The Central Role of Cytogenetics in Radiation Biology.

Radiation cytogenetics has a rich history seldom appreciated by those outside the field. Early radiobiology was dominated by physics and biophysical concepts that borrowed heavily from the study of radiation-induced chromosome aberrations. From such studies, quantitative relationships between biological effect and changes in absorbed dose, dose rate and ionization density were codified into key concepts of radiobiological theory that have persisted for nearly a century. This review aims to provide a historical perspective of some of these concepts, including evidence supporting the contention that chromosome aberrations underlie development of many, if not most, of the biological effects of concern for humans exposed to ionizing radiations including cancer induction, on the one hand, and tumor eradication on the other. The significance of discoveries originating from these studies has widened and extended far beyond their original scope. Chromosome structural rearrangements viewed in mitotic cells were first attributed to the production of breaks by the radiations during interphase, followed by the rejoining or mis-rejoining among ends of other nearby breaks. These relatively modest beginnings eventually led to the discovery and characterization of DNA repair of double-strand breaks by non-homologous end joining, whose importance to various biological processes is now widely appreciated. Two examples, among many, are V(D)J recombination and speciation. Rapid technological advancements in cytogenetics, the burgeoning fields of molecular radiobiology and third-generation sequencing served as a point of confluence between the old and new. As a result, the emergent field of "cytogenomics" now becomes uniquely positioned for the purpose of more fully understanding mechanisms underlying the biological effects of ionizing radiation exposure.

A Second Space Age Spanning Omics, Platforms, and Medicine Across Orbits.

The recent acceleration of commercial, private, and multi-national spaceflight has created an unprecedented level of activity in low Earth orbit (LEO), concomitant with the highest-ever number of crewed missions entering space and preparations for exploration-class (>1 year) missions. Such rapid advancement into space from many new companies, countries, and space-related entities has enabled a"Second Space Age." This new era is also poised to leverage, for the first time, modern tools and methods of molecular biology and precision medicine, thus enabling precision aerospace medicine for the crews. The applications of these biomedical technologies and algorithms are diverse, encompassing multi-omic, single-cell, and spatial biology tools to investigate human and microbial responses to spaceflight. Additionally, they extend to the development of new imaging techniques, real-time cognitive assessments, physiological monitoring, and personalized risk profiles tailored for astronauts. Furthermore, these technologies enable advancements in pharmacogenomics (PGx), as well as the identification of novel spaceflight biomarkers and the development of corresponding countermeasures. In this review, we highlight some of the recent biomedical research from the National Aeronautics and Space Administration (NASA), Japan Aerospace Exploration Agency (JAXA), European Space Agency (ESA), and other space agencies, and also detail the commercial spaceflight sector's (e.g. SpaceX, Blue Origin, Axiom, Sierra Space) entrance into aerospace medicine and space biology, the first aerospace medicine biobank, and the myriad upcoming missions that will utilize these tools to ensure a permanent human presence beyond LEO, venturing out to other planets and moons.

Telomeric RNA (TERRA) increases in response to spaceflight and high-altitude climbing.

Telomeres are repetitive nucleoprotein complexes at chromosomal termini essential for maintaining genome stability. Telomeric RNA, or TERRA, is a previously presumed long noncoding RNA of heterogeneous lengths that contributes to end-capping structure and function, and facilitates telomeric recombination in tumors that maintain telomere length via the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. Here, we investigated TERRA in the radiation-induced DNA damage response (DDR) across astronauts, high-altitude climbers, healthy donors, and cellular models. Similar to astronauts in the space radiation environment and climbers of Mt. Everest, in vitro radiation exposure prompted increased transcription of TERRA, while simulated microgravity did not. Data suggest a specific TERRA DDR to telomeric double-strand breaks (DSBs), and provide direct demonstration of hybridized TERRA at telomere-specific DSB sites, indicative of protective TERRA:telomeric DNA hybrid formation. Targeted telomeric DSBs also resulted in accumulation of TERRA foci in G2-phase, supportive of TERRA's role in facilitating recombination-mediated telomere elongation. Results have important implications for scenarios involving persistent telomeric DNA damage, such as those associated with chronic oxidative stress (e.g., aging, systemic inflammation, environmental and occupational radiation exposures), which can trigger transient ALT in normal human cells, as well as for targeting TERRA as a therapeutic strategy against ALT-positive tumors.

