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At least 19 records

Decoding the effects of synonymous variants

Synonymous single nucleotide variants (sSNVs) are common in the human genome but are often overlooked. However, sSNVs can have significant biological impact and may lead to disease. Existing computational methods for evaluating the effect of sSNVs suffer from the lack of gold-standard training/evaluation data and exhibit over-reliance on sequence conservation signals. We developed synVep (synonymous Variant effect predictor), a machine learning-based method that overcomes both of these limitations. Our training data was a combination of variants reported by gnomAD (observed) and those unreported, but possible in the human genome (generated). We used positive-unlabeled learning to purify the generated variant set of any likely unobservable variants. We then trained two sequential extreme gradient boosting models to identify subsets of the remaining variants putatively enriched and depleted in effect. Our method attained 90% precision/recall on a previously unseen set of variants. Furthermore, although synVep does not explicitly use conservation, its scores correlated with evolutionary distances between orthologs in cross-species variation analysis. synVep was also able to differentiate pathogenic vs. benign variants, as well as splice-site disrupting variants (SDV) vs. non-SDVs. Thus, synVep provides an important improvement in annotation of sSNVs, allowing users to focus on variants that most likely harbor effects.

Zishuo Zeng↗

The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

Reinsch, S. S.↗

Abundant raw material for cis-regulatory evolution in humans

Changes in gene expression and regulation--due in particular to the evolution of cis-regulatory DNA sequences--may underlie many evolutionary changes in phenotypes, yet little is known about the distribution of such variation in populations. We present in this study the first survey of experimentally validated functional cis-regulatory polymorphism. These data are derived from more than 140 polymorphisms involved in the regulation of 107 genes in Homo sapiens, the eukaryote species with the most available data. We find that functional cis-regulatory variation is widespread in the human genome and that the consequent variation in gene expression is twofold or greater for 63% of the genes surveyed. Transcription factor-DNA interactions are highly polymorphic, and regulatory interactions have been gained and lost within human populations. On average, humans are heterozygous at more functional cis-regulatory sites (>16,000) than at amino acid positions (<13,000), in part because of an overrepresentation among the former in multiallelic tandem repeat variation, especially (AC)(n) dinucleotide microsatellites. The role of microsatellites in gene expression variation may provide a larger store of heritable phenotypic variation, and a more rapid mutational input of such variation, than has been realized. Finally, we outline the distinctive consequences of cis-regulatory variation for the genotype-phenotype relationship, including ubiquitous epistasis and genotype-by-environment interactions, as well as underappreciated modes of pleiotropy and overdominance. Ordinary small-scale mutations contribute to pervasive variation in transcription rates and consequently to patterns of human phenotypic variation.

NASA Discipline Evolutionary Biology↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. The human genome project is expected to determine the complete sequence of all human genes, and the genomes of several other organisms are already completely sequenced.

gene↗

TPSAS-NF1676L-17800-DND

Currently, there are two national challenge problems that guide much of the research in durability and damage tolerance at NASA Langley. The first, Airframe Digital Twin, is a concept that combines as-built vehicle components, as-experienced loads and environments, and other vehicle-specific characteristics to enable ultrahigh fidelity modeling of aircraft and spacecraft throughout their service lives. The second, Materials Genome Initiative, is an analog to the Human Genome Project, and is intended to improve the rate at which materials scientists can discover, understand fundamental physics, and improve material systems. This presentation will highlight several research projects ongoing at NASA Langley that are in support of the above challenge problems. Two of those topics will be the subject of detailed discussion. First, investigations of microstructurally-small fatigue cracking (MSFC) in Al-2Cu and Al-4Cu, fabricated in-house, will be presented. Single- and oligo-crystals of Al-Cu specimens were loaded in uniaxial fatigue, while high-resolution in-situ measurements of deformation were made using image correlation (IC) in a scanning-electron microscope (SEM). The Al-Cu specimens were then replicated as crystal plasticity finite element models (CPFEM), where evolution of slip localization near grain boundaries was computed. Comparison among experiment and CPFEM is made. In addition, XRay diffraction measurements of the as-fabricated specimens were carried out, where direct measurements of the embedded copper precipitates were made, and their influence on growing MSFCs were directly observed. The second main topic will illustrate ongoing work in the area of so-called damage-sensing particles. In this work, shape-memory alloys are embedded in an aluminum alloy matrix. Upon the propagation of a fatigue crack, these particles undergo a strain-induced phase transformation which is detected using an acoustic sensor, providing real-time information on propagating cracks. Experiments and simulations regarding the development of this system will also be detailed.

