Herein, we are performing studies in humanized mice to enable omics capability for in-flight measurements of radiation/stress blood biomarkers (human) using RNASeq and microfluidic-based transcriptomic/proteomic biomarker detection platforms. We are also utilizing the Human-Microbial Cross-Talk model (HuMiX) gut-on-a-chip to perform the 1st studies monitoring the effects of simulated space radiation on the human GI tract. Based on exciting preliminary data, we will also examine the ability of the dietary supplement curcumin to prevent and/or mitigate the effects of space radiation on the hematopoietic and GI systems, and to determine the optimal working concentration for maximal radioprotective/radiomitigating effects. Given curcumin's poor water solubility, we are also performing studies to validate the ability of nanolipoprotein particles (NLP) loaded with curcumin (cNLPs) to serve as an effective countermeasure against the effects of SEP/GCR radiation in both the human hematopoietic and GI systems. Once validated as an effective countermeasure, we will also assess the stability of these cNLPs for long-term storage aboard ISS/long duration missions, suitability for lyophilization/resuspension for oral delivery. If successful, these cNLPs could readily be implemented as a dietary supplement during prolonged missions, protecting astronauts from the deleterious effects of space radiation on the hematopoietic and GI systems.

Herein, we are performing studies in humanized mice to enable omics capability for in-flight measurements of radiation/stress blood biomarkers (human) using RNASeq and microfluidic-based transcriptomic/proteomic biomarker detection platforms. We are also utilizing the Human-Microbial Cross-Talk model (HuMiX) gut-on-a-chip to perform the 1st studies monitoring the effects of simulated space radiation on the human GI tract. Based on exciting preliminary data, we will also examine the ability of the dietary supplement curcumin to prevent and/or mitigate the effects of space radiation on the hematopoietic and GI systems, and to determine the optimal working concentration for maximal radioprotective/radiomitigating effects. Given curcumin's poor water solubility, we are also performing studies to validate the ability of nanolipoprotein particles (NLP) loaded with curcumin (cNLPs) to serve as an effective countermeasure against the effects of SEP/GCR radiation in both the human hematopoietic and GI systems. Once validated as an effective countermeasure, we will also assess the stability of these cNLPs for long-term storage aboard ISS/long duration missions, suitability for lyophilization/resuspension for oral delivery. If successful, these cNLPs could readily be implemented as a dietary supplement during prolonged missions, protecting astronauts from the deleterious effects of space radiation on the hematopoietic and GI systems.

During this third year of funding, we have made significant progress towards achieving these goals. During this time, we have:

• Performed H+, 56Fe ion, 16O, ?28Si, ?-ray, and simplified 5-ion galactic cosmic ray (GCR) simulator irradiations on both wild-type mice, as requested by TRISH (Translational Research Institute for Space Health), to provide an in vivo comparator/validation of the in vitro HuMiX human gut-on-a-chip system, and on NSG mice whose hematopoietic systems were first humanized by repopulation with human hematopoietic stem cells (HSC) isolated from the bone marrow of healthy, astronaut-age donors.

• Collected tissues (at multiple times post-irradiation) and are analyzing GI system, blood for biomarker identification by omics, and hematopoietic system for lineage alterations and DNA damage.

• Found marked histological alterations in small intestine in response to protons and both 16O and 56Fe ions.

• Discovered that exposure to Mars mission-relevant doses of protons and 56Fe ions leads to disruption of the epithelial barrier of the small intestine, as assessed by immunofluorescent staining for Claudin-3.

• Humanized the hematopoietic system of over 200 NSG mice, installed RFI-tracking p-Chips, and irradiated these mice during NASA Space Radiation Laboratory (NSRL)-19C and the abbreviated 20A/20B run. All mice from NSRL19A have now been sacrificed and tissues are being analyzed. Mice from NSRL 19C are being sacrificed and analysis has begun.

• Have begun analyzing the various tissues from the 96 wild-type C57Bl/6 mice we irradiated during NSRL-19B by both immunohistochemistry and multi-omics approaches to define the effects that solar energetic particles (SEP) and GCR radiation exert on the various tissues/organ systems of the body and the ability of our curcumin-laden NLPs to protect against these effects.

• We have found that pre-treatment of the wild-type mice with cNLPs appears to reduce the amount of apoptosis seen in the small intestine following exposure to SEP/GCR radiation.

• The HuMix gut-on-a-chip platform has been further optimized and limited bacterial species have been incorporated into the HuMiX chip. This new optimized system with a simplified microbiome was exposed to various ions during NSRL-19C and will be returning for NSRL 20C.