Genome and clonal hematopoiesis stability contrasts with immune, cfDNA, mitochondrial, and telomere length changes during short duration spaceflight.

Background: The Inspiration4 (I4) mission, the first all-civilian orbital flight mission, investigated the physiological effects of short-duration spaceflight through a multi-omic approach. Despite advances, there remains much to learn about human adaptation to spaceflight's unique challenges, including microgravity, immune system perturbations, and radiation exposure. Methods: To provide a detailed genetics analysis of the mission, we collected dried blood spots pre-, during, and post-flight for DNA extraction. Telomere length was measured by quantitative PCR, while whole genome and cfDNA sequencing provided insight into genomic stability and immune adaptations. A robust bioinformatic pipeline was used for data analysis, including variant calling to assess mutational burden. Result: Telomere elongation occurred during spaceflight and shortened after return to Earth. Cell-free DNA analysis revealed increased immune cell signatures post-flight. No significant clonal hematopoiesis of indeterminate potential (CHIP) or whole-genome instability was observed. The long-term gene expression changes across immune cells suggested cellular adaptations to the space environment persisting months post-flight. Conclusion: Our findings provide valuable insights into the physiological consequences of short-duration spaceflight, with telomere dynamics and immune cell gene expression adapting to spaceflight and persisting after return to Earth. CHIP sequencing data will serve as a reference point for studying the early development of CHIP in astronauts, an understudied phenomenon as previous studies have focused on career astronauts. This study will serve as a reference point for future commercial and non-commercial spaceflight, low Earth orbit (LEO) missions, and deep-space exploration.

Telomeres and aging: on and off the planet!

Improving human healthspan in our rapidly aging population has never been more imperative. Telomeres, protective "caps" at the ends of linear chromosomes, are essential for maintaining genome stability of eukaryotic genomes. Due to their physical location and the "end-replication problem" first envisioned by Dr. Alexey Olovnikov, telomeres shorten with cell division, the implications of which are remarkably profound. Telomeres are hallmarks and molecular drivers of aging, as well as fundamental integrating components of the cumulative effects of genetic, lifestyle, and environmental factors that erode telomere length over time. Ongoing telomere attrition and the resulting limit to replicative potential imposed by cellular senescence serves a powerful tumor suppressor function, and also underlies aging and a spectrum of age-related degenerative pathologies, including reduced fertility, dementias, cardiovascular disease and cancer. However, very little data exists regarding the extraordinary stressors and exposures associated with long-duration space exploration and eventual habitation of other planets, nor how such missions will influence telomeres, reproduction, health, disease risk, and aging. Here, we briefly review our current understanding, which has advanced significantly in recent years as a result of the NASA Twins Study, the most comprehensive evaluation of human health effects associated with spaceflight ever conducted. Thus, the Twins Study is at the forefront of personalized space medicine approaches for astronauts and sets the stage for subsequent missions. We also extrapolate from current understanding to future missions, highlighting potential biological and biochemical strategies that may enable human survival, and consider the prospect of longevity in the extreme environment of space.

Monitoring Genomic Structural Rearrangements Resulting from Gene Editing.

The cytogenomics-based methodology of directional genomic hybridization (dGH) enables the detection and quantification of a more comprehensive spectrum of genomic structural variants than any other approach currently available, and importantly, does so on a single-cell basis. Thus, dGH is well-suited for testing and/or validating new advancements in CRISPR-Cas9 gene editing systems. In addition to aberrations detected by traditional cytogenetic approaches, the strand specificity of dGH facilitates detection of otherwise cryptic intra-chromosomal rearrangements, specifically small inversions. As such, dGH represents a powerful, high-resolution approach for the quantitative monitoring of potentially detrimental genomic structural rearrangements resulting from exposure to agents that induce DNA double-strand breaks (DSBs), including restriction endonucleases and ionizing radiations. For intentional genome editing strategies, it is critical that any undesired effects of DSBs induced either by the editing system itself or by mis-repair with other endogenous DSBs are recognized and minimized. In this paper, we discuss the application of dGH for assessing gene editing-associated structural variants and the potential heterogeneity of such rearrangements among cells within an edited population, highlighting its relevance to personalized medicine strategies.

