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1.  Quantitative characteristics of clustered DNA damage in irradiated cells by heavy ion beams 
Journal of Radiation Research  2014;55(Suppl 1):i89-i90.
Heavy ion beam as typical high linear energy transfer (LET) radiation produces more expanding ionization domain around their tracks than low LET radiation such as X-rays and gamma rays. Thus, heavy ion beam can cause more densely accumulated damage cluster in the target DNA, termed clustered DNA damage. This damage exhibits difficulty for repair and inhibition of DNA replication with its complex structure [ 1]. So, clustered DNA damage is thought to be strongly involved in the biological effectiveness of heavy ion beam. However, a lot of studies have presented no certain correlation between yields of clustered DNA damage and severity of radiation effect. We previously indicated that the yields of clustered DNA damage decreased with increasing LET in the DNA molecules irradiated in test tubes with gamma rays, and carbon and iron ion beams whose showed different LET, respectively [ 2]. In this study, we aimed to reveal correlation between clustered DNA damage and the LET of heavy ion beam in the irradiated cells.
In the experiments, Chinese hamster ovary AA8 cells growing exponentially were irradiated by carbon, silicon, argon and iron ion beams from Heavy Ion Medical Accelerator in Chiba (HIMAC) of the National Institute of Radiological Sciences, Japan. These LETs were 13, 55, 90 and 200 keV/µm, respectively. For comparison, we used gamma rays from 137Cs-gamma source, Gammacell 40 (Atomic Energy of Canada Ltd), at Saga University. The irradiated cells were subjected by static-field gel electrophoresis to quantify clustered DNA damage of the genomic DNA. For this analysis, we used Fpg and endonuclease III for clustered DNA damage including oxidative purine and pyrimidine lesions, respectively. We also analysed the corresponding isolated DNA damages by aldehyde reactive probe method [ 3], and the surviving fractions of the irradiated cells in this study.
The electrophoretic results indicated that total yields of clustered DNA damage in the irradiated cells decreased with increasing LET, including the double-strand break (DSB) and the respective clustered base damages (Fig. 1). This result conforms to our previous study with the irradiated DNA molecules [ 2]. The damage kinetics is thought to be mainly derived from two reasons: decreasing fluxes and increasing reaction with reactive oxygen species each other in increase in LET. In the clustered DNA damage induced by each radiation, the most decremental fraction was clustered base damage, but not DSB. The isolated DNA damages decreased with increasing LET like clustered DNA damage in this study (data not shown). These results make us realize the degree of contribution of direct and indirect effects of ionizing radiation. The certain amount of DSB were derived from the direct effect and showed less reactivity to LET. In contrast, oxidative base lesions were mainly generated by indirect effect with reactive oxygen species, which sensitively responded to LET change. We also found seemingly conflicted result of the relationship between LET and RBE (data not shown). We need further study to elucidate act of clustered DNA damage in radiobiological effect with heavy ion beams. Fig. 1.The yields of clustered DNA damages in the cells irradiated with respective ionizing radiations. Each clustered DNA damage consists of DSB (open bar) and clustered base damage (closed bar), and calculated from the strength of released band on electrophoretic gel.
Clinical trial registration number if required: None.
doi:10.1093/jrr/rrt173
PMCID: PMC3941507
heavy ion beam; clustered DNA damage; LET; RBE
2.  Nanopore sculpting with noble gas ions 
Journal of applied physics  2006;100(2):024914-024914-6.
We demonstrate that 3 keV ion beams, formed from the common noble gasses, He, Ne, Ar, Kr, and Xe, can controllably “sculpt” nanometer scale pores in silicon nitride films. Single nanometer control of structural dimensions in nanopores can be achieved with all ion species despite a very wide range of sputtering yields and surface energy depositions. Heavy ions shrink pores more efficiently and make thinner pores than lighter ions. The dynamics of nanopore closing is reported for each ion species and the results are fitted to an adatom diffusion model with excellent success. We also present an experimental method for profiling the thickness of the local membrane around the nanopore based on low temperature sputtering and data is presented that provides quantitative measurements of the thickness and its dependence on ion beam species.
doi:10.1063/1.2216880
PMCID: PMC3039599  PMID: 21331305
3.  Co-localization of the Ganglioside GM1 and Cholesterol Detected by Secondary Ion Mass Spectrometry 
The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remains a major challenge. The concept of lipid rafts is widely invoked, however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of Secondary Ion Mass Spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano and micro-scale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the co-localization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d>3 μm); and, (3) nanometer-scale domains (d=100 nm − 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.
doi:10.1021/ja310831m
PMCID: PMC3639293  PMID: 23514537
4.  DNA damage intensity in fibroblasts in a 3-dimensional collagen matrix correlates with the Bragg curve energy distribution of a high LET particle 
Purpose
The DNA double-strand break (DSB) damage response induced by high energy charged particles on lung fibroblast cells embedded in a 3-dimensional (3-D) collagen tissue equivalents was investigated using antibodies to the DNA damage response proteins gamma-histone 2AX (γ-H2AX) and phosphorylated DNA-PKcs (p-DNA-PKcs).