• Have now shown that the presence of representative species from the microbiome in the HuMiX system appears to provide some degree of protection against the deleterious effects of space radiation. Further studies are now underway to define the mechanistic basis for these observations.

• Have shown cNLPs can mediate protection against SEP and GCR radiation in vivo, at omics level.

• We have shown that the cNLPs provide markedly better protection against all varieties of ions tested than simply giving curcumin in DMSO (dimethylsulfoxide), using human fibroblasts.

• Have generated a wealth of data on the cellular/molecular pathways that are altered by both SEP and GCR radiation and the impact that cNLPs (and DMSO-curcumin) have on these alterations, and have shown that curcumin delivered packaged in NLPs is far more effective, and induces transcriptomic changes that differ markedly from, than curcumin suspended in DMSO.

• We have shown, for the first time ever, that the exposure of mice harboring human hematopoietic systems to GCR radiation (56Fe ions, thus far, other ions now being tested) leads to enlargement of the spleens to roughly 30 times the normal size (in several mice; not all). Immunohistology exam by clinical hematopathologists has preliminarily concluded this is due to induction of human hematological malignancy, as we observed in our prior studies in which human HSC were exposed to these same ions in vitro and then used to repopulate NSG mice. Extensive analyses are underway to define the nature of the malignancy.

Our progress to-date has enabled us to begin rigorously testing our hypotheses regarding the effects of space radiation (SEP and GCR) on the human hematopoietic and GI systems, and has provided compelling evidence to support our hypothesis that exposure to space radiation at Mars mission-relevant doses, produces marked deleterious effects on both of these human systems. These data have also supported our premise that a novel formulation of a readily available dietary supplement (curcumin) may have the ability to serve as an easy to administer countermeasure that can protect astronauts from at least some of the deleterious effects of space radiation during prolonged missions beyond Low Earth Orbit (LEO). Our omics work thus far has also begun to shed light on the molecular/ biological pathways that are altered within multiple tissues as a result of exposure of a living organism to ?-ray radiation and to simulated SEP and GCR radiation, and it is beginning to provide mechanistic clues regarding the means by which curcumin-NLPs mediate their protective effects. Our newly formulated HuMiX chip is performing well, and it has begun providing a wealth of information (the first of its kind) on the response of the human GI tract to space radiation and the role played by the gut microbiota in this response.

The original Aims of this project were to: 1) use mice with humanized hematopoiesis to define changes in radiation/stress blood biomarkers in response to mission-relevant doses of simulated space radiation; 2) use the innovative Human-Microbial Cross-Talk human gut-on-a-chip model (HuMiX) to perform studies defining the effects of mission-relevant doses of simulated space radiation on the human GI tract; 3) validate the ability of nanolipoprotein particles (NLP) loaded with curcumin to serve as an effective countermeasure against the effects of simulated space radiation in both the human hematopoietic and GI models; and 4) assess the suitability of curcumin-NLPs as space radiation countermeasures.

During this second year of funding, we have made significant progress towards achieving these goals. During this time, we have:

• Performed H+, 56Fe ion, 16O, gamma-ray, and simplified 5-ion GCR simulator irradiations on both wild-type mice, as requested by TRISH, to provide an in vivo comparator/validation of the in vitro HuMiX human gut-on-a-chip system, and on NSG mice whose hematopoietic systems were first humanized by repopulation with human hematopoietic stem cells (HSC) isolated from the bone marrow of healthy, astronaut-age donors.

• Collected tissues (at multiple times post-IR) and are analyzing GI system, blood for biomarker identification by omics, and hematopoietic system for lineage alterations and DNA damage.

• Found marked histological alterations in small intestine in response to protons and iron ions.

• Discovered that exposure to Mars mission-relevant doses of protons, iron ions, and oxygen ions leads to disruption of the epithelial barrier of the small intestine, as assessed by immunofluorescent staining for Claudin-3.

• Humanized the hematopoietic system of the 1st cohort of 72 NSG mice, installed RFI-tracking p-Chips, and irradiated these mice during NSRL-18C. All mice have now been sacrificed and tissues are being analyzed.

• Switched back to NSG mice (in lieu of NSG-SG3) as the SG3 provided higher short-term engraftment at the cost of long-term repopulation.

• Humanized 84 NSG mice and irradiated during NSRL-19A.