Arabidopsis telomerase takes off by uncoupling enzyme activity from telomere length maintenance in space.

Spaceflight-induced changes in astronaut telomeres have garnered significant attention in recent years. While plants represent an essential component of future long-duration space travel, the impacts of spaceflight on plant telomeres and telomerase have not been examined. Here we report on the telomere dynamics of Arabidopsis thaliana grown aboard the International Space Station. We observe no changes in telomere length in space-flown Arabidopsis seedlings, despite a dramatic increase in telomerase activity (up to 150-fold in roots), as well as elevated genome oxidation. Ground-based follow up studies provide further evidence that telomerase is induced by different environmental stressors, but its activity is uncoupled from telomere length. Supporting this conclusion, genetically engineered super-telomerase lines with enhanced telomerase activity maintain wildtype telomere length. Finally, genome oxidation is inversely correlated with telomerase activity levels. We propose a redox protective capacity for Arabidopsis telomerase that may promote survivability in harsh environments.

Exploring the Relationship between Spontaneous Sister Chromatid Exchange and Genome Instability in Two Cryptic Species of Non-Human Primates.

There are extensive studies on chromosome morphology and karyotype diversity in primates, yet we still lack insight into genomic instability as a key factor underlying the enormous interspecies chromosomal variability and its potential contribution to evolutionary dynamics. In this sense, the assessment of spontaneous sister chromatid exchange (SCE) frequencies represents a powerful tool for evaluating genome stability. Here, we employed G-banding, fluorescence plus Giemsa (FPG), and chromosome orientation fluorescence in situ hybridization (CO-FISH) methodologies to characterize both chromosome-specific frequencies of spontaneously occurring SCE throughout the genome (G-SCE) and telomere-specific SCE (T-SCE). We analyzed primary fibroblast cultures from two male species of Ateles living in captivity: Ateles paniscus (APA) and Ateles chamek (ACH). High frequencies of G-SCEs were observed in both species. Interestingly, G-SCEs clustered on evolutionary relevant chromosome pairs: ACH chromosomes 1, 2, 3, 4, and 7, and APA chromosomes 1, 2, 3, 4/12, 7, and 10. Furthermore, a statistically significant difference between the observed and expected G-SCE frequencies, not correlated with chromosome size, was also detected. CO-FISH analyses revealed the presence of telomere-specific recombination events in both species, which included T-SCE, as well as interstitial telomere signals and telomere duplications, with APA chromosomes displaying higher frequencies, compared to ACH. Our analyses support the hypothesis that regions of Ateles chromosomes susceptible to recombination events are fragile sites and evolutionary hot spots. Thus, we propose SCE analyses as a valuable indicator of genome instability in non-human primates.

Ad Astra - telomeres in space!

PURPOSE: My journey to the stars began as I - along with the whole world - stood still and watched Neil Armstrong take those first small steps on the Moon. Fast forward 50 years and NASA astronauts Scott Kelly and Christina Koch each spend nearly a year in space aboard the International Space Station (ISS), a remarkable multinational collaborative project and floating U.S. National Laboratory that has supported continuous human presence in low Earth orbit for the past 20 years. Marking a new era of human space exploration, the first commercial rocket, SpaceX Falcon 9, recently launched NASA astronauts Doug Hurley and Bob Behnken in the Crew Dragon spacecraft Endeavor to the ISS and returned safely to Earth. NASA and its commercial partners are rapidly advancing innovative space technologies, and with the recently announced Artemis team of astronauts, plans to send the first woman and next man back to the moon and establish sustainable exploration by the end of the decade. Humankind will then be poised to take the next giant leap - pioneering human exploration of Mars. CONCLUSIONS: Historically, fewer than 600 individuals have participated in spaceflight, the vast majority of whom have been middle aged males (35-55 years) on short duration missions (less than 20 days). Thus, as the number and diversity of space travelers increase, a better understanding of how long-duration spaceflight affects human health is essential to maintaining individual astronaut performance during, and improving disease and aging trajectories following, future exploration missions. Here, I review findings from our NASA Twins Study and Telomeres investigations, highlighting potential mechanistic roles of chronic space radiation exposure in changes in telomere length and persistent DNA damage responses associated with long-duration spaceflight. Importantly, similar trends were observed in prostate cancer patients undergoing intensity-modulated radiation therapy (IMRT), additional support specifically for the role of radiation exposure. Individual differences in response were also observed in both cohorts, underscoring the importance of developing personalized approaches for evaluating human health effects and long-term outcomes associated with radiation exposures, whether on Earth or living in the extreme environment of space.

