, quality controls were performed on the instrument with three-color fluorescent beads, followed by assay of five control tubes: (1) no dyes, (2) Alexa fluor 488 annexin binding alone, (3) propidium iodide alone, (4) Alexa fluor 488 annexin binding with propidium iodide, and (5) Nile Red alone.\u003c/p\u003e \u003cp name=\"removable\"\u003eFlow cytometric and spectrophotometric data were evaluated by two-tailed Student's t-test comparing heat- or cold-exposed \u003cem\u003eChlamydomonas\u003c/em\u003e to the relevant room temperature control.\u003c/p\u003e \u003cp name=\"removable\"\u003e \u003cb\u003eSingle-cell confocal Raman spectroscopy of\u003c/b\u003e \u003cb\u003eChlamydomonas reinhardtii\u003c/b\u003e\u003c/p\u003e \u003cp name=\"removable\"\u003eMultiple colonies of \u003cem\u003eChlamydomonas\u003c/em\u003e were harvested from a ground plate, and flight plate. The samples included colonies flown live and colonies pretreated with azide before flight. The colonies were harvested into 1.5 ml Eppendorf tubes with 500 µL TAP and resuspended by pipetting and vortexing. The resultant \u003cem\u003eChlamydomonas\u003c/em\u003e solution was filtered through a 70 µm mesh into 12 x 74 mm polypropylene tubes to remove multicellular aggregates. The remaining solution of cells was divided into two 500 µL Eppendorf tubes, spun at 3000g for 1 minute to pellet the cells, and the supernatant aspirated. One tube was left to air dry. The second tube had 100 µL of 10% electron microscopy paraformaldehyde added, incubated for 10 min, and then was spun to pellet the cells before the supernatant was aspirated. This paraformaldehyde-fixed sample was washed with 500 µL deionized water, spun, and the supernatant aspirated.\u003c/p\u003e \u003cp name=\"removable\"\u003eA 2 µl aliquot of each sample rehydrated with distilled water was spotted on a quartz slide (25 mm x 25 mm x 1 mm) that was kept on a microscopic glass slide covered with aluminum foil. This substrate showed minimal background signal compared to various other substrates we tried such as glass, CaF\u003csub\u003e2\u003c/sub\u003e and Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. A silicon wafer was not used as it has a strong Raman band at 521 cm\u003csup\u003e− 1\u003c/sup\u003e and interfered with the current analysis. The samples were air dried at room temperature and were analyzed. A Horiba LabRAM Odyssey Raman microscope was used for the Raman spectral characterization of these samples. Laser power density was optimized in order to achieve better spectral intensity with characteristic Raman bands, which were not present at lower laser power densities. Exposure time and laser intensity were optimized by conducting a series of experiments to prevent charring of the samples to eliminate any unwanted signals due to laser-induced biomolecule denaturation. A 785 nm laser was used to acquire Raman spectra (acquisition time of 30 s with 300 lines/mm grating). First, white light focused on the individual algal cells at a magnification of 100X. Then, Raman spectra were collected in the range of 400–1800 cm\u003csup\u003e− 1\u003c/sup\u003e. The spectral baselines were pre-processed by polynomial fitting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2 name=\"removable\"\u003eStatistics\u003c/h2\u003e \u003cp name=\"removable\"\u003eFlow cytometric and spectrophotometric data are presented as geometric mean ± standard error with six replicates (unless otherwise noted). Correlations were analyzed by Statistica 6.1 (StatSoft Inc. Tulsa OK) using correlation matrix product moment and partial correlations.\u003c/p\u003e \u003cp name=\"removable\"\u003eFor the Raman spectral analysis: Spectra were collected from five or more algal samples across three different colonies exposed to various conditions and subsequently averaged to better represent statistical variations in the intensities of vibrations corresponding to various biomolecular components within the spectra. The standard deviation of these intensities under different conditions was illustrated using error bars in the histogram to provide a clear visualization of the variability and reliability of our measurements.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods and materials","content":"\u003ch2\u003eChlamydomonas reinhardtii\u003c/h2\u003e\u003cp\u003e \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e (CC-125 wild type mt+ [137c]), were purchased from the University of Minnesota \u003cem\u003eChlamydomonas\u003c/em\u003e collection (Minneapolis, MN).