Materials and methods
3-D tissue equivalents were irradiated in positions across the linear distribution of the Bragg curve profiles of 307.7 MeV/nucleon, 556.9 MeV/nucleon, or 967.0 MeV/nucleon 56Fe ions at a dose of 0.30 Gy.
Results
Patterns of discrete DNA damage streaks across nuclei or saturated nuclear damage were observed, with saturated nuclear damage being more predominant as samples were positioned closer to the physical Bragg peak. Quantification of the DNA damage signal intensities at each distance for each of the examined energies revealed a biological Bragg curve profile with a pattern of DNA damage intensity similar to the physical Bragg curve for the particular energy. Deconvolution microscopy of nuclei with streaked or saturated nuclear damage pattern revealed more details of the damage, with evidence of double-strand breaks radially distributed from the main particle track as well as multiple discrete tracks within saturated damage nuclei.
Conclusions
These 3-D culture systems can be used as a biological substrate to better understand the interaction of heavy charged particles of different energies with tissue and could serve as a basis to model space-radiation-induced cancer initiation and progression.
doi:10.3109/09553000903418603
PMCID: PMC3382085  PMID: 20201648
heavy ion irradiation; DNA damage; DNA double-strand break repair; 3-D tissue equivalents
5.  Primary and secondary clustering of DSB repair foci and repair kinetics compared for γ-rays, protons of different energies and high-LET 20Ne ions 
Journal of Radiation Research  2014;55(Suppl 1):i79-i80.
Purpose: Ionizing radiations of different qualities (e.g. high-LET and low-LET) might differently interact with structurally and functionally distinct higher order chromatin domains (discussed in [ 1] and citations therein); this might be reflected by DNA double strand break (DSB) repair efficiency and the mechanism of how cancerogenous chromosomal translocations (CHT) form. Therefore, we compared the DSB repair kinetics and formation of γH2AX/p53BP1 repair clusters upon the action of γ-rays [ 2, 3], protons (15 and 30 MeV) [ 4], and 20Ne ions (preliminary data). Consequently, we discuss biological impacts of these clusters.
Material and methods: Immunostaining methods in combination with high-resolution confocal microscopy, performed on 3D-fixed normal human skin fibroblasts [ 2– 4], were used to study initial distributions of γH2AX and p53BP1 repair foci and their changes during the post-irradiation (PI) time, with a special concern on foci clustering. Irradiations with γ-rays, protons of different energies (15 and 30 MeV), and high-LET 20Ne ions was performed in IBP ASCR Brno (CR), NPI AVCR Řež (CR) and JINR Dubna (Russia), respectively.
Results: Upon irradiating cells with 20Ne ions, tracks of multiple clustered γH2AX and p53BP1 repair foci appeared immediately after the irradiation; these clusters, called here as the ‘primary clusters’, were rare in cells irradiated with γ-rays or protons (submitted). Though γH2AX/p53BP1 foci were positionally quite stable [ 2], ‘secondary clusters’ occasionally appeared after all kinds of irradiation during about 30 min PI. The formation of secondary clusters usually appeared due to the heterochromatin decondensation at the sites of heterochromatic DNA double-strand breaks (hcDSBs), followed by their protrusion into a limited space of nuclear subdomains of low density-chromatin (discussed in [ 1, 2, 5]).
Conclusions: Primary clusters appear in cell nuclei immediately PI as the consequence of highly localized energy deposition, while secondary clusters develop during (and because of) DSB repair. Primary DSB clusters probably represent the main cause of chromosomal translocations induced with high-LET radiations while secondary clusters seem to be more important for low-LET γ-rays and protons. Secondary clusters of primary clusters (higher-order clusters) observed for 20Ne ions might explain frequent formation of complex translocations upon the action of high-LET radiations. Finally, we suggest [ 1, 2, 4] a model that describes the relationship between the higher order chromatin structure, DSB formation, repair and misrepair.
doi:10.1093/jrr/rrt210
PMCID: PMC3941551
DNA double-strand breaks (DSB); DSB repair; chromosomal translocations; primary and secondary DSB clusters; higher order chromatin structure; ionizing radiation of different quality
6.  Chromatin differentiation of white blood cells decreases DSB damage induction, prevents functional assembly of repair foci, but has no influence on protrusion of heterochromatic DSBs into the low-dense chromatin 
Journal of Radiation Research  2014;55(Suppl 1):i81-i82.