Destabilizing Effects of Ionizing Radiation on Chromosomes: Sizing up the Damage.

For long-term survival and evolution, all organisms have depended on a delicate balance between processes involved in maintaining stability of their genomes and opposing processes that lead toward destabilization. At the level of mammalian somatic cells in renewal tissues, events or conditions that can tip this balance toward instability have attracted special interest in connection with carcinogenesis. Mutations affecting DNA (and its subsequent repair) would, of course, be a major consideration here. These may occur spontaneously through endogenous cellular processes or as a result of exposure to mutagenic environmental agents. It is in this context that we discuss the rather unique destabilizing effects of ionizing radiation (IR) in terms of its ability to cause large-scale structural rearrangements to the genome. We present arguments supporting the conclusion that these and other important effects of IR originate largely from microscopically visible chromosome aberrations.

Evaluation of DNA damage and stress in wildlife chronically exposed to low-dose, low-dose rate radiation from the Fukushima Dai-ichi Nuclear Power Plant accident.

The health effects associated with chronic low-dose, low-dose rate (LD-LDR) exposures to environmental radiation are uncertain. All dose-effect studies conducted outside controlled laboratory conditions are challenged by inherent complexities of ecological systems and difficulties quantifying dose to free-ranging organisms in natural environments. Consequently, the effects of chronic LD-LDR radiation exposures on wildlife health remain poorly understood and much debated. Here, samples from wild boar (Sus scrofa leucomystax) and rat snakes (Elaphe spp.) were collected between 2016 and 2018 across a gradient of radiation exposures in Fukushima, Japan. In vivo biomarkers of DNA damage and stress were evaluated as a function of multiple measurements of radiation dose. Specifically, we assessed frequencies of dicentric chromosomes (Telomere-Centromere Fluorescence in situ Hybridization: TC-FISH), telomere length (Telo-FISH, qPCR), and cortisol hormone levels (Enzyme Immunoassay: EIA) in wild boar, and telomere length (qPCR) in snakes. These biological parameters were then correlated to robust calculations of radiation dose rate at the time of capture and plausible upper bound lifetime dose, both of which incorporated internal and external dose. No significant relationships were observed between dicentric chromosome frequencies or telomere length and dose rate at capture or lifetime dose (p value range: 0.20-0.97). Radiation exposure significantly associated only with cortisol, where lower concentrations were associated with higher dose rates (r2 = 0.58; p < 0.0001), a relationship that was likely due to other (unmeasured) factors. Our results suggest that wild boar and snakes chronically exposed to LD-LDR radiation sufficient to prohibit human occupancy were not experiencing significant adverse health effects as assessed by biomarkers of DNA damage and stress.

Haplotype diversity and sequence heterogeneity of human telomeres.

Telomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, and whose lengths can be correlated with age and adverse health risk factors. Yet, given their length and repetitive nature, telomeric regions are not easily reconstructed from short-read sequencing, thus making telomere sequencing, mapping, and variant resolution challenging problems. Recently, long-read sequencing, with read lengths measuring in hundreds of kilobase pairs, has made it possible to routinely read into telomeric regions and inspect their sequence structure. Here, we describe a framework for extracting telomeric reads from whole-genome single-molecule sequencing experiments, including de novo identification of telomere repeat motifs and repeat types, and also describe their sequence variation. We find that long, complex telomeric stretches and repeats can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize noncanonical telomere sequence motifs (both previously reported, as well as novel), and validate them in short-read sequence data. These data reveal extensive intra- and inter-population diversity of repeats in telomeric haplotypes, reveal higher paternal inheritance of telomeric variants, and represent the first motif composition maps of multi-kilobase-pair human telomeric haplotypes across three distinct ancestries (Ashkenazi, Chinese, and Utah), which can aid in future studies of genetic variation, aging, and genome biology.