• Irradiated 96 wild-type C57Bl/6 mice during NSRL-19B, as detailed in bullet #1 above, have collected serum and all major tissues, which are currently being analyzed by both immunohistochemistry and multi-omics approaches to define the effects that SEP and GCR radiation exert on the various tissues/organ systems of the body and the ability of our curcumin-laden NLPs to protect against these effects.

• Miniaturized and optimized the HuMix gut-on-a-chip platform to enable its use in the NSRL beamline and have exposed this new optimized system to various ions during NSRL-19A and NSRL-19B.

• Have now shown that the presence of representative species from the microbiome in the HuMiX system appears to provide some degree of protection against the deleterious effects of space radiation. Further studies are now underway to define the mechanistic basis for these observations.

• Designed, formulated, and began testing curcumin-loaded NLPs for their ability to provide radioprotection in vivo in normal/wildtype mice and in NSG mice with humanized hematopoietic systems (NSRL runs 18C, 19A, and 19B).

• Shown cNLPs can mediate protection against SEP and GCR radiation in vivo, at omics level.

• We have shown that the cNLPs provide markedly better protection against all varieties of ions tested than simply giving curcumin in DMSO, using human fibroblasts.

• Have generated a wealth of data on the cellular/molecular pathways that are altered by both SEP and GCR radiation and the impact that cNLPs (and DMSO (dimethyl sulfoxide)-curcumin)) have on these alterations.

• We have shown, for the first time ever, that the exposure of mice harboring human hematopoietic systems to GCR radiation (56Fe ions, thus far; other ions now being tested) leads to enlargement of the spleens to roughly 30 times the normal size (in several mice; not all). Immunohistology exam by clinical hematopathologists have preliminary concluded this is due to induction of human hematological malignancy, as we observed in our prior studies in which human HSC were exposed to these same ions in vitro and then used to repopulate NSG mice. Needless to say, extensive analyses are underway to define the nature of the malignancy.

Our progress to-date has enabled us to begin rigorously testing our hypotheses regarding the effects of space radiation (SEP and GCR) on the human hematopoietic and GI systems, and has provided compelling evidence to support our hypothesis that exposure to space radiation at Mars mission-relevant doses produces marked deleterious effects on both of these human systems. These data have also supported our premise that a novel formulation of a readily available dietary supplement (curcumin) may have the ability to serve as an easy to administer countermeasure that can protect astronauts from at least some of the deleterious effects of space radiation during prolonged missions beyond LEO. Our omics work thus far has also begun to shed light on the molecular/biological pathways that are altered within multiple tissues as a result of exposure of a living organism to gamma-ray radiation and to simulated SEP and GCR radiation, and it is beginning to provide mechanistic clues regarding the means by which curcumin-NLPs mediate their protective effects. Our newly formulated HuMiX chip is performing exceptionally well, and it has begun providing a wealth of information (the first of its kind) on the response of the human GI tract to space radiation and the role played by the gut microbiota in this response.

Aim 1: We will utilize mice with 'humanized' hematopoietic systems to define changes in human and mouse radiation/stress blood biomarkers in response to mission-relevant doses of simulated space radiation employing a microfluidic-based transcriptomic/proteomic biomarker detection platform; Aim 2: Validate the ability of nanoparticles (nanolipoproteins; NLPs) loaded with curcumin as effective countermeasures against the effects of simulated space radiation in both the human hematopoietic and mouse GI (gastroinstestinal) systems; Aim 3: Assess the suitability of curcumin-NLPs for use as radiation countermeasures in the space environment by: a) assessing their long-term stability for storage/use aboard the International Space Station (ISS) and long duration deep-space missions; b) evaluating their suitability for lyophilization/resuspension for oral delivery; and c) supplementing the diet of a small cohort of "humanized" mice with an optimized formulation to further evaluate their potential for both radioprotection (pre-irradiation supplementation) and radiation mitigation (post-irradiation supplementation). Aim 4: Use the innovative Human-Microbial Cross-Talk human 'gut-on-a-chip' model (HuMiX) to perform the first studies defining critical biomarker responses of mission-relevant doses of simulated space radiation on the human GI tract.