Purpose: Higher order chromatin structure progressively changes with cell differentiation and seems to play an important role in DNA double-strand break (DSB) induction and repair (reviewed in [1]). We compared DNA damage in heterochromatin (Hc) upon the action of qualitatively different radiations. We also studied, how is the sensitivity to DSB induction, assembly of repair foci and processing of DSBs influenced by the differentiation-induced changes in chromatin structure and composition.
Materials and methods: Formation, localization (relative to higher-order chromatin domains) and mutual colocalization of γH2AX and p53BP1 repair foci have been studied together with DSB repair kinetics in spatially fixed human skin fibroblast and differently differentiated white blood cells (WBC) irradiated with gamma rays, protons of different energies [2, 3], and 20Ne ions (submitted). Immunostaining and ImmunoFISH were used in combination with high-resolution confocal microscopy [2, 3] and living cell imaging [4].
Results: We found that less DSBs appear in Hc after irradiating cells with gamma rays and protons but not 20Ne ions (preliminary results). In addition, contrary to γ-irradiated human skin fibroblasts and lymphocytes, mature granulocytes neither express DSB repair proteins nor form functional repair foci [5]. At least some DSB repair proteins (e.g. 53BP1) are expressed and γH2AX foci still occur in immature granulocytes and monocytes [2, 5]; however, the colocalization of γH2AX with 53BP1 is low and the majority of DSBs are not repaired. Despite this fact, γH2AX foci protrude from Hc into nuclear subcompartments with low chromatin density. Our living cell observations suggest that 53BP1 can penetrate into the interior of dense Hc domains only after their decondensation [2].
Conclusions: We show that Hc is less sensitive to DSB induction by gamma rays but not heavy ions; lower Hc hydratation and higher protein density (when compared with euchromatin) probably reduce formation of free radicals and increase their sequestration, respectively. This mechanism can protect cells against the indirect effect of ionizing radiation (marked for gamma rays and protons but not heavy ions). Hc features, however, preclude DSB repair, which is best illustrated by its absence in differentiated WBC but not their immature precursors. The protrusion of Hc-DSBs into low-density chromatin nuclear subdomains, however, appears also in differentiated WBC, so the process might simply follow physical forces (e.g. as suggested by M Durante's group).
There is no Clinical Trial Registration number.
doi:10.1093/jrr/rrt194
PMCID: PMC3941545
DNA double-strand breaks (DSB); DSB repair; white blood cells differentiation; higher-order chromatin structure; ionizing radiations of different quality; ionizing radiation-induced repair foci (IRIF)
7.  Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation 
Journal of Radiation Research  2015;56(3):446-455.
Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm−1, respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process.
doi:10.1093/jrr/rru122
PMCID: PMC4426916  PMID: 25717060
clustered DNA damage; oxidative base lesion; heavy ion beam; high-LET radiation; relative biological effectiveness (RBE)
8.  Absolute cross section for low-energy-electron damage to condensed macromolecules: A case study of DNA 
Cross sections (CSs) for the interaction of low-energy electrons (LEE) with condensed macromolecules are essential parameters for accurate modeling of radiation-induced molecular decomposition and chemical synthesis. Electron irradiation of dry nanometer-scale macromolecular solid films has often been employed to measure CSs and other quantitative parameters for LEE interactions. Since such films have thicknesses comparable with electron thermalization distances, energy deposition varies throughout the film. Moreover, charge accumulation occurring inside the films shields a proportion of the macromolecules from electron irradiation. Such effects complicate the quantitative comparison of the CSs obtained in films of different thicknesses and limit the applicability of such measurements. Here, we develop a simple mathematical model, termed the molecular survival model, that employs a CS for a particular damage process together with an attenuation length related to the total CS, to investigate how a measured CS might be expected to vary with experimental conditions. As a case study, we measure the absolute CS for the formation of DNA strand breaks (SBs) by electron irradiation at 10 and 100 eV of lyophilized plasmid DNA films with thicknesses between 10 and 30 nm. The measurements are shown to depend strongly on the thickness and charging condition of the nanometer-scale films. Such behaviors are in accord with the model and support its validity. Via this analysis, the CS obtained for SB damage is nearly independent of film thickness and charging effects. In principle, this model can be adapted to provide absolute CSs for electron-induced damage or reactions occurring in other molecular solids across a wider range of experimental conditions.
PMCID: PMC3815646  PMID: 23030950 CAMSID: cams3603
9.  Model Assembly for Estimating Cell Surviving Fraction for Both Targeted and Nontargeted Effects Based on Microdosimetric Probability Densities 
PLoS ONE  2014;9(11):e114056.
