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.

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.

Telomere Length Dynamics and DNA Damage Responses Associated with Long-Duration Spaceflight.

Telomere length dynamics and DNA damage responses were assessed before, during, and after one-year or shorter duration missions aboard the International Space Station (ISS) in a comparatively large cohort of astronauts (n = 11). Although generally healthy individuals, astronauts tended to have significantly shorter telomeres and lower telomerase activity than age- and sex-matched ground controls before and after spaceflight. Although telomeres were longer during spaceflight irrespective of mission duration, telomere length shortened rapidly upon return to Earth, and overall astronauts had shorter telomeres after spaceflight than they did before; inter-individual differences were identified. During spaceflight, all crewmembers experienced oxidative stress, which positively correlated with telomere length dynamics. Significantly increased frequencies of chromosomal inversions were observed during and after spaceflight; changes in cell populations were also detected. We propose a telomeric adaptive response to chronic oxidative damage in extreme environments, whereby the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway is transiently activated in normal somatic cells.

Temporal Telomere and DNA Damage Responses in the Space Radiation Environment.

Telomeres, repetitive terminal features of chromosomes essential for maintaining genome integrity, shorten with cell division, lifestyle factors and stresses, and environmental exposures, and so they provide a robust biomarker of health, aging, and age-related diseases. We assessed telomere length dynamics (changes over time) in three unrelated astronauts before, during, and after 1-year or 6-month missions aboard the International Space Station (ISS). Similar to our results for National Aeronautics and Space Administration's (NASA's) One-Year Mission twin astronaut (Garrett-Bakelman et al., 2019), significantly longer telomeres were observed during spaceflight for two 6-month mission astronauts. Furthermore, telomere length shortened rapidly after return to Earth for all three crewmembers and, overall, telomere length tended to be shorter after spaceflight than before spaceflight. Consistent with chronic exposure to the space radiation environment, signatures of persistent DNA damage responses were also detected, including mitochondrial and oxidative stress, inflammation, and telomeric and chromosomal aberrations, which together provide potential mechanistic insight into spaceflight-specific telomere elongation.

Directional Genomic Hybridization (dGH) for Detection of Intrachromosomal Rearrangements.

Fluorescence in situ Hybridization (FISH) techniques, including whole chromosome painting (WCP), spectral karyotyping (SKY), and multicolor FISH (mFISH), are used extensively to characterize and enumerate inter-chromosomal rearrangements (e.g., translocations). Directional genomic hybridization (dGH) is a relatively new cytogenomics-based methodology that combines the strand-specific strategy of Chromosome Orientation-FISH (CO-FISH) with bioinformatics-driven design of single-stranded DNA probe sets that are unique and of like orientation. Such a strategy produces directional probe sets that hybridize to one-and only one-chromatid of prepared (single-stranded) metaphase chromosomes, thereby facilitating high-resolution visualization of intra-chromosomal rearrangements, specifically inversions, and greatly improving our ability to detect such otherwise cryptic structural variants within the genome. In addition to its usefulness in the study of various disease states, including cancer, relevant applications of dGH include monitoring cytogenetic damage caused by exposure to clastogenic agents (e.g., ionizing radiation). dGH can be applied as a discovery tool to globally assess the integrity of the genome, but it can also be used in a more targeted fashion to interrogate fine structural changes at the kilobase level. Consequently, dGH is capable of providing significant mechanistic insight and information not easily obtainable by other approaches.

The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight.

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

Estimation of Radiation Doses to U.S. Military Test Participants from Nuclear Testing: A Comparison of Historical Film-Badge Measurements, Dose Reconstruction and Retrospective Biodosimetry.