Jacob Hochhalter↗

The Analysis of the Patterns of Radiation-Induced DNA Damage Foci by a Stochastic Monte Carlo Model of DNA Double Strand Breaks Induction by Heavy Ions and Image Segmentation Software

To create a generalized mechanistic model of DNA damage in human cells that will generate analytical and image data corresponding to experimentally observed DNA damage foci and will help to improve the experimental foci yields by simulating spatial foci patterns and resolving problems with quantitative image analysis. Material and Methods: The analysis of patterns of RIFs (radiation-induced foci) produced by low- and high-LET (linear energy transfer) radiation was conducted by using a Monte Carlo model that combines the heavy ion track structure with characteristics of the human genome on the level of chromosomes. The foci patterns were also simulated in the maximum projection plane for flat nuclei. Some data analysis was done with the help of image segmentation software that identifies individual classes of RIFs and colocolized RIFs, which is of importance to some experimental assays that assign DNA damage a dual phosphorescent signal. Results: The model predicts the spatial and genomic distributions of DNA DSBs (double strand breaks) and associated RIFs in a human cell nucleus for a particular dose of either low- or high-LET radiation. We used the model to do analyses for different irradiation scenarios. In the beam-parallel-to-the-disk-of-a-flattened-nucleus scenario we found that the foci appeared to be merged due to their high density, while, in the perpendicular-beam scenario, the foci appeared as one bright spot per hit. The statistics and spatial distribution of regions of densely arranged foci, termed DNA foci chains, were predicted numerically using this model. Another analysis was done to evaluate the number of ion hits per nucleus, which were visible from streaks of closely located foci. In another analysis, our image segmentaiton software determined foci yields directly from images with single-class or colocolized foci. Conclusions: We showed that DSB clustering needs to be taken into account to determine the true DNA damage foci yield, which helps to determine the DSB yield. Using the model analysis, a researcher can refine the DSB yield per nucleus per particle. We showed that purely geometric artifacts, present in the experimental images, can be analytically resolved with the model, and that the quantization of track hits and DSB yields can be provided to the experimentalists who use enumeration of radiation-induced foci in immunofluorescence experiments using proteins that detect DNA damage. An automated image segmentaiton software can prove useful in a faster and more precise object counting for colocolized foci images.

Ponomarev, Artem↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.

Mjolsness, Eric↗

Final Report - Hypergravity Effects on Chromatin Conformation and Nuclear Structure in Cultured Cells using High-throughput Sequencing

The conformation of the human genome is known to play an important role in transcriptional control of gene expression. Our aim is to assess whether exposure of cultured human cardiomyocytes to hypergravity can induce changes in chromatin organization as assayed by Hi-C proximity ligation technique. Hi-C is used to analyze chromatin interactions by using formaldehyde to crosslink regions of chromatin that are in close proximity. The DNA is then fragmented using a restriction enzyme and ligated under dilute conditions to favor intramolecular ligation of cross-linked fragments. Finally, the DNA is sequenced, allowing reconstruction of genomic structure. iCell cardiomyocyte cultures will be exposed to 40 g via an engineered device (incu-fuge) that allows for the chemical fixation of cells on a spinning centrifuge. Additionally, we plan on using fluorescence microscopy to analyze the nuclear and actin cytoskeletal conformation of cardiomyocytes post hypergravity exposure. We hypothesize that chromatin will rearrange in hypergravity conditions, both rapidly due to direct mechanical forces, as well as over longer time frames due to changes to structures necessary for transcriptional responses such as the de novo formation of promoter-enhancer loops. A negative result–no change between 1 g and hypergravity conditions–would nevertheless be an important data point in our understanding of the rheology of the nucleus, and how cells and cellular structures respond to different gravity fields. The cellular physiology of hypergravity has clinical relevance for spaceflight, and can further inform our understanding of microgravity physiology.