We here propose a new model assembly for estimating the surviving fraction of cells irradiated with various types of ionizing radiation, considering both targeted and nontargeted effects in the same framework. The probability densities of specific energies in two scales, which are the cell nucleus and its substructure called a domain, were employed as the physical index for characterizing the radiation fields. In the model assembly, our previously established double stochastic microdosimetric kinetic (DSMK) model was used to express the targeted effect, whereas a newly developed model was used to express the nontargeted effect. The radioresistance caused by overexpression of anti-apoptotic protein Bcl-2 known to frequently occur in human cancer was also considered by introducing the concept of the adaptive response in the DSMK model. The accuracy of the model assembly was examined by comparing the computationally and experimentally determined surviving fraction of Bcl-2 cells (Bcl-2 overexpressing HeLa cells) and Neo cells (neomycin resistant gene-expressing HeLa cells) irradiated with microbeam or broadbeam of energetic heavy ions, as well as the WI-38 normal human fibroblasts irradiated with X-ray microbeam. The model assembly reproduced very well the experimentally determined surviving fraction over a wide range of dose and linear energy transfer (LET) values. Our newly established model assembly will be worth being incorporated into treatment planning systems for heavy-ion therapy, brachytherapy, and boron neutron capture therapy, given critical roles of the frequent Bcl-2 overexpression and the nontargeted effect in estimating therapeutic outcomes and harmful effects of such advanced therapeutic modalities.
doi:10.1371/journal.pone.0114056
PMCID: PMC4245256  PMID: 25426641
10.  Surface Morphology Evolution of GaAs by Low Energy Ion Sputtering 
Nanoscale Research Letters  2007;2(10):504-508.
Low energy Ar+ion sputtering, typically below 1,200 eV, of GaAs at normal beam incident angle is investigated. Surface morphology development with respect to varying energy is analyzed and discussed. Dot-like patterns in the nanometer scale are obtained above 600 eV. As the energy approaches upper eV range regular dots have evolved. The energy dependent dot evolution is evaluated based on solutions of the isotropic Kuramoto-Sivashinsky equation. The results are in agreement with the theoretical model which describes a power law dependency of the characteristic wavelength on ion energy in the ion-induced diffusion regime.
doi:10.1007/s11671-007-9090-4
PMCID: PMC3246606
Low energy; Ion sputtering; Surface morphology; GaAs quantum dot
11.  Genotoxicity of charged particles of importance in space flight using murine kidney epithelial cells 
Journal of Radiation Research  2014;55(Suppl 1):i77-i78.
Ionizing radiation presents significant challenges for human space flight including an increased cancer risk. High-energy heavy ions in the galactic cosmic radiation can produce qualitative and quantitative differences in biological effects when compared with sparsely ionizing radiations. Mutations are induced by charged particle exposure and are integral to the formation and/or progression of human cancers. Most cancer-associated mutations occur on autosomal chromosomes, and most solid cancers occur in epithelial tissues. Here, a combined in vitro/in vivo approach was used to evaluate cell killing and the induction of mutations at a model autosomal locus, Aprt, in mouse kidney epithelium. For in vitro exposures, Aprt heterozygous kidney cells (clones 1a, 4a or 6a) were used from C57BL/6×DBA/2 mice. Additional experiments were performed using whole body irradiation of mice with the same genotype. Both males and females were irradiated in approximately equal numbers. Irradiations were performed at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. For in vitro studies, cells from primary kidney clones were irradiated and seeded at limiting dilution immediately post-irradiation to determine the toxicity of the treatment. The irradiated kidney cells were also seeded in mutation assays within 1 week post-irradiation to determine the Aprt mutant fraction at the earliest time post-exposure. This work was complemented by studies wherein mice were exposed to the same ions with kidneys harvested several months post-irradiation to determine the residual toxicity and the Aprt mutant fraction. Our previous studies focused on sparsely ionizing 1 GeV protons (LET = 0.24 keV/µm) and densely ionizing 1 GeV/amu Fe ions (LET = 151 keV/µm). Our most recent studies have included work with Si ions (240 MeV/amu for in vitro studies, LET = 78 keV/µm; 263 MeV/amu initial energy for in vivo studies to achieve 78 keV/µm near the midline of the animal) and O ions (250 MeV/amu in vitro studies only, LET = 25 keV/µm). Toxicity for the cultured kidney cells in vitro follows this pattern: Fe > Si > O > protons when the results are expressed per unit dose. D0 values were 92 cGy for Fe ions, 103 cGy for Si ions, 192 cGy for O ions and 340 cGy for protons. With regard to the induction of Aprt mutations, Fe ions were more mutagenic than protons. Si ions were also quite mutagenic with evidence for a linear dose–response for Aprt mutations in kidney cells exposed in vitro or in kidneys harvested from mice irradiated several months earlier. These results are consistent with the linear dose–response data obtained previously for Aprt mutation induction following Fe ion exposure in vitro or in vivo, but the results for Si ions differ from the curvilinear dose–response data we recently published following similar exposures to energetic protons [ 1, 2]. Our most recent studies examined the molecular characteristics of Si ion-induced Aprt mutants following in vitro exposure. A dose of 160 cGy was used to collect 58 Aprt kidney cell mutants. Mutational events were classified as follows based on PCR-based analyses of polymorphic markers along mouse chromosome 8: intragenic events, apparent mitotic recombination, interstitial deletions of Aprt only, multilocus deletions, discontinuous loss of heterozygosity or whole chromosome loss. The results for this group of mutants will be compared against our previous studies on Aprt mutants arising after exposure to sparsely ionizing 1 GeV protons or densely ionizing 1 GeV/amu Fe ions. Additional studies are ongoing to define mutational spectra following Si ion exposure to kidney epithelium in vivo.