Retrospective radiation dose estimations, whether based on physical or biological measurements, or on theoretical dose reconstruction, are limited in their precision and reliability, particularly for exposures that occurred many decades ago. Here, we studied living U.S. military test participants, believed to have received high-dose radiation exposures during nuclear testing-related activities approximately six decades ago, with two primary goals in mind. The first was to compare three different approaches of assessing past radiation exposures: 1. Historical personnel monitoring data alone; 2. Dose reconstruction based on varying levels of completeness of individual information, which can include film badge data; and 3. Retrospective biodosimetry using chromosome aberrations in peripheral blood lymphocytes. The second goal was to use the collected data to make the best possible estimates of bone marrow dose received by a group with the highest military recorded radiation doses of any currently living military test participants. Six nuclear test participants studied had been on Rongerik Atoll during the 1954 CASTLE Bravo nuclear test. Another six were present at the Nevada Test Site (NTS) and/or Pacific Proving Ground (PPG) and were believed to have received relatively high-dose exposures at those locations. All were interviewed, and all provided a blood sample for cytogenetic analysis. Military dose records for each test participant, as recorded in the Defense Threat Reduction Agency's Nuclear Test Review and Information System, were used as the basis for historical film badge records and provided exposure scenario information to estimate dose via dose reconstruction. Dose to bone marrow was also estimated utilizing directional genomic hybridization (dGH) for high-resolution detection of radiation-induced chromosomal translocations and inversions, the latter being demonstrated for the first time for the purpose of retrospective biodosimetry. As the true dose for each test participant is not known these many decades after exposure, this study gauged the congruence of different methods by assessing the degree of correlation and degree of systematic differences. Overall, the best agreement between methods, defined by statistically significant correlations and small systematic differences, was between doses estimated by a dose reconstruction methodology that exploited all the available individual detail and the biodosimetry methodology derived from a weighted average dose determined from chromosomal translocation and inversion rates. Employing such a strategy, we found that the Rongerik veterans who participated in this study appear to have received, on average, bone marrow equivalent doses on the order of 300-400 mSv, while the NTS/ PPG participants appear to have received approximately 250-300 mSv. The results show that even for nuclear events that occurred six decades in the past, biological signatures of exposure are still present, and when taken together, chromosomal translocations and inversions can serve as reliable retrospective biodosimeters, particularly on a group-average basis, when doses received are greater than statistically-determined detection limits for the biological assays used.

Chromosome Translocations, Inversions and Telomere Length for Retrospective Biodosimetry on Exposed U.S. Atomic Veterans.

It has now been over 60 years since U.S. nuclear testing was conducted in the Pacific islands and Nevada, exposing military personnel to varying levels of ionizing radiation. Actual doses are not well-established, as film badges in the 1950s had many limitations. We sought a means of independently assessing dose for comparison with historical film badge records and dose reconstruction conducted in parallel. For the purpose of quantitative retrospective biodosimetry, peripheral blood samples from 12 exposed veterans and 12 age-matched (>80 years) veteran controls were collected and evaluated for radiation-induced chromosome damage utilizing directional genomic hybridization (dGH), a cytogenomics-based methodology that facilitates simultaneous detection of translocations and inversions. Standard calibration curves were constructed from six male volunteers in their mid-20s to reflect the age range of the veterans at time of exposure. Doses were estimated for each veteran using translocation and inversion rates independently; however, combining them by a weighted-average generally improved the accuracy of dose estimations. Various confounding factors were also evaluated for potential effects on chromosome aberration frequencies. Perhaps not surprisingly, smoking and age-associated increases in background frequencies of inversions were observed. Telomere length was also measured, and inverse relationships with both age and combined weighted dose estimates were observed. Interestingly, smokers in the non-exposed control veteran cohort displayed similar telomere lengths as those in the never-smoker exposed veteran group, suggesting that chronic smoking had as much effect on telomere length as a single exposure to radioactive fallout. Taken together, we find that our approach of combined chromosome aberration-based retrospective biodosimetry provided reliable dose estimation capability, particularly on a group average basis, for exposures above statistical detection limits.

Telomeres and NextGen CO-FISH: Directional Genomic Hybridization (Telo-dGH™).

The cytogenomics-based methodology of Directional Genomic Hybridization (dGH™) emerged from the concept of strand-specific hybridization, first made possible by Chromosome Orientation FISH (CO-FISH), the utility of which was demonstrated in a variety of early applications, often involving telomeres. Similar to standard whole chromosome painting (FISH), dGH™ is capable of identifying inter-chromosomal rearrangements (translocations between chromosomes), but its distinctive strength stems from its ability to detect intra-chromosomal rearrangements (inversions within chromosomes), and to do so at higher resolution than previously possible. dGH™ brings together the strand specificity and directionality of CO-FISH with sophisticated bioinformatics-based oligonucleotide probe design to unique sequences. dGH™ serves not only as a powerful discovery tool-capable of interrogating the entire genome at the megabase level-it can also be used for high-resolution targeted detection of known inversions, a valuable attribute in both research and clinical settings. Detection of chromosomal inversions, particularly small ones, poses a formidable challenge for more traditional cytogenetic approaches, especially when they occur near the ends or telomeric regions. Here, we describe Telo-dGH™, a strand-specific scheme that utilizes dGH™ in combination with telomere CO-FISH to differentiate between terminal exchange events, specifically terminal inversions, and an altogether different form of genetic recombination that often occurs near the telomere, namely sister chromatid exchange (SCE).

Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis.

Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks) and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore, telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR) exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles, telomeres and telomerase play in the response of human cells to IRs of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET) gamma(γ)-rays or high LET, high charge, high energy (HZE) particles, delivered either acutely or at low dose rates. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprograming. Taken together, the results reported here establish the critical importance of telomeres and telomerase in the radiation response and, as such, have compelling implications not only for accelerated tumor repopulation following radiation therapy but also for carcinogenic potential following low dose exposures as well, including those of relevance to spaceflight-associated galactic cosmic radiations.

Geographic and taxonomic disparities in species diversity: dispersal and diversification rates across Wallace's line.

Broad-scale patterns of species diversity have received much attention in the literature, yet the mechanisms behind their formation may not explain species richness disparities across small spatial scales. Few taxa display high species diversity on either side of Wallace's Line and our understanding of the processes causing this biogeographical pattern remains limited, particularly in plant lineages. To understand the evolution of this biogeographical pattern, a time-calibrated molecular phylogeny of Livistoninae palms (Arecaceae) was used to infer the colonization history of the Sahul tectonic plate region and to test for disparities in diversification rates across taxa and across each side of Wallace's Line. Our analyses allowed us to examine how timing, migration history, and shifts in diversification rates have contributed to shape the biogeographical pattern observed in Livistoninae. We inferred that each of the three genera found in Sahul crossed Wallace's Line only once and relatively recently. In addition, at least two of the three dispersing genera underwent an elevation in their diversification rate leading to high species richness on each side of Wallacea. The correspondence of our results with Southeast Asian geologic and climatic history show how palms emerge as excellent models for understanding the historical formation of fine-scale biogeographic patterns in a phylogenetic framework.

Evaluating multiple criteria for species delimitation: an empirical example using Hawaiian palms (Arecaceae: Pritchardia).

BACKGROUND: Robust species delimitations are fundamental for conservation, evolutionary, and systematic studies, but they can be difficult to estimate, particularly in rapid and recent radiations. The consensus that species concepts aim to identify evolutionarily distinct lineages is clear, but the criteria used to distinguish evolutionary lineages differ based on the perceived importance of the various characteristics of evolving populations. We examined three different species-delimitation criteria (monophyly, absence of genetic intermediates, and diagnosability) to determine whether currently recognized species of Hawaiian Pritchardia are distinct lineages. RESULTS: Data from plastid and nuclear genes, microsatellite loci, and morphological characters resulted in various levels of lineage subdivision that were likely caused by differing evolutionary rates between data sources. Additionally, taxonomic entities may be confounded because of the effects of incomplete lineage sorting and/or gene flow. A coalescent species tree was largely congruent with the simultaneous analysis, consistent with the idea that incomplete lineage sorting did not mislead our results. Furthermore, gene flow among populations of sympatric lineages likely explains the admixture and lack of resolution between those groups. CONCLUSIONS: Delimiting Hawaiian Pritchardia species remains difficult but the ability to understand the influence of the evolutionary processes of incomplete lineage sorting and hybridization allow for mechanisms driving species diversity to be inferred. These processes likely extend to speciation in other Hawaiian angiosperm groups and the biota in general and must be explicitly accounted for in species delimitation.

Phylogeny of Celastraceae tribe Euonymeae inferred from morphological characters and nuclear and plastid genes.

The phylogeny of Celastraceae tribe Euonymeae (≈ 230 species in eight genera in both the Old and New Worlds) was inferred using morphological characters together with plastid (matK, trnL-F) and nuclear (ITS and 26S rDNA) genes. Tribe Euonymeae has been defined as those genera of Celastraceae with generally opposite leaves, isomerous carpels, loculicidally dehiscent capsules, and arillate seeds (except Microtropis). Euonymus is the most diverse (129 species) and widely cultivated genus in the tribe. We infer that tribe Euonymeae consists of at least six separate lineages within Celastraceae and that a revised natural classification of the family is needed. Microtropis and Quetzalia are inferred to be distinct sister groups that together are sister to Zinowiewia. The endangered Monimopetalum chinense is an isolated and early derived lineage of Celastraceae that represents an important component of phylogenetic diversity within the family. Hedraianthera is sister to Brassiantha, and we describe a second species (Brassiantha hedraiantheroides A.J. Ford) that represents the first reported occurrence of this genus in Australia. Euonymus globularis, from eastern Australia, is sister to Menepetalum, which is endemic to New Caledonia, and we erect a new genus (Dinghoua R.H. Archer) for it. The Madagascan species of Euonymus are sister to Pleurostylia and recognized as a distinct genus (Astrocassine ined.). Glyptopetalum, Torralbasia, and Xylonymus are all closely related to Euonymus sensu stricto and are questionably distinct from it. Current intrageneric classifications of Euonymus are not completely natural and require revision.