Multiwell-based G0-PCC assay for radiation biodosimetry.

In cytogenetic biodosimetry, assessing radiation exposure typically requires over 48 hours for cells to reach mitosis, significantly delaying the administration of crucial radiation countermeasures needed within the first 24 hours post-exposure. To improve medical response times, we incorporated the G0-Premature Chromosome Condensation (G0-PCC) technique with the Rapid Automated Biodosimetry Tool-II (RABiT-II), creating a faster alternative for large-scale radiation emergencies. Our findings revealed that using a lower concentration of Calyculin A (Cal A) than recommended effectively increased the yield of highly-condensed G0-PCC cells (hPCC). However, integrating recombinant CDK1/Cyclin B kinase, vital for chromosome condensation, proved challenging due to the properties of these proteins affecting interactions with cellular membranes. Interestingly, Cal A alone was capable of inducing chromosome compaction in some G0 cells even in the absence of mitotic kinases, although these chromosomes displayed atypical morphologies. This suggests that Cal A mechanism for compacting G0 chromatin may differ from condensation driven by mitotic kinases. Additionally, we observed a correlation between radiation dose and extent of hPCC chromosome fragmentation, which allowed us to automate radiation damage quantification using a Convolutional Neural Network (CNN). Our method can address the need for a same-day cytogenetic biodosimetry test in radiation emergency situations.

Sex and dose rate effects in automated cytogenetics.

Testing and validation of biodosimetry assays is routinely performed using conventional dose rate irradiation platforms, at a dose rate of approximately 1 Gy/min. In contrast, the exposures from an improvised nuclear device will be delivered over a large range of dose rates with a prompt irradiation component, delivered in less than 1 µs, and a protracted component delivered over hours and days. We present preliminary data from a large demographic study we have undertaken for investigation of age, sex and dose rate effects on dicentric and micronucleus yields. Our data demonstrate reduced dicentric and micronucleus yields at very high dose rates. Additionally, we have seen small differences between males and females, with males having slightly fewer micronuclei and slightly more dicentrics than females, at high doses.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

A machine learning method for improving the accuracy of radiation biodosimetry by combining data from the dicentric chromosomes and micronucleus assays.

A large-scale malicious or accidental radiological event can expose vast numbers of people to ionizing radiation. The dicentric chromosome (DCA) and cytokinesis-block micronucleus (CBMN) assays are well-established biodosimetry methods for estimating individual absorbed doses after radiation exposure. Here we used machine learning (ML) to test the hypothesis that combining automated DCA and CBMN assays will improve dose reconstruction accuracy, compared with using either cytogenetic assay alone. We analyzed 1349 blood sample aliquots from 155 donors of different ages (3-69 years) and sexes (49.1% males), ex vivo irradiated with 0-8 Gy at dose rates from 0.08 Gy/day to ≥ 600 Gy/s. We compared the performances of several state-of-the-art ensemble ML methods and found that random forest generated the best results, with R2 for actual vs. reconstructed doses on a testing data subset = 0.845, and mean absolute error = 0.628 Gy. The most important predictor variables were CBMN and DCA frequencies, and age. Removing CBMN or DCA data from the model significantly increased squared errors on testing data (p-values 3.4 × 10-8 and 1.1 × 10-6, respectively). These findings demonstrate the promising potential of combining CBMN and DCA assay data to reconstruct radiation doses in realistic scenarios of heterogeneous populations exposed to a mass-casualty radiological event.

Ultra-high dose rate FLASH irradiator at the radiological research accelerator facility.

The Radiological Research Accelerator Facility has modified a decommissioned Varian Clinac to deliver ultra-high dose rates: operating in 9 MeV electron mode (FLASH mode), samples can be irradiated at a Source-Surface Distance (SSD) of 20 cm at average dose rates of up to 600 Gy/s (3.3 Gy per 0.13 µs pulse, 180 pulses per second). In this mode multiple pulses are required for most irradiations. By modulating pulse repetition rate and irradiating at SSD = 171 cm, dose rates below 1 Gy/min can be achieved, allowing comparison of FLASH and conventional irradiations with the same beam. Operating in 6 MV photon mode, with the conversion target removed (SuperFLASH mode), samples are irradiated at higher dose rates (0.2-150 Gy per 5 µs pulse, 360 pulses per second) and most irradiations can be performed with a single very high dose rate pulse. In both modes we have seen the expected inverse relation between dose rate and irradiated area, with the highest dose rates obtained for beams with a FWHM of about 2 cm and ± 10% uniformity over 1 cm diameter. As an example of operation of the ultra-high dose rate FLASH irradiator, we present dose rate dependence of dicentric chromosome yields.

