Here, we hypothesize during spaceflight and regolith exposure the increase in oxidative phosphorylation (OXPHOS) in the liver is due to increases in mitochondrial DNA (mtDNA) driven by microRNAs (miRNAs) creating systemic impact on the body. By inhibiting these key miRNAs associated with changes in the body during spaceflight, we will be able to mitigate the damage caused to the liver and improve immune and mitochondrial functions. In addition, we have shown that inhibition of these miRNAs can lead to rescue of inflammatory, immune, and OXPHOS functions and improved efficiency with DNA double strand break (DSB) repair. To show the relationship of the impact this will have in the liver and link to the pathogen increase in the gut, we will use archived liver tissue for a baseline without regolith exposure from our previous experiments, which will include: 1) young and old mice from the Rodent Research Reference Mission-1 (RRRM-1) and Rodent Research Reference Mission-2 (RRRM-2) International Space Station (ISS) missions; and 2) mice irradiated with 0.5Gy galactic cosmic radiation (GCR) simulated beam with and without simulated microgravity, and with and without miRNA-based countermeasures. In addition, we will correlate omics data from the liver tissues available on NASA’s Open Science Data Repository and astronaut data from Japan Aerospace Exploration Agency (JAXA) missions, the NASA Twins Study, and commercial spaceflight missions (i.e., Inspiration4 and Polaris Dawn). We will then utilize 3D human liver and microvasculature tissues exposed to regolith and GCR radiation to determine the additional molecular changes occurring. Lastly, we will test potential countermeasures in the 3D human tissues determined through established machine learning techniques to target the key miRNAs.

• Beamline Experiments: We successfully completed two rounds of beam time at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), first in Fall 2024 and then in Spring 2025. During these sessions, 3D human liver organoid models were exposed to simulated lunar regolith, combined with a simplified GCR (Galactic Cosmic Ray) radiation profile.

• Countermeasure Testing: Two countermeasures targeting mitochondrial function were tested during the beamline experiments: o Kaempferol: a flavonoid previously shown to enhance mitochondrial biogenesis. o MitoQ: a mitochondria-targeted antioxidant.

• Data Generation: Post-irradiation, the samples are currently undergoing initial assessments for viability, mtDNA quantification, and preliminary quality control. We are preparing for comprehensive transcriptomic and small RNA sequencing (RNA-seq and miRNA-seq), expected to commence in the upcoming months.

• Administrative Update: Due to my transition from Blue Marble Space Institute of Science/NASA Ames Research Center to the University of Pittsburgh in September 2024, the transfer of award funds has encountered administrative delays. While this has impact on the pace of downstream analyses, we secured bridge funding that enabled the completion of the critical beamline experiments as originally planned.

Here, we hypothesize during spaceflight and regolith exposure the increase in oxidative phosphorylation (OXPHOS) in the liver is due to increases in mitochondrial DNA (mtDNA) driven by microRNAs (miRNAs) creating systemic impact on the body. By inhibiting these key miRNAs associated with changes in the body during spaceflight, we will be able to mitigate the damage caused to the liver and improve immune and mitochondrial functions. In addition, we have shown that inhibition of these miRNAs can lead to rescue of inflammatory, immune, and OXPHOS functions and improved efficiency with DNA double strand break (DSB) repair. To show the relationship of the impact this will have in the liver and link to the pathogen increase in the gut, we will use archived liver tissue for a baseline without regolith exposure from our previous experiments, which will include: 1) young and old mice from the Rodent Research Reference Mission-1 (RRRM-1) and Rodent Research Reference Mission-2 (RRRM-2) International Space Station (ISS) missions; and 2) mice irradiated with 0.5Gy galactic cosmic radiation (GCR) simulated beam with and without simulated microgravity, and with and without miRNA-based countermeasures. In addition, we will correlate omics data from the liver tissues available on NASA’s Open Science Data Repository and astronaut data from Japan Aerospace Exploration Agency (JAXA) missions, the NASA Twins Study, and commercial spaceflight missions (i.e., Inspiration4 and Polaris Dawn). We will then utilize 3D human liver and microvasculature tissues exposed to regolith and GCR radiation to determine the additional molecular changes occurring. Lastly, we will test potential countermeasures in the 3D human tissues determined through established machine learning techniques to target the key miRNAs.