Telomere Length Dynamics and Chromosomal Instability for Predicting Individual Radiosensitivity and Risk via Machine Learning.

The ability to predict a cancer patient's response to radiotherapy and risk of developing adverse late health effects would greatly improve personalized treatment regimens and individual outcomes. Telomeres represent a compelling biomarker of individual radiosensitivity and risk, as exposure can result in dysfunctional telomere pathologies that coincidentally overlap with many radiation-induced late effects, ranging from degenerative conditions like fibrosis and cardiovascular disease to proliferative pathologies like cancer. Here, telomere length was longitudinally assessed in a cohort of fifteen prostate cancer patients undergoing Intensity Modulated Radiation Therapy (IMRT) utilizing Telomere Fluorescence in situ Hybridization (Telo-FISH). To evaluate genome instability and enhance predictions for individual patient risk of secondary malignancy, chromosome aberrations were assessed utilizing directional Genomic Hybridization (dGH) for high-resolution inversion detection. We present the first implementation of individual telomere length data in a machine learning model, XGBoost, trained on pre-radiotherapy (baseline) and in vitro exposed (4 Gy γ-rays) telomere length measurements, to predict post radiotherapy telomeric outcomes, which together with chromosomal instability provide insight into individual radiosensitivity and risk for radiation-induced late effects.

Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5' C-Rich Overhangs and Recruitment of TERRA.

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5' C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5' C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

Twins, Telomeres, and Aging-in Space!

BACKGROUND: The landmark National Aeronautics and Space Administration Twins Study represented an integrated effort to launch human space life science research into the modern age of molecular- and "omics"-based studies. As part of the first One-Year Mission aboard the International Space Station, identical twin astronauts Scott and Mark Kelly were the subjects of this "out of this world" research opportunity. Telomeres, the natural ends of chromosomes that shorten with cell division and a host of lifestyle factors and stresses, are key molecular determinants of aging and aging trajectories. METHODS: We proposed that telomere length dynamics (changes over time) represent a particularly relevant and integrative biomarker for astronauts, as they reflect the combined experiences and environmental exposures encountered during spaceflight. Telomere length (quantitative polymerase chain reaction and telomere fluorescence in situ hybridization) and telomerase activity (quantitative polymerase chain reaction -telomere repeat amplification protocol) were longitudinally assessed in the space- and earth-bound twins. Chromosome aberrations (directional genomic hybridization), signatures of radiation exposure, were also evaluated. RESULTS: The twins had relatively similar telomere lengths before spaceflight, and the earth-bound twins' telomeres remained relatively stable over the course of the study. Surprisingly, the space twins' telomeres were longer during spaceflight, and upon return to Earth shortened rapidly, resulting in many more short telomeres after spaceflight than before. Chromosomal signatures of space radiation exposure were also elevated during spaceflight, and increased inversion frequencies persisted after spaceflight, suggestive of ongoing genome instability. CONCLUSION: Although the definitive mechanisms underlying such dramatic spaceflight-associated shifts in telomere length remain unclear, improved maintenance of telomere length has important implications for aging science and improving healthspan for those on Earth, as well.

Cell-free DNA (cfDNA) and Exosome Profiling from a Year-Long Human Spaceflight Reveals Circulating Biomarkers.

Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and the other unique environmental conditions of spaceflight.

Clonal Hematopoiesis Before, During, and After Human Spaceflight.

Clonal hematopoiesis (CH) occurs when blood cells harboring an advantageous mutation propagate faster than others. These mutations confer a risk for hematological cancers and cardiovascular disease. Here, we analyze CH in blood samples from a pair of twin astronauts over 4 years in bulk and fractionated cell populations using a targeted CH panel, linked-read whole-genome sequencing, and deep RNA sequencing. We show CH with distinct mutational profiles and increasing allelic fraction that includes a high-risk, TET2 clone in one subject and two DNMT3A mutations on distinct alleles in the other twin. These astronauts exhibit CH almost two decades prior to the mean age at which it is typically detected and show larger shifts in clone size than age-matched controls or radiotherapy patients, based on a longitudinal cohort of 157 cancer patients. As such, longitudinal monitoring of CH may serve as an important metric for overall cancer and cardiovascular risk in astronauts.