Inflammasome Fingerprint: Mapping Tissue-Specific Responses to Cosmic Radiation for Precision Risk Modeling

Quantifying how different radiation types (protons vs heavy ions)drive inflammatory damage at mission-relevant doses is challenging.After cosmic ray exposure, unique inflammasome "fingerprints"(NLRP3/AIM2 patterns) exists in heart, brain and blood vessels. AIM2detects broken dsDNA in cytoplasm, leading to secretion of cytokines(especially IL-1β), whose receptors (IL-1R) activate the canonicalNF-κB pathway, creating a positive feedback loop of inflammation.NF-κB is a key activator that primes the NLRP3 inflammasome. Itacts as a primary signaling step (Signal 1) by driving the transcriptionand expression of NLRP3 and pro-IL-1β. A novel molecular bindingscheme for the detection of NF-κB was investigated for its affinity toIg-κB DNA composed by dye and quencher fluorophores, and thisspecificity is confirmed by competing with the DNA sequence that iscomplementary to the Ig-κB DNA. We create a normalizationequation to remove the negative effects from the various initialfluorophore concentrations and the background noise. We also foundthat a periodic shaking at a frequency could help to stabilize theDNA–protein binding. The calibration experiment shows that thismolecular probe biosensor has a detection limit on the order ofnanomolar. The specificity experiment also shows that p50/p65heterodimer has the highest affinity for Ig-κB DNA; p65 homodimerbinds with intermediate affinity, whereas p50 shows the lowestbinding affinity, and Ig-κB DNA is not sensitive to BSA (bovinealbumin serum). The experiment of HeLa nuclear extract shows thatTNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κBDNA than non-TNF-stimulated HeLa nuclear extract (4-h serumresponse). Therefore, the molecular binding scheme provides arapid, quantitative, high throughput, and automated measurement ofthe DNA-binding protein NF-κB at low cost, which is beneficial forautomated drug screening systems.