Clinical Trial Registration number: not applicable.
doi:10.1093/jrr/rrt191
PMCID: PMC3941538
charged particles; heavy ions; mutation; cell killing; epithelium
12.  Focused electron beam induced deposition: A perspective 
Summary
Background: Focused electron beam induced deposition (FEBID) is a direct-writing technique with nanometer resolution, which has received strongly increasing attention within the last decade. In FEBID a precursor previously adsorbed on a substrate surface is dissociated in the focus of an electron beam. After 20 years of continuous development FEBID has reached a stage at which this technique is now particularly attractive for several areas in both, basic and applied research. The present topical review addresses selected examples that highlight this development in the areas of charge-transport regimes in nanogranular metals close to an insulator-to-metal transition, the use of these materials for strain- and magnetic-field sensing, and the prospect of extending FEBID to multicomponent systems, such as binary alloys and intermetallic compounds with cooperative ground states.
Results: After a brief introduction to the technique, recent work concerning FEBID of Pt–Si alloys and (hard-magnetic) Co–Pt intermetallic compounds on the nanometer scale is reviewed. The growth process in the presence of two precursors, whose flux is independently controlled, is analyzed within a continuum model of FEBID that employs rate equations. Predictions are made for the tunability of the composition of the Co–Pt system by simply changing the dwell time of the electron beam during the writing process. The charge-transport regimes of nanogranular metals are reviewed next with a focus on recent theoretical advancements in the field. As a case study the transport properties of Pt–C nanogranular FEBID structures are discussed. It is shown that by means of a post-growth electron-irradiation treatment the electronic intergrain-coupling strength can be continuously tuned over a wide range. This provides unique access to the transport properties of this material close to the insulator-to-metal transition. In the last part of the review, recent developments in mechanical strain-sensing and the detection of small, inhomogeneous magnetic fields by employing nanogranular FEBID structures are highlighted.
Conclusion: FEBID has now reached a state of maturity that allows a shift of the focus towards the development of new application fields, be it in basic research or applied. This is shown for selected examples in the present review. At the same time, when seen from a broader perspective, FEBID still has to live up to the original idea of providing a tool for electron-controlled chemistry on the nanometer scale. This has to be understood in the sense that, by providing a suitable environment during the FEBID process, the outcome of the electron-induced reactions can be steered in a controlled way towards yielding the desired composition of the products. The development of a FEBID-specialized surface chemistry is mostly still in its infancy. Next to application development, it is this aspect that will likely be a guiding light for the future development of the field of focused electron beam induced deposition.
doi:10.3762/bjnano.3.70
PMCID: PMC3458607  PMID: 23019557
atomic force microscopy; binary systems; electron beam induced deposition; granular metals; micro Hall magnetometry; radiation-induced nanostructures; strain sensing
13.  Issues for Simulation of Galactic Cosmic Ray Exposures for Radiobiological Research at Ground-Based Accelerators 
Frontiers in Oncology  2015;5:122.