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eChlamydomonas\u003c/em\u003e were spotted on Tris-acetate-phosphate (TAP) agar plates, and aliquots reseeded onto fresh plates robotically every 2 weeks. TAP was 20 ml 1M Tris base, 1 ml phosphate K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e/KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e buffer, 1 ml Hunter’s trace metals, 10 ml of solution A [NH\u003csub\u003e4\u003c/sub\u003eCl, MgSP\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 1 ml CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO], glacial acetic acid pH 7 made up to 1 liter with distilled water.\u003c/p\u003e\u003ch2\u003eChemicals\u003c/h2\u003e\u003cp\u003eLife Technologies was the vendor for Annexin V, Alexa Fluor 488 conjugate, and Annexin-binding buffer – “5X concentrate” for flow cytometry. All other chemicals were purchased from Sigma/Aldrich, now Millipore-Sigma (Burlington, MA). Hutner’s trace elements were purchased from the University of Minnesota (Minneapolis, MN).\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEquipment\u003c/h2\u003e \u003cp\u003eSpectrophotometry was performed on a Molecular Dynamics (San Jose CA) M5e Spectrophotometer. Flow cytometry was performed on a Becton-Dickinson Accuri C + Plus flow cytometer (Franklin Lakes, NJ). Raman spectra were collected using a Renishaw inVia Raman Microscope coupled with Leica DM2500M microscope at the Dana-Farber Cancer Institute/Harvard Med. School, Boston, MA. A 785 nm diode laser was used for the surface enhanced Raman spectroscopy measurements.\u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMoonshot hardware was designed and built by The Bionetics Corporation, (Kennedy Space Center, Brevard County, FL) as an ultralow-power growth system for use on Artemis-1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Each of the three growth modules holds one standard 100 mm diameter circular Petri dish. The plates are illuminated with blue (∼450 nm) and red (∼660 nm) light-emitting diode (LED) lights continuously for 6 h in every 24 h. The combination of red and blue light is optimum for \u003cem\u003eChlamydomonas\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The hardware monitors and records the temperature of the experiment, the power level, and draw from the batteries (as an indication that the lights were turned on and off), as well as the acceleration levels in three axes.\u003c/p\u003e\u003cp\u003eOnce activated and loaded with the biological samples, the flight hardware was placed in a custom lathed form-fitting Styrofoam support and encased in a Nitex bag. The assembly was termed the BioExpt-01 Science Bag.\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003cp\u003eCrew Active Dosimeter (CAD) and Radiation Area Monitor (RAM) radiation detectors were placed adjacent to the biological flight hardware in the BioExpt-01 Science Bag. The detectors had been assembled and delivered to NASA/Kennedy Space Center prior to flight by the Space Radiation Analysis Group based at NASA Johnson Space Center (JSC) by Human Health and Performance Directorate/Leidos personnel. The detectors were collected post-flight and data was analyzed at NASA JSC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eEquipment\u003c/h2\u003e\u003cp\u003eSpectrophotometry was performed on a Molecular Dynamics (San Jose CA) M5e Spectrophotometer. Flow cytometry was performed on a Becton-Dickinson Accuri C + Plus flow cytometer (Franklin Lakes, NJ). Raman spectra were collected using a Renishaw inVia Raman Microscope coupled with Leica DM2500M microscope at the Dana-Farber Cancer Institute/Harvard Med. School, Boston, MA. A 785 nm diode laser was used for the surface enhanced Raman spectroscopy measurements.\u003c/p\u003e\u003cp\u003eMoonshot hardware was designed and built by The Bionetics Corporation, (Kennedy Space Center, Brevard County, FL) as an ultralow-power growth system for use on Artemis-1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Each of the three growth modules holds one standard 100 mm diameter circular Petri dish. The plates are illuminated with blue (∼450 nm) and red (∼660 nm) light-emitting diode (LED) lights continuously for 6 h in every 24 h. The combination of red and blue light is optimum for \u003cem\u003eChlamydomonas\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The hardware monitors and records the temperature of the experiment, the power level, and draw from the batteries (as an indication that the lights were turned on and off), as well as the acceleration levels in three axes.\u003c/p\u003e\u003cp\u003eOnce activated and loaded with the biological samples, the flight hardware was placed in a custom lathed form-fitting Styrofoam support and encased in a Nitex bag. The assembly was termed the BioExpt-01 Science Bag.