Task Progress:
|
The experimental work on the project has been significantly delayed by the ongoing Covid-19 pandemic. However, we were allowed to return to hands-on laboratory research in March 2021 and finished all particle irradiation experiments at Brookhaven National Laboratory (BNL) BNL21B run in June 2021.
We have irradiated the peripheral blood mononuclear cells (PBMCs) from all remaining subjects, including the repeats of previous BNL experiments that we were unable to analyze previously due to Covid restrictions. We have analyzed their DNA repair kinetics using immunostaining with fluorescently-tagged 53BP1 antibody followed by semi-automated high throughput microscopy, image processing, and quantification. Currently, we are collaborating with the lab of Dr. Christopher Mason at Weill Cornell Medicine to match the phenotypic outcomes of DNA repair with genotypes based on low coverage whole genome sequencing.
In summary, for this project we have collected DNA repair data from 750 subjects, whose PBMCs have been irradiated ex vivo with 3 types of particle radiation (350 MeV/n 28Si, 350 MeV/n 40Ar, 600 MeV/n 56Fe) as well as gamma rays, at 2 doses each (1.1 and 3 particle/100 micrometers2 fluence, which translates to 0.1 Gy and 0.3 Gy for 28Si, 0.18 Gy and 0.5 Gy for 40Ar, and 0.3 Gy and 0.82 Gy for 56Fe respectively; and 0.1 Gy and 1 Gy doses of gamma rays), and at 2 timepoints post irradiation: 4 and 24 hours; as well as at baseline that represents the time of collecting PBMCs. To our knowledge this is the largest such dataset of human ex vivo responses to simulated space radiation. We anticipate that our data, which we will publish open access on NASA GeneLab, will serve as a useful resource for multiple future investigations.
Furthermore, we have collected data on oxidative stress and cell death from a subset of ~400 subjects, analyzed additional responses to 5-ion simplified simulated GCRs (0.25 Gy and 0.5 Gy doses, 4 h and 24 h post irradiation) and 250 MeV/n 4He (0.15 Gy and 0.5 Gy doses, 4 h and 24 h post irradiation) as part of piggyback experiments at NASA Space Radiation Laboratory (NSRL), and collected supernatant for quantifying secreted factors from all our samples for follow-up studies that will be available for potential collaborations. Additional supernatant samples from 24 most variable subjects in response to 56Fe irradiation have also been collected for exosome quantification as part of a Human Research Program (HRP) Augmentation Award to our former postdoctoral scholar Eloise Pariset (awarded January 2021).
Finally, as part of a collaboration with the Mason lab, we have selected 96 subjects with maximal variability in DNA repair responses and collected their RNA for transcriptomic analysis after 56Fe and gamma irradiation (funded by the Mason lab) to validate whether genomic associations with radiosensitivity are reflected in changes in gene expression.
This year we have one review article on DNA damage markers in press (Penninckx et al., Quantification of radiation-induced DNA repair foci to evaluate and predict biological responses to ionizing radiation, NAR Cancer--ed. note: see Bibliography section) and one primary research article under revision (Cekanaviciute et al., Mouse Genomic Associations with Ex Vivo Sensitivity to Simulated Space Radiation). Our research was presented at the Human Research Program Investigators’ Workshop, the National Council on Radiation Protection (NCRP): Biomarkers & Countermeasures and Conclusions conference, the Radiation Research Society Annual Meeting, and the American Society for Gravitational and Space Research Annual Meeting (all Sylvain Costes, oral presentations) and will be presented at COSPAR 2022 (invited presentation, Sylvain Costes)).
Our main future directions for this project are reflected in two key collaborations that we have started this year, with the goals to use the data from this project to generate new results: a) applying AI/ML (artificial intelligence/machine learning) methods to analyze our data (with NASA GeneLab and Frontier Development Lab); and b) using network analysis to compare the effects of spaceflight stressors and terrestrial diseases and repurpose Food & Drug Administration (FDA)-approved therapeutics as countermeasures for space radiation (with the SPOKE project and Baranzini Lab at the University of California, San Francisco, on which we have published a pilot study: Nelson et al., Life 2021).
|