For radiobiology research on the health risks of galactic cosmic rays (GCR) ground-based accelerators have been used with mono-energetic beams of single high charge, Z and energy, E (HZE) particles. In this paper, we consider the pros and cons of a GCR reference field at a particle accelerator. At the NASA Space Radiation Laboratory (NSRL), we have proposed a GCR simulator, which implements a new rapid switching mode and higher energy beam extraction to 1.5 GeV/u, in order to integrate multiple ions into a single simulation within hours or longer for chronic exposures. After considering the GCR environment and energy limitations of NSRL, we performed extensive simulation studies using the stochastic transport code, GERMcode (GCR Event Risk Model) to define a GCR reference field using 9 HZE particle beam–energy combinations each with a unique absorber thickness to provide fragmentation and 10 or more energies of proton and 4He beams. The reference field is shown to well represent the charge dependence of GCR dose in several energy bins behind shielding compared to a simulated GCR environment. However, a more significant challenge for space radiobiology research is to consider chronic GCR exposure of up to 3 years in relation to simulations with animal models of human risks. We discuss issues in approaches to map important biological time scales in experimental models using ground-based simulation, with extended exposure of up to a few weeks using chronic or fractionation exposures. A kinetics model of HZE particle hit probabilities suggests that experimental simulations of several weeks will be needed to avoid high fluence rate artifacts, which places limitations on the experiments to be performed. Ultimately risk estimates are limited by theoretical understanding, and focus on improving knowledge of mechanisms and development of experimental models to improve this understanding should remain the highest priority for space radiobiology research.
doi:10.3389/fonc.2015.00122
PMCID: PMC4455530  PMID: 26090339
space radiobiology; galactic cosmic rays; cancer risk; central nervous system risk; radiation transport; shielding
14.  Modeling Gastrulation in the Chick Embryo: Formation of the Primitive Streak 
PLoS ONE  2010;5(5):e10571.
The body plan of all higher organisms develops during gastrulation. Gastrulation results from the integration of cell proliferation, differentiation and migration of thousands of cells. In the chick embryo gastrulation starts with the formation of the primitive streak, the site of invagination of mesoderm and endoderm cells, from cells overlaying Koller's Sickle. Streak formation is associated with large-scale cell flows that carry the mesoderm cells overlying Koller's sickle into the central midline region of the embryo. We use multi-cell computer simulations to investigate possible mechanisms underlying the formation of the primitive streak in the chick embryo. Our simulations suggest that the formation of the primitive streak employs chemotactic movement of a subpopulation of streak cells, as well as differential adhesion between the mesoderm cells and the other cells in the epiblast. Both chemo-attraction and chemo-repulsion between various combinations of cell types can create a streak. However, only one combination successfully reproduces experimental observations of the manner in which two streaks in the same embryo interact. This finding supports a mechanism in which streak tip cells produce a diffusible morphogen which repels cells in the surrounding epiblast. On the other hand, chemotactic interaction alone does not reproduce the experimental observation that the large-scale vortical cell flows develop simultaneously with streak initiation. In our model the formation of large scale cell flows requires an additional mechanism that coordinates and aligns the motion of neighboring cells.
doi:10.1371/journal.pone.0010571
PMCID: PMC2868022  PMID: 20485500
15.  Radiation-quality-dependent bystander effects induced by the microbeams with different radiation sources 
Journal of Radiation Research  2014;55(Suppl 1):i54.
A central paradigm in radiation biology has been that only cells ‘hit’ by a track of radiation would be affected to induce radiobiological consequences, and cells ‘not hit’ should not be. This is the basis of the current system for risk estimation of radiobiological effects. However, it has recently been challenged by so-called non-targeted effects, such as bystander effect, and such radiation-induced cellular responses may have important implications for risk evaluation of low-dose-rate radiations as well as in tumor radiotherapy. Our group has been studying radiation-quality bystander cellular effects using the microbeams with different radiation sources.
It is essentially important for evaluating risk such a low-dose-rate exposure as the accident of Fukushima Daiichi Nuclear Power Plants to examine bystander effects induced by low-LET electromagnetic radiations, such as X or gamma rays. We have been studying the cellular responses in normal human fibroblasts by targeted cell nucleus irradiations with monochromatic X-ray microbeams (5.35 keV) produced by Photon Factory in High Energy Accelerator Research Organization. The results indicated that the bystander effect in cell- killing effect was observed in the targeted cell nucleus irradiation, not in the random irradiation containing both cell nucleus and cytoplasm by Poisson distribution. The results suggest that energy deposition in cytoplasm is an important role of inducing bystander effects in case of low-LET radiations.
We have also been investigating high-LET-radiation induced bystander effects using the heavy-ion microbeams at Takasaki Ion Accelerators for Advanced Radiation Application in Japan Atomic Energy Agency. Only 0.04% of the total numbers of normal human fibroblasts were irradiated with C-ion (220 MeV), Ne-ion (260 MeV) and Ar-ion (460 MeV) microbeams collimated at 20 μm in diameter. Cell-killing effect and gene mutation at HPRT locus in the cells irradiated with C ions were higher beyond our expectations and returned the estimated values that only 0.04% of the total cells were irradiated when using the specific inhibitor of gap junctions. On the other hand, no induced biological effects were observed in Ne and Ar ions whether the inhibitor was applied or not. The result suggested that the C-ion microbeam was capable of inducing bystander cellular effects via gap junction-mediated cell-cell communication. There is clear evidence that bystander cellular effects are dependent on radiation quality.