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCrew Active Dosimeter (CAD) and Radiation Area Monitor (RAM) radiation detectors were placed adjacent to the biological flight hardware in the BioExpt-01 Science Bag. The detectors had been assembled and delivered to NASA/Kennedy Space Center prior to flight by the Space Radiation Analysis Group based at NASA Johnson Space Center (JSC) by Human Health and Performance Directorate/Leidos personnel. The detectors were collected post-flight and data was analyzed at NASA JSC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eArtemis-1 flight\u003c/h2\u003e\u003cp\u003eAfter an initial delay of 19 days on the launch pad, the Artemis − 1 mission was launched from Kennedy Space Center (KSC) on November 16, 2022 for a planned 25-day space mission. Orion completed one flyby of the Moon on November 21, followed by a distant retrograde orbit for six days and then a second flyby of the Moon on November 25, and subsequently returned to Earth. The Orion capsule was recovered from the Pacific Ocean, returned to California, and transported back to KSC by truck. However, the Space Biology experiments were first removed from Orion in California, flown on an accompanied commercial flight to Kennedy Space Center at ambient temperature (without security radiation scanning) and the Moonshot hardware containing the samples, released to the investigators 9 days after the landing. The sample arrived back in the investigator’s lab three days later, after decommissioning of the hardware at Bionetics facility adjacent to KSC. The samples for the payload described in the report were in the hardware for a total of 56.5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The flight samples were exposed to microgravity in space, but also gravity transitions during launch and re-entry.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eAnalysis\u003c/h2\u003e\u003ch2\u003eSpectrophotometry\u003c/h2\u003e\u003cp\u003e \u003cem\u003eChlamydomonas\u003c/em\u003e colonies were scraped from the agar, washed three times in phosphate buffered saline, resuspended in TAP buffer and assayed by spectrophotometry for chlorophyll a (absorbance at 436 nm), chlorophyll b (absorbance at 472 nm), photosystem I and II (absorbance at 700 nm and 680nm respectively but the peaks are too broad to separate), cytochrome f (absorbance at 554 nm), and protein content (absorption at 800 nm). Net absorbance for each wavelength was calculated by subtracting the background absorbance of TAP buffer alone. The net absorbance values were then normalized to the protein content as measured by absorbance at 800 nm.\u003c/p\u003e\u003ch2\u003eFlow Cytometry\u003c/h2\u003e\u003cp\u003eSix colonies from flight and ground plates, plus four \u003cem\u003eDsup\u003c/em\u003e colonies from flight and ground plates, were harvested and resuspended in annexin-binding buffer, which is a phosphate buffer with calcium. The aggregates were disrupted by vortexing and then filtered through a 70µm nylon mesh to remove clumps that would plug the flow cytometer. The resuspended \u003cem\u003eChlamydomonas\u003c/em\u003e samples were aliquoted 100 µl per well into V-bottom microtiter plates. Individual aliquots were either stained with Alexa Fluor 488-annexin (5 µl/well) and propidium iodide (PI) (1 µl) to identify PCD and necrosis, or Nile Red (1:100 dilution from a stock solution of 100 µg ml\u003csup\u003e− 1\u003c/sup\u003e in methanol) with 0.005% Triton-X (1:100 dilution from a stock solution of 0.5% in water) permeabilization to measure lipid content.\u003c/p\u003e\u003cp\u003eWe counted 2,000 \u003cem\u003eChlamydomonas\u003c/em\u003e cells from each sample. Background signal was estimated by appropriate no-dye controls. In every flow cytometry run, quality controls were performed on the instrument with three-color fluorescent beads, followed by assay of five control tubes: (1) no dyes, (2) Alexa fluor 488 annexin binding alone, (3) propidium iodide alone, (4) Alexa fluor 488 annexin binding with propidium iodide, and (5) Nile Red alone.\u003c/p\u003e\u003cp\u003eFlow cytometric and spectrophotometric data were evaluated by two-tailed Student's t-test comparing heat- or cold-exposed \u003cem\u003eChlamydomonas\u003c/em\u003e to the relevant room temperature control.