NBS1 I171V variant underlies individual differences in chromosomal radiosensitivity within human populations.

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.

The RABiT-II DCA in the Rhesus Macaque Model.

An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool II (RABiT-II)," adapts the dicentric chromosome assay (DCA) for high-throughput mass-screening of the population after a large-scale radiological event. To validate this test, the U.S. Federal Drug Administration (FDA) recommends demonstrating that the high-throughput biodosimetric assay in question correctly reports the dose in an in vivo model. Here we describe the use of rhesus macaques (Macaca mulatta) to augment human studies and validate the accuracy of the high-throughput version of the DCA. To perform analysis, we developed the 17/22-mer peptide nucleic acid (PNA) probes that bind to the rhesus macaque's centromeres. To our knowledge, these are the first custom PNA probes with high specificity that can be used for chromosome analysis in M. mulatta. The accuracy of fully-automated chromosome analysis was improved by optimizing a low-temperature telomere PNA FISH staining in multiwell plates and adding the telomere detection feature to our custom chromosome detection software, FluorQuantDic V4. The dicentric frequencies estimated from in vitro irradiated rhesus macaque samples were compared to human blood samples of individuals subjected to the same ex vivo irradiation conditions. The results of the RABiT-II DCA analysis suggest that, in the lymphocyte system, the dose responses to gamma radiation in the rhesus macaques were similar to those in humans, with small but statistically significant differences between these two model systems.

RABiT-II-DCA: A Fully-automated Dicentric Chromosome Assay in Multiwell Plates.

We developed a fully-automated dicentric chromosome assay (DCA) in multiwell plates. All operations, from sample loading to chromosome scoring, are performed, without human intervention, by the second-generation Rapid Automated Biodosimetry Tool II (RABiT-II) robotic system, a plate imager and custom software, FluorQuantDic. The system requires small volumes of blood (30 µl per individual) to determine radiation dose received as a result of a radiation accident or terrorist attack. To visualize dicentrics in multiwell plates, we implemented a non-classical protocol for centromere FISH staining at 37°C. The RABiT-II performs rapid analysis of chromosomes after extracting them from metaphase cells. With the use of multiwell plates, many samples can be screened at the same time. Thus, the RABiT-II DCA provides an advantage during triage when risk-based stratification and medical management are required for a large population exposed to unknown levels of ionizing radiation.

Evaluation of ATM heterozygous mutations underlying individual differences in radiosensitivity using genome editing in human cultured cells.

Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), which are an initial step towards chromosomal aberrations and cell death. It has been suggested that there are individual differences in radiosensitivity within human populations, and that the variations in DNA repair genes might determine this heterogeneity. However, it is difficult to quantify the effect of genetic variants on the individual differences in radiosensitivity, since confounding factors such as smoking and the diverse genetic backgrounds within human populations affect radiosensitivity. To precisely quantify the effect of a genetic variation on radiosensitivity, we here used the CRISPR-ObLiGaRe (Obligate Ligation-Gated Recombination) method combined with the CRISPR/Cas9 system and a nonhomologous end joining (NHEJ)-mediated knock-in technique in human cultured cells with a uniform genetic background. We generated ATM heterozygous knock-out (ATM +/-) cell clones as a carrier model of a radiation-hypersensitive autosomal-recessive disorder, ataxia-telangiectasia (A-T). Cytokinesis-blocked micronucleus assay and chromosome aberration assay showed that the radiosensitivity of ATM +/- cell clones was significantly higher than that of ATM +/+ cells, suggesting that ATM gene variants are indeed involved in determining individual radiosensitivity. Importantly, the differences in radiosensitivity among the same genotype clones were small, unlike the individual differences in fibroblasts derived from A-T-affected family members.

The Microtubule-Depolymerizing Activity of a Mitotic Kinesin Protein KIF2A Drives Primary Cilia Disassembly Coupled with Cell Proliferation.

The primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although primary cilia form in the quiescent phase, ciliary disassembly occurs when quiescent cells re-enter the proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for cell-proliferation-coupled primary cilia disassembly. Here, we report that kinesin superfamily protein 2A (KIF2A), phosphorylated at T554 by PLK1, exhibits microtubule-depolymerizing activity at the mother centriole to disassemble the primary cilium in a growth-signal-dependent manner. KIF2A-deficient hTERT-RPE1 cells showed the impairment of primary cilia disassembly following growth stimulation. It was also found that the PLK1-KIF2A pathway is constitutively active in cells from patients with premature chromatid separation (PCS) syndrome and is responsible for defective ciliogenesis in this syndrome. These findings provide insights into the roles of the PLK1-KIF2A pathway in physiological cilia disassembly and cilia-associated disorders.