It is also important for highly developed heavy-ion radiotherapy to identify bystander effects induced by spatially low-fluence irradiations with heavy-ion beams. We have been investigating the biological effects using human tumor cell lines. The results clearly showed that bystander effects were observed in the carbon-ion irradiation but not in other ions as well as the effects in normal fibroblasts. Furthermore, the bystander cell-killing effect in tumor cell lines was strongly induced in the cells harboring wild-type P53 not in mutated-type P53 cells. The results provide the important implication for a tailor-made therapy using carbon ions.
doi:10.1093/jrr/rrt219
PMCID: PMC3941530
Bystander effect; Microbeam; Gap junction, P53, HPRT
16.  Energy deposition by heavy ions: Additivity of kinetic and potential energy contributions in hillock formation on CaF2 
Scientific Reports  2014;4:5742.
Modification of surface and bulk properties of solids by irradiation with ion beams is a widely used technique with many applications in material science. In this study, we show that nano-hillocks on CaF2 crystal surfaces can be formed by individual impact of medium energy (3 and 5 MeV) highly charged ions (Xe22+ to Xe30+) as well as swift (kinetic energies between 12 and 58 MeV) heavy xenon ions. For very slow highly charged ions the appearance of hillocks is known to be linked to a threshold in potential energy (Ep) while for swift heavy ions a minimum electronic energy loss per unit length (Se) is necessary. With our results we bridge the gap between these two extreme cases and demonstrate, that with increasing energy deposition via Se the Ep-threshold for hillock production can be lowered substantially. Surprisingly, both mechanisms of energy deposition in the target surface seem to contribute in an additive way, which can be visualized in a phase diagram. We show that the inelastic thermal spike model, originally developed to describe such material modifications for swift heavy ions, can be extended to the case where both kinetic and potential energies are deposited into the surface.
doi:10.1038/srep05742
PMCID: PMC4102904  PMID: 25034006
17.  Dependence of the yields of AP sites and AP clusters produced in plasmid DNA on scavenging capacity and LET 
Journal of Radiation Research  2014;55(Suppl 1):i15-i16.
An apurinic or apyrimidinic site (AP site) has known to be one of the typical DNA lesions induced by ionizing irradiation to cells. A clustered DNA damage site composed of AP sites (AP clusters) can be visualized as a double-strand break (DSB) by a treatment of DNA sample with endonuclease IV proteins as an enzymatic probe. AP clusters are efficiently induced in human cells by low LET γ- and X-irradiation [ 1] with the similar yields with those for the clusters which contain pyrimidine or purine base lesions. However, there has been very little knowledge of the mechanistic aspects of the production of AP sites and AP clusters. Recently, we reported the dependence of the yields of AP sites and AP clusters, as well as base lesions, strand breaks and base lesion clusters induced by irradiation of carbon ions (LET: 13 and 60 keV/μm) obtained from HIMAC (NIRS, Chiba, Japan) on radical scavenging capacity in the DNA samples [ 2].
In the present study, we have performed theoretical calculation for the production of AP sites as well as those of strand breaks and base lesions by the carbon ion exposure (13 keV/μm). A Monte Carlo track structure simulation code for ion tracks, TRACION, was used for this work according to our previous study [ 3]. As a DNA model molecule, simple linear DNA with 150 bp was applied for the calculation. Several scavenging capacities of the samples are tested to estimate the effect of indirect action of diffusible OH radicals in the damage induction in the experimental conditions.
We assumed that the AP site formation pathway originates from a common intermediate of an OH radical adduct with that of single-strand breaks (SSBs) or base lesions (Scheme 1). In order to reproduce the experimentally obtained yields of AP sites, we newly determined the branching ratio of induction of AP site to that of SSBs or base lesions to be 17:33 or 2:48, respectively, in the simulation. The calculated yields of AP site, as well as SSB, were well consistent with those for experimentally obtained (Fig. 1). However, the calculated yields for AP clusters were one-fifth of those for experimental data. The yields for DSBs in lower scavenging capacities were also one-fifth of those for experimental ones in a lower scavenging capacity, thought they were well consistent with each other above a cell mimetic condition (>3 × 108 s). In order to bridge a gap between theoretical and experimental yields, we need a novel mechanism of damage-clustering such as dissociative low energy electron attachment or hole-migration onto a DNA molecule. Fig. 1.Dependence of the yield of AP site or AP cluster (A) and prompt SSB or prompt DSB (B) on scavenging capacity in the sample. The experimental data below a scavenging capacity of 109 s are cited from [ 2] and those at 1010 s in (B) are from [ 4]. Scheme 1.Reactions of DNA with an OH radical and the branching ratios among the damage induction pathways.