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSingle-cell confocal Raman spectroscopy of\u003c/b\u003e \u003cb\u003eChlamydomonas reinhardtii\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMultiple colonies of \u003cem\u003eChlamydomonas\u003c/em\u003e were harvested from a ground plate, and flight plate. The samples included colonies flown live and colonies pretreated with azide before flight. The colonies were harvested into 1.5 ml Eppendorf tubes with 500 µL TAP and resuspended by pipetting and vortexing. The resultant \u003cem\u003eChlamydomonas\u003c/em\u003e solution was filtered through a 70 µm mesh into 12 x 74 mm polypropylene tubes to remove multicellular aggregates. The remaining solution of cells was divided into two 500 µL Eppendorf tubes, spun at 3000g for 1 minute to pellet the cells, and the supernatant aspirated. One tube was left to air dry. The second tube had 100 µL of 10% electron microscopy paraformaldehyde added, incubated for 10 min, and then was spun to pellet the cells before the supernatant was aspirated. This paraformaldehyde-fixed sample was washed with 500 µL deionized water, spun, and the supernatant aspirated.\u003c/p\u003e\u003cp\u003eA 2 µl aliquot of each sample rehydrated with distilled water was spotted on a quartz slide (25 mm x 25 mm x 1 mm) that was kept on a microscopic glass slide covered with aluminum foil. This substrate showed minimal background signal compared to various other substrates we tried such as glass, CaF\u003csub\u003e2\u003c/sub\u003e and Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. A silicon wafer was not used as it has a strong Raman band at 521 cm\u003csup\u003e− 1\u003c/sup\u003e and interfered with the current analysis. The samples were air dried at room temperature and were analyzed. A Horiba LabRAM Odyssey Raman microscope was used for the Raman spectral characterization of these samples. Laser power density was optimized in order to achieve better spectral intensity with characteristic Raman bands, which were not present at lower laser power densities. Exposure time and laser intensity were optimized by conducting a series of experiments to prevent charring of the samples to eliminate any unwanted signals due to laser-induced biomolecule denaturation. A 785 nm laser was used to acquire Raman spectra (acquisition time of 30 s with 300 lines/mm grating). First, white light focused on the individual algal cells at a magnification of 100X. Then, Raman spectra were collected in the range of 400–1800 cm\u003csup\u003e− 1\u003c/sup\u003e. The spectral baselines were pre-processed by polynomial fitting.\u003c/p\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eFlow cytometric and spectrophotometric data are presented as geometric mean ± standard error with six replicates (unless otherwise noted). Correlations were analyzed by Statistica 6.1 (StatSoft Inc. Tulsa OK) using correlation matrix product moment and partial correlations.\u003c/p\u003e\u003cp\u003eFor the Raman spectral analysis: Spectra were collected from five or more algal samples across three different colonies exposed to various conditions and subsequently averaged to better represent statistical variations in the intensities of vibrations corresponding to various biomolecular components within the spectra. The standard deviation of these intensities under different conditions was illustrated using error bars in the histogram to provide a clear visualization of the variability and reliability of our measurements.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMoonshot hardware\u003c/h2\u003e \u003cp\u003eThe Moonshot hardware performed nominally throughout the flight and ground control experiments. The maximum temperature delta between flight and ground control hardware copies was 2.5\u003csup\u003eo\u003c/sup\u003eF, well within the envelope of tolerable experimental conditions. At the end of the 25.5-day flight, the batteries still had more than 50% of power remaining. The power profile validated that the lights came on for 6 out of 24 h daily, as planned. Acceleration recordings show serial measurements of gravity in the z axis, but no acceleration in other directions as expected. The hardware met all the validation criteria for power use, power cycling, temperature control, g-force recording in three axes, data capture, and downloading for analysis, plus materials, and biological compatibility. The Moonshot hardware is now not only flight certified but flight proven.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSpectrophotometry of chlorophyll a and b, cytochrome f, and photosystem levels\u003c/h2\u003e \u003cp\u003eLevels of the light absorbing pigments chlorophyll a and b, the mitochondrial heme protein cytochrome f, and the photon harvesting pigment photosystems were no different between ground and flight samples (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eRadiation exposure in assorted environments in space and on Earth\u003c/h2\u003e \u003cp\u003eThe total radiation exposure during the flight as measured in the RAM and CAD are reported in Table II. Comparison of the exposure levels during flight to other environmental systems (as shown in Table II) is examined in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003ediscussion\u003c/span\u003e section.