chromatin↗

Scheme for Entering Binary Data Into a Quantum Computer

A quantum algorithm provides for the encoding of an exponentially large number of classical data bits by use of a smaller (polynomially large) number of quantum bits (qubits). The development of this algorithm was prompted by the need, heretofore not satisfied, for a means of entering real-world binary data into a quantum computer. The data format provided by this algorithm is suitable for subsequent ultrafast quantum processing of the entered data. Potential applications lie in disciplines (e.g., genomics) in which one needs to search for matches between parts of very long sequences of data. For example, the algorithm could be used to encode the N-bit-long human genome in only log2N qubits. The resulting log2N-qubit state could then be used for subsequent quantum data processing - for example, to perform rapid comparisons of sequences.

Williams, Colin↗

Simulation of Radiation-Induced DNA Damage With the Code RITRACKS

INTRODUCTION DNA damage is one of the most physiologically important effects of ionizing radiation. Clustered DNA damage events, like double-strand breaks (DSBs), have the most notable biological consequences. DNA damage types depend on both the track structure of the radiation and the spatial organization of the DNA. High linear energy transfer (LET) charged nuclei, found in galactic cosmic rays (GCR), are known to produce large numbers of complex DNA damage events. The human genome is packaged into chromatin, which can take on locus-dependent and cell type-dependent spatial conformations that correspond to epigenetic states, such as more open, extended structures in transcriptionally active chromatin. These epigenetic differences can affect DNA break patterns in response to ionizing radiation, potentially creating distinct DNA repair and signaling outcomes across the genome in different cells. MATERIAL AND METHODS The code RITRACKS (Relativistic Ion Tracks), which simulates stochastic radiation track structures and radiation chemistry, was used to model damage on isolated and histone-bound DNA by various types of ions and photons. The changes made to the code to perform radiation-induced DNA damage, and simulation results on single nucleosomes are given in our recent paper. In this work, the DNA building capabilities of RITRACKS have been extended to simulate more complex DNA structures build on the coarse-grain simulation framework meso-WLCsim. This code can sample generic chromatin fiber conformation ensembles based on the geometry of nucleosomes and mechanical properties of DNA. Using RITRACKS, we simulated the fragment length distributions (FLD) of irradiated DNA structures built using the chromatin conformations of WLCsim and obtained results representative of those obtained with Radiation-Induced Correlated Cleavage with sequencing (RICC-Seq) experiments [6]. We have also performed Fe ion and photon irradiations of K562, IMR90, BJ and RPE-1 cells at NSRL to experimentally validate results. Sample processing and data analysis are in progress and any available preliminary results will be discussed. DISCUSSION The recent updates in the code RITRACKS allow the calculation of several quantities such as the DNA damage yield and the FLD. This approach can be used to model epigenetic state-specific chromatin structure parameters to leverage the epigenetic state data available for many human cell types to infer relative DNA damage sensitivity among genomic loci.