Approach: We will use 'humanized' immunodeficient (NSG) mice (huMice) whose hematopoietic system has been repopulated with human hematopoietic stem cells (HSC) from astronaut-age M/F donors. Using these huMice (3-4 months post-repopulation) as our experimental model, we will measure space radiation-induced human and mouse blood transcriptomic and proteomic changes using our low LET (linear energy transfer) photon-validated radiation biomarker detection panel and microfluidic-based detection platform. We will also test a promising curcumin-based nanolipoprotein (NLP)-based countermeasure that we have recently shown significantly improves human HSC proliferation and differentiation in vitro following low doses of high-energy proton and iron ions delivered at NASA Space Radiation Laboratory (NSRL). The huMice will serve as 'avatars' allowing us to study in vivo responses and leukemogenic potential of human hematopoietic systems exposed to modeled space radiation. HuMice will be developed and matured at Wake Forest Institute for Regenerative Medicine (WFIRM) and transported to Brookhaven National Laboratory (BNL), as will HuMiX 'gut-on-a-chip' populated with intestinal cells from healthy human donors (both sexes) of typical astronaut age. At NSRL, the huMice 'avatars' and HuMiX chips will be exposed to mission-relevant doses of high-energy protons, intermediate and high LET ions, and the GCR (galactic cosmic radiation) simulator. HuMiX chips will be monitored for short-term human GI cell biomarker responses, and the animals will be monitored for both short and long-term human and mouse biomarker responses, using our established low LET cesium-137 gamma radiation-specific protein biomarkers coupled to an ELISA-based microfluidic device that we will further optimize for ISS/in-flight use. Biomarker responses measured in these devices will be validated using qPCR, RNASeq, Western blotting, and several traditional and molecular radiobiological techniques. We will serially monitor animals for short and long term blood-based biomarker responses and physiological radiation effects (specifically leukemogenesis). We will submit tissue/organ samples of huMice to the NASA Human Research Program (HRP) Shared Tissue Repository, and our biomarker datasets will be deposited into the NASA GeneLab database.

Herein, we will perform studies in humanized mice to enable omics capability for in-flight measurements of radiation/stress blood biomarkers (human) using RNASeq and microfluidic-based transcriptomic/proteomic biomarker detection platforms. We will also utilize the highly innovative Human-Microbial Cross-Talk model (HuMiX) gut-on-a-chip to perform the first studies monitoring the effects of simulated space radiation on the human GI tract. Based on exciting preliminary data, we will also use RNASeq, the refined biomarker panels, and the HuMiX chip to examine the ability of the dietary supplement curcumin to prevent and/or mitigate the effects of space radiation on the hematopoietic and GI systems, and to determine the optimal working concentration for maximal radioprotective/radiomitigating effects. Given curcumin's poor water solubility, we will also perform studies to validate the ability of nanolipoprotein particles (NLP) loaded with curcumin (cNLPs) to serve as an effective countermeasure against the effects of SEP/GCR radiation in both the human hematopoietic and GI systems. We will also assess the stability of these cNLPs for long-term storage aboard ISS/long duration deep-space missions, suitability for lyophilization/resuspension for oral delivery, and as a potential radiomitigator post-exposure. If successful, these cNLPs could readily be implemented as a dietary supplement during prolonged missions, protecting astronauts from the deleterious effects space radiation exerts upon the hematopoietic and GI systems.

1) use mice with "humanized" hematopoiesis to define changes in radiation/stress blood biomarkers in response to mission-relevant doses of simulated space radiation; 2) use the innovative Human-Microbial Cross-Talk human "gut-on-a-chip" model (HuMiX) to perform studies defining the effects of mission-relevant doses of simulated space radiation on the human GI tract; 3) validate the ability of nanolipoprotein particles (NLP) loaded with curcumin to serve as an effective countermeasure against the effects of simulated space radiation in both the human hematopoietic and GI models; and 4) assess the suitability of curcumin-NLPs as space radiation countermeasures.

During the first partial year of funding, we have made significant progress towards achieving these goals. In the 8 months, we have: a) Performed H-plus 56Fe ion and gamma-ray irradiations on 84 wild-type mice, as requested by The Translational Research Institute for Space Health (TRISH), to provide in vivo comparator/validation of the in vitro HuMiX human gut-on-a-chip system; b) Collected tissues (at multiple times post-IR and are analyzing GI system, blood for biomarker identification by omics, and hematopoietic system for lineage alterations and DNA damage; c) Found marked histological alterations in small intestine in response to protons and iron ions; d) Discovered that exposure to Mars mission-relevant doses of protons and iron ions leads to disruption of the epithelial barrier of the small intestine, as assessed by immunofluorescent staining for Claudin-3; e) Humanized the hematopoietic system of the 1st cohort of 72 NSG mice and installed radio frequency identification (RFI)-tracking p-Chips for NSRL-18C; f) Optimized staining protocols for detecting human hematopoietic cells/lineages in humanized murine hosts; g) Miniaturized and optimized the HuMiX gut-on-a-chip platform to enable its use in the NSRL beamline; h) Performed pilot x-ray irradiations on HuMiX to validate this platform and optimize downstream analyses of radiation-induced molecular alterations and DNA damage; i) Successfully adapted HuMiX to enable use of primary human intestinal epithelial cells; j) Designed, formulated, and began testing curcumin-loaded NLPs for their ability to provide radioprotection in vivo in normal/wild-type mice; k) Showed cNLPs can mediate protection against SEP and GCR radiation in vivo, at omics level.