doi:10.1093/jrr/rrt160
PMCID: PMC3941554
AP site; track structure; Monte Carlo simulation; carbon ion beam
18.  Quantum dynamics in continuum for proton transport II: Variational solvent-solute interface 
Proton transport plays an important role in biological energy transduction and sensory systems. Therefore it has attracted much attention in biological science and biomedical engineering in the past few decades. The present work proposes a multiscale/multiphysics model for the understanding of the molecular mechanism of proton transport in transmembrane proteins involving continuum, atomic and quantum descriptions, assisted with the evolution, formation and visualization of membrane channel surfaces. We describe proton dynamics quantum mechanically via a new density functional theory based on the Boltzmann statistics, while implicitly model numerous solvent molecules as a dielectric continuum to reduce the number of degrees of freedom. The density of all other ions in the solvent is assumed to obey the Boltzmann distribution in a dynamic manner. The impact of protein molecular structure and its charge polarization on the proton transport is considered explicitly at the atomic scale. A variational solute-solvent interface is designed to separate the explicit molecule and implicit solvent regions. We formulate a total free energy functional to put proton kinetic and potential energies, the free energy of all other ions, the polar and nonpolar energies of the whole system on an equal footing. The variational principle is employed to derive coupled governing equations for the proton transport system. Generalized Laplace-Beltrami equation, generalized Poisson-Boltzmann equation and generalized Kohn-Sham equation are obtained from the present variational framework. The variational solvent-solute interface is generated and visualized to facilitate the multiscale discrete/continuum/quantum descriptions. Theoretical formulations for the proton density and conductance are constructed based on fundamental laws of physics. A number of mathematical algorithms, including the Dirichlet to Neumann mapping (DNM), matched interface and boundary (MIB) method, Gummel iteration, and Krylov space techniques are utilized to implement the proposed model in a computationally efficient manner. The Gramicidin A (GA) channel is used to validate the performance of the proposed proton transport model and demonstrate the efficiency of the proposed mathematical algorithms. The proton channel conductances are studied over a number of applied voltages and reference concentrations. A comparison with experimental data verifies the present model predictions and confirms the proposed model.
doi:10.1002/cnm.1458
PMCID: PMC3274368  PMID: 22328970
Proton transport; Quantum dynamics in continuum; Multiscale model; Laplace-Beltrami equation; Poisson-Boltzmann equation; Kohn-Sham equation; Variational principle
19.  Suppression of E. multilocularis Hydatid Cysts after Ionizing Radiation Exposure 
Background
Heavy-ion therapy has an advantage over conventional radiotherapy due to its superb biological effectiveness and dose conformity in cancer therapy. It could be a potential alternate approach for hydatid cyst treatment. However, there is no information currently available on the cellular and molecular basis for heavy-ion irradiation induced cell death in cystic echinococcosis.
Methododology/Principal Findings
LD50 was scored by protoscolex death. Cellular and ultrastructural changes within the parasite were studied by light and electron microscopy, mitochondrial DNA (mtDNA) damage and copy number were measured by QPCR, and apoptosis was determined by caspase 3 expression and caspase 3 activity. Ionizing radiation induced sparse cytoplasm, disorganized and clumped organelles, large vacuoles and devoid of villi. The initial mtDNA damage caused by ionizing radiation increased in a dose-dependent manner. The kinetic of DNA repair was slower after carbon-ion radiation than that after X-rays radiation. High dose carbon-ion radiation caused irreversible mtDNA degradation. Cysts apoptosis was pronounced after radiation. Carbon-ion radiation was more effective to suppress hydatid cysts than X-rays.
Conclusions
These studies provide a framework to the evaluation of attenuation effect of heavy-ion radiation on cystic echinococcosis in vitro. Carbon-ion radiation is more effective to suppress E. multilocularis than X-rays.
Author Summary
Surgical removal of cysts may be impractical in cases that cysts are in multiple organs or tissues, or in risky locations. In that case, alternative treatment should be employed. Heavy-ion radiation could be an effective way for treatment of hydatid cysts, taking its full advantage of well-defined range, small lateral beam spread and an enhanced biological effectiveness. In this study, we found that carbon-ion radiation could result in extensive mitochondrial DNA damage and apoptosis in hydatid cysts. Cellular and ultrastructural changes were observed after ionizing radiation, which were indicative of cysts growth inhibition. To our knowledge, this