I Plante↗

The role of clinical pharmacology in molecular genetics

PROBLEM: Discovering the causes of unusual phenotypes in human subjects is an important aspect of patient-oriented research. MATERIAL: The tools of clinical pharmacology are uniquely useful in addressing these problems. PATIENTS, SUBJECTS, OR CASE HISTORIES: We evaluated a 42-year-old patient with lifelong orthostatic hypotension and ptosis of the eyelids. He underwent a series of biochemical, physiological, and pharmacological tests outlined in this article. RESULTS: These studies indicated that sympathetic innervation was intact but that the sympathetic neurotransmitter was dopamine rather than norepinephrine. These results demonstrated that dopamine-beta-hydroxylase deficiency underlies the clinical abnormalities of this patient. CONCLUSION: In selected individuals with unusual phenotypes, the techniques of clinical chemistry and clinical pharmacology can define the nature of the defect at almost the resolution of the human genome.

NASA Discipline Regulatory Physiology↗

Overview of NASARTI (NASA Radiation Track Image) Program: Highlights of the Model Improvement and the New Results

This presentation summarizes several years of research done by the co-authors developing the NASARTI (NASA Radiation Track Image) program and supporting it with scientific data. The goal of the program is to support NASA mission to achieve a safe space travel for humans despite the perils of space radiation. The program focuses on selected topics in radiation biology that were deemed important throughout this period of time, both for the NASA human space flight program and to academic radiation research. Besides scientific support to develop strategies protecting humans against an exposure to deep space radiation during space missions, and understanding health effects from space radiation on astronauts, other important ramifications of the ionizing radiation were studied with the applicability to greater human needs: understanding the origins of cancer, the impact on human genome, and the application of computer technology to biological research addressing the health of general population. The models under NASARTI project include: the general properties of ionizing radiation, such as particular track structure, the effects of radiation on human DNA, visualization and the statistical properties of DSBs (DNA double-strand breaks), DNA damage and repair pathways models and cell phenotypes, chromosomal aberrations, microscopy data analysis and the application to human tissue damage and cancer models. The development of the GUI and the interactive website, as deliverables to NASA operations teams and tools for a broader research community, is discussed. Most recent findings in the area of chromosomal aberrations and the application of the stochastic track structure are also presented.

Ponomarev, Artem L.↗

Genomic instability in human lymphoid cells exposed to 1 GeV/amu Fe ions

The goal of this study was to assess whether charged particle radiations of importance to spaceflight elicit genomic instability in human TK6 lymphoblasts. The incidence of genomic instability in TK6 cells was assessed ~21 days after exposure to 2, 4, or 6 Fe ions (1 GeV/amu, LET= 146 keV/micrometers). Three indices of instability were used: intraclonal karyotypic heterogeneity, mutation rate analysis at the thymidine kinase (TK1) locus, and re-cloning efficiency. Fifteen of sixty clones demonstrated karyotypic heterogeneity. Five clones had multiple indicators of karyotypic change. One clone was markedly hypomutable and polyploid. Six clones were hypomutable, while 21 clones were mutators. Of these, seven were karyotypically unstable. Six clones had low re-cloning efficiencies, one of which was a mutator. All had normal karyotypes. In summary, many clones that survived exposure to a low fluence of Fe ions manifested one or more forms of genomic instability that may hasten the development of neoplasia through deletion or by recombination.

Non-NASA Center↗

The 'Biologically-Inspired Computing' Column

The field of Biology changed dramatically in 1953, with the determination by Francis Crick and James Dewey Watson of the double helix structure of DNA. This discovery changed Biology for ever, allowing the sequencing of the human genome, and the emergence of a "new Biology" focused on DNA, genes, proteins, data, and search. Computational Biology and Bioinformatics heavily rely on computing to facilitate research into life and development. Simultaneously, an understanding of the biology of living organisms indicates a parallel with computing systems: molecules in living cells interact, grow, and transform according to the "program" dictated by DNA. Moreover, paradigms of Computing are emerging based on modelling and developing computer-based systems exploiting ideas that are observed in nature. This includes building into computer systems self-management and self-governance mechanisms that are inspired by the human body's autonomic nervous system, modelling evolutionary systems analogous to colonies of ants or other insects, and developing highly-efficient and highly-complex distributed systems from large numbers of (often quite simple) largely homogeneous components to reflect the behaviour of flocks of birds, swarms of bees, herds of animals, or schools of fish. This new field of "Biologically-Inspired Computing", often known in other incarnations by other names, such as: Autonomic Computing, Pervasive Computing, Organic Computing, Biomimetics, and Artificial Life, amongst others, is poised at the intersection of Computer Science, Engineering, Mathematics, and the Life Sciences. Successes have been reported in the fields of drug discovery, data communications, computer animation, control and command, exploration systems for space, undersea, and harsh environments, to name but a few, and augur much promise for future progress.