Our progress to-date has provided us the technical tools we need to begin rigorously testing our hypotheses regarding the effects of space radiation (SEP and GCR) on the human hematopoietic and GI systems, and has provided preliminary evidence to support our hypothesis that a novel formulation of a readily available dietary supplement may have the ability to serve as an easy to administer countermeasure that can protect astronauts from at least some of the deleterious effects of space radiation during prolonged missions beyond LEO. Our omics work thus far has also begun to shed light on the molecular/biological pathways that are altered within multiple tissues as a result of exposure of a living organism to gamma-ray radiation and to simulated SEP and GCR radiation, and it is beginning to provide mechanistic clues regarding the means by which curcumin-NLPs mediate their protective effects.

Aim 1: We will utilize mice with “humanized” hematopoietic systems to define changes in human and mouse radiation/stress blood biomarkers in response to mission-relevant doses of simulated space radiation employing a microfluidic-based transcriptomic/proteomic biomarker detection platform; Aim 2: Validate the ability of nanoparticles (nanolipoproteins; NLPs) loaded with curcumin as effective countermeasures against the effects of simulated space radiation in both the human hematopoietic and mouse GI systems; Aim 3: Assess the suitability of curcumin-NLPs for use as radiation countermeasures in the space environment by: a) assessing their long-term stability for storage/use aboard the International Space Station (ISS) and long duration deep-space missions; b) evaluating their suitability for lyophilization/resuspension for oral delivery; and c) supplementing the diet of a small cohort of “humanized” mice with an optimized formulation to further evaluate their potential for both radioprotection (pre-irradiation supplementation) and radiation mitigation (post-irradiation supplementation). Aim 4: Use the innovative Human-Microbial Cross-Talk human “gut-on-a-chip” model (HuMiX) to perform the first studies defining critical biomarker responses of mission-relevant doses of simulated space radiation on the human GI tract.

Approach: We will use “humanized” immunodeficient (NSG) mice (huMice) whose hematopoietic system has been repopulated with human hematopoietic stem cells (HSC) from astronaut-age M/F donors. Using these huMice (3–4 months post-repopulation) as our experimental model, we will measure space radiation-induced human and mouse blood transcriptomic and proteomic changes using our low LET (linear energy transfer) photon-validated radiation biomarker detection panel and microfluidic-based detection platform. We will also test a promising curcumin-based nanolipoprotein (NLP)-based countermeasure that we have recently shown significantly improves human HSC proliferation and differentiation in vitro following low doses of high-energy proton and iron ions delivered at NASA Space Radiation Laboratory (NSRL). The huMice will serve as “avatars” allowing us to study in vivo responses and leukemogenic potential of human hematopoietic systems exposed to modeled space radiation. HuMice will be developed and matured at Wake Forest Institute for Regenerative Medicine (WFIRM) and transported to Brookhaven National Laboratory (BNL), as will HuMiX “gut-on-a-chip” populated with intestinal cells from healthy human donors (both sexes) of typical astronaut age. At NSRL, the huMice “avatars” and HuMiX chips will be exposed to mission-relevant doses of high-energy protons, intermediate and high LET ions, and the GCR (galactic cosmic radiation) simulator. HuMiX chips will be monitored for short-term human GI cell biomarker responses, and the animals will be monitored for both short and long-term human and mouse biomarker responses, using our established low LET cesium-137 gamma radiation-specific protein biomarkers coupled to an ELISA-based microfluidic device that we will further optimize for ISS/in-flight use. Biomarker responses measured in these devices will be validated using qPCR, RNASeq, Western blotting, and several traditional and molecular radiobiological techniques. We will serially monitor animals for short- and long-term blood-based biomarker responses and physiological radiation effects (specifically leukemogenesis). We will submit tissue/organ samples of huMice to the NASA Human Research Program (HRP) Shared Tissue Repository, and our biomarker datasets will be deposited into the NASA GeneLab database.