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry assay of viability and PCD of\u003c/b\u003e \u003cb\u003eChlamydomonas\u003c/b\u003e \u003cb\u003ein Flight vs Ground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe percentages of PCD cells (Annexin V positive) and dead cells (propidium iodide positive) in the flight and ground control samples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. 45.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4% of the cells in flight had undergone PCD compared to 31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0% of the cells in the ground control (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). 53.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3% of the cells in flight were had undergone PCD, compared to 67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% of the cells in the ground control (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry assay of the effect of\u003c/b\u003e \u003cb\u003eDsup\u003c/b\u003e \u003cb\u003eon the viability of\u003c/b\u003e \u003cb\u003eChlamydomonas\u003c/b\u003e \u003cb\u003ein Flight vs Ground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe percentages of live cells (PI negative) in flown and ground control \u003cem\u003eChlamydomonas\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. 41.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% of the flown cells with random gene inserts were living compared with 46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% of the flown cells with \u003cem\u003eDsup\u003c/em\u003e insertion (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). 16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% of the ground control cells with random gene inserts were living compared with 53.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6% of the ground control cells with \u003cem\u003eDsup\u003c/em\u003e insertion (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, n\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSingle-cell confocal Raman spectroscopy of\u003c/b\u003e \u003cb\u003eChlamydomonas reinhardtii\u003c/b\u003e \u003cb\u003ein Flight vs Ground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this pioneering use of Raman spectroscopy for the analysis of space biology samples, we noticed a differential survival strategy among \u003cem\u003eChlamydomonas\u003c/em\u003e exposed to cosmic rays and/or microgravity. Specifically, we found two different cell types (Type I and Type II) with distinct Raman spectral features in flight samples compared to the ground samples. The intensity of Raman vibration corresponding to carotenoids in Type I cells (FL-Type I) was much weaker. Whereas, in Type II cells (FL-Type II), characteristic carotenoid vibrations were enhanced significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In Raman spectra of \u003cem\u003eChlamydomonas\u003c/em\u003e, the presence of prominent vibrations at 1001, 1156, and 1523 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate the presence of carotenoids [\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], which are attributed to C\u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e deformation, C-C stretching, and C\u0026thinsp;=\u0026thinsp;C stretching vibrations of polyene, respectively [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The significant variation in these carotenoid Raman vibrations found in the spectral profiles between Type I and Type II algal cells offers a compelling insight into the heterogeneity of stress responses within a single-cell population in two-cell populations. The lack of significant carotenoid vibrations in the Type I flight sample implies that these cells may not have been able to induce the same protective response and might have yielded to radiation-induced damage. Moreover, the azide-killed flight samples exhibited nearly identical Raman spectra with weak carotenoid signal as in Type I cells, corroborating our proposed link between the absence of carotenoid and cell death pathways. The weaker carotenoid signals in these samples suggest that the living cells in the flight environment actively maintain or increase their carotenoid content. In contrast, the azide treatment or cell death ceases metabolic activity, thereby reducing the carotenoid levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor better understanding the relative increase in the carotenoid content at various conditions, the intensity ratio (I\u003csub\u003eCaro/Chlo\u003c/sub\u003e) of carotenoid-chlorophyll vibration (1523 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1359 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively) was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also used Raman spectroscopy to identify any significant changes in chlorophyll composition by analyzing prominent and characteristic Raman bands of chlorophylls found at 1359 (vibrations of C-C and C-N bonds within the porphyrin ring) and 1549 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e1 (primarily involves the C\u0026thinsp;=\u0026thinsp;C stretching vibrations within the porphyrin ring) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. We noticed that the intensity ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) of these bands (I\u003csub\u003e1549/1359\u003c/sub\u003e) in flight samples (FL-Type I and FL-Type II) as well as in azide-killed samples, showed no significant differences compared to the ground sample, suggesting a comparable level chlorophyll levels across these conditions. This was in concordance with the data obtained from the spectrophotometry. A slight variation in the I\u003csub\u003e1549/1359\u003c/sub\u003e found in FL-Type II could potentially be due to the variation in chlorophyll a to chlorophyll b ratio as vibration corresponds to the stretching vibrations of the carbonyl (C\u0026thinsp;=\u0026thinsp;O) group in the aldehyde group present in Chlorophyll b also contributes to the intensity of Raman vibration at 1549 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (this ratio has slightly increased in FL-Type II samples). In addition, a minor shift in the 1549 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band to 1546 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in FL-Type II samples was also found, which could possibly be attributed to the change in the electronic environment around the chlorophyll molecules as an adaptive response to radiation stress and subsequent variation in chlorophyll b content.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eAuthor contributions were: Hardware design and build HWW, JMR, and TGH; Hands on biology experiments TGH, PLA, and HHB; Strain cloning HK, CN, and GG; Data management and statistics TGH and HHB; Administration services TGH, HLG, YZ, and HHB; Radiation dosimetry selection and management DD, and RG; Raman spectroscopy SP. Prose drafting TGH, SP, PD and HHB. All authors reviewed, edited, and approved multiple manuscript versions.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions were: Hardware design and build HWW, JMR, and TGH; Hands on biology experiments TGH, PLA, and HHB; Strain cloning HK, CN, and GG; Data management and statistics TGH and HHB; Administration services TGH, HLG, YZ, and HHB; Radiation dosimetry selection and management DD, and RG; Raman spectroscopy SP. Prose drafting TGH, SP, PD and HHB. All authors reviewed, edited, and approved multiple manuscript versions.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis material is the result of work supported with resources and the use of facilities at the Durham Veterans Affairs Health Care System and Duke University School of Medicine. Contents do not represent the views of the Department of Veterans Affairs or the United States of America. CN and GG are both supported as Tier 1 Canadian Research Chairs.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data is available in the NASA Research Repository. Contact Tim Hammond, the corresponding author, for information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBokharia, R. S. et al. Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel. \u003cem\u003eSci. Direct\u003c/em\u003e. \u003cb\u003e35\u003c/b\u003e, 105\u0026ndash;112 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorzillo, G., Scoma, A., Faraloni, C. \u0026amp; Giannelli, L. Advances in the biotechnology of hydrogen production with the microalga Chlamydomonas reinhardtii. \u003cem\u003eCrit. Rev. 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Carotenes and carotenoids in natural biological samples: a Raman spectroscopic analysis. \u003cem\u003eJ. Raman Spectrosc.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e (6), 642\u0026ndash;650 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUdensi, J., Loughman, J., Loskutova, E. \u0026amp; Byrne, H. J. Raman spectroscopy of carotenoid compounds for clinical applications-A review. \u003cem\u003eMolecules\u003c/em\u003e. \u003cb\u003e27\u003c/b\u003e, 9017. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules27249017\u003c/span\u003e\u003cspan address=\"10.3390/molecules27249017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026iacute;tek, P., Osterrothov\u0026aacute;, K. \u0026amp; Jehlička, J. Beta-carotene\u0026mdash;A possible biomarker in the Martian evaporitic environment: Raman micro-spectroscopic study. \u003cem\u003ePlanet. Space Sci.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e (4), 454\u0026ndash;459 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai, Z. L., Zeng, H., Chen, M. \u0026amp; Larkum, A. W. D. Raman spectroscopy of chlorophyll d from Acaryochloris marina. \u003cem\u003eBiochim. et Biophys. Acta (BBA) - Bioenergetics\u003c/em\u003e. \u003cb\u003e1556\u003c/b\u003e (2), 89\u0026ndash;91 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassler, D. M. et al. Mars' surface radiation environment measured with the Mars Science Laboratory's Curiosity rover. \u003cem\u003eScience\u003c/em\u003e. \u003cb\u003e343\u003c/b\u003e, 1244797. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1244797\u003c/span\u003e\u003cspan address=\"10.1126/science.1244797\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eRelative quantities of protein in flown vs. ground control \u003cem\u003eChlamydomonas \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eProtein (OD 800)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFlight\u003c/p\u003e\n\u003cp\u003eAvg\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFlight\u003c/p\u003e\n\u003cp\u003eSEM\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGround\u003c/p\u003e\n\u003cp\u003eAvg\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGround\u003c/p\u003e\n\u003cp\u003eSEM\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChlorophyll a (OD 436)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.244\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.049\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.257\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.038\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChlorophyll b (OD 472)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.273\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.044\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.266\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.030\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCytochrome f (OD 554)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.027\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.119\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePhotosystem I and II (OD 700)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.153\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.141\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003eTable 1.\u0026nbsp; Aliquots of flown and ground control \u003cem\u003eChlamydomonas\u003c/em\u003e were suspended in TAP and the optical densities (ODs) for chlorophyll a (OD 436), chlorophyll b (OD 472), cytochrome f (OD 554) and photosystem I and II (OD 700) were measured by spectrophotometry.\u0026nbsp; Values shown are the net OD above background absorbance for TAP media alone, normalized to the total quantity of protein measured as OD 800.\u0026nbsp; Values are the mean and SEM of six replicates.\u0026nbsp; Significance was estimated by unpaired t-test, with non-significant differences (NS) defined as p\u0026gt;0.05.\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable II\u003c/strong\u003e\u003cstrong\u003e Radiation exposure in assorted environments in space and on Earth\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eExposure\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRadiation (mGy)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRef.\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRadiation Area Monitor Artemis I Mission Dose\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCrew Active Dosimeter Artemis I Mission Dose\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNaturally occurring \"background\" for 25.5 days (Earth)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCAT-Scan of Abdomen \u0026amp; Pelvis, \u003cspan class=\"Underline\"\u003e\u0026plusmn;\u003c/span\u003e contrast\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eExposure on Mars for 25.5 days (Curiosity rover)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMars transit for 25.5 days (Curiosity rover)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInternational Space Station for 25.5 days\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable II. Comparison of total radiation doses for 25.5 days during the Artemis I mission, plus 25.5 days of exposure to background radiation on Earth, the Curiosity rover in transit to Mars, and on Mars, and 25.5 days on the ISS. CAT-scan exposure is a one-time dose.\u003c/p\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cosmic radiation, Chlamydomonas reinhardtii, protection, β-carotene, Dsup, tardigrade, spaceflight, redox, Artemis-1","lastPublishedDoi":"10.21203/rs.3.rs-5268750/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5268750/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe hypothesized that on the Artemis I mission, exposure to the galactic cosmic environment, specifically microgravity, lack of convection, and galactic cosmic radiation, would reduce survival of \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e, a green unicellular flagellate alga, through the process of necrosis. An alternative hypothesis was that the radiation stimulus would induce a population survival response via a programmed cell death pathway.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eChlamydomonas\u003c/em\u003e strains were spotted on nutrient agar acetate plates and flown on Artemis I in the new Moonshot hardware that provided six hours of light daily to synchronize the algal cell cycle and tracked temperature, power use, and gravity over time. Synchronous ground controls were run in parallel. Analysis included spectrophotometry of chlorophylls and photosystems, flow cytometry of cell viability and lipid content, and Raman spectroscopy to identify DNA damage and cellular proteins. To test for radiation protection, select strains carried the tardigrade damage suppressor (\u003cem\u003eDsup\u003c/em\u003e) gene known to protect animal cells against gamma and ionizing radiation, dehydration, and temperature extremes in ground-based studies.\u003c/p\u003e \u003cp\u003eA new flight hardware termed \u0026ldquo;Moonshot\u0026rdquo; was designed, built, and flown. \u0026ldquo;Moonshot\u0026rdquo; performed flawlessly, and is now available as flight-certified, flight-proven hardware for timed illumination and monitoring for flight and terrestrial applications.\u003c/p\u003e \u003cp\u003eWithin the limitations of the physical handling of specimens necessitated by the Artemis-1 mission flight around the Moon with exposure to the galactic cosmic environment:\u003c/p\u003e \u003cp\u003e1. Flown samples exposed to cosmic radiation were more viable than ground controls, with increased programmed cell death and decreased necrosis.\u003c/p\u003e \u003cp\u003e2. There was no difference in \u003cem\u003eChlamydomonas\u003c/em\u003e growth or content of chlorophylls and photosystems in flown versus ground controls, ruling out energy metabolism as the mediator of cell death.\u003c/p\u003e \u003cp\u003e3. Raman spectroscopy analysis showed that the redox-protective protein beta-carotene, a known cell death mediator, was increased during flight around the moon.\u003c/p\u003e \u003cp\u003e4. Compared to control inserts, insertion of the \u003cem\u003eDsup\u003c/em\u003e tardigrade gene was protective both on the ground and in flight.\u003c/p\u003e","manuscriptTitle":"Programmed cell death and redox metabolism protect Chlamydomonas reinhardtii populations from the galactic cosmic environment on the Artemis-1 mission","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 05:36:28","doi":"10.21203/rs.3.rs-5268750/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-03T08:34:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T15:08:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296331974700036720370550449499041741536","date":"2024-11-05T10:06:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-04T15:32:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-04T13:15:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-04T13:05:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-30T04:04:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-15T12:22:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fe13928-864f-4677-a542-fb56dc4fcdaf","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39962685,"name":"Biological sciences/Cell biology"},{"id":39962686,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-07-07T16:19:57+00:00","versionOfRecord":{"articleIdentity":"rs-5268750","link":"https://doi.org/10.1038/s41598-025-05419-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-02 15:58:04","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2024-11-11 05:36:28","video":"","vorDoi":"10.1038/s41598-025-05419-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05419-w","workflowStages":[]},"version":"v1","identity":"rs-5268750","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5268750","identity":"rs-5268750","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}