Hinchey, Mike↗

Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

NASA Discipline Cardiopulmonary↗

The Single Nucleotide Polymorphism Consortium

I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

Morgan, Michael↗

Predicting Cancer Risk from Ionizing Radiation

The ability to predict cancer risk associated with exposure to low doses of high-LET ionizing radiation (IR) remains a challenge. Epidemiological methods lack the sensitivity and power to provide detailed risk estimates for cancer and ignore individual variance in IR sensitivity. We have hypothesized that DNA repair capacity can be used as a marker to evaluate and differentiate individual radiation sensitivity. More specifically, this work is based on the concept that the combined time-dose dependence of radiation-induced foci (RIF) of p53-binding protein 1 (53BP1) following low-LET exposure contains sufficient information to infer sensitivity to any other LET. Our hypothesis was tested in 15 different mouse strains as well as in primary human immune cells. We first approached individual ionizing radiation sensitivity in a mouse model by culturing primary skin fibroblasts extracted from 76 mice of 15 different genetic backgrounds and exposing them to HZE particles and X-rays. This work is one of the most extensive studies on the kinetics and possible genetic underpinnings of radiation-induced DNA damage and repair. Our results is in agreement with a DNA repair model we previously postulated, where nearby DNA double strand breaks (DSB) in the nucleus are brought together for more efficient repair, leading to RIF clustering. Such mechanism was evidenced by a specific dose and LET dependence of RIF numbers. Briefly, RIF quantification after low-LET X-ray exposure showed an asymptotic saturation for doses between 1 Gy and 4 Gy 4 hours post-irradiation across all 15 strains. The clustering of DSB across all strains also led to more RIF/Gy for lower LET (X-ray and 350 MeV/n Ar) than for higher LET (600 MeV/n Fe) 4 hours post-exposure. Considering the fact that the number of DSB/Gy should be independent of LET, our data suggest there are more DSB in individual RIF as the LET increases. RIF numbers for 24 and 48 hours post-exposure led to the inverse trend, with more remaining RIF/Gy for higher LET (by 600 MeV/n Fe). This result suggests cells have more difficulty resolving RIF from higher LET as they the number DSB/RIF increases. Note that for most conditions, the variance of RIF/Gy was small within individual animals of the same strain and large between strains, suggesting a strong genetics component. Furthermore, we present our preliminary data from an ongoing study on human genetic associations with IR sensitivity. To address the human variability in responses to HZE particle irradiation in a maximally comprehensive manner, we are in the process of collecting and isolating primary blood mononuclear cells from 768 healthy subjects of European descent, 18-75 years of age, 50/50 male/female distribution. We have analyzed 53BP1+ RIF formation as well as oxidative stress and cell death in primary cells from 192 subjects in response to the same HZE particles as used in mice: 600 MeV/n Fe, 350 MeV/n Ar and 350 MeV/n Si, 1.1 and 3 particles/100m2, 4 and 24 hours after irradiation. We will next complete the quantification of HZE particle-induced DNA and cellular damage in the remaining subjects and compare it to their responses to low-LET irradiation. Finally, we will perform GWAS analysis to identify human genomic associations with IR sensitivity and potential targets for biomarker development.

High-LET radiation↗

Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.

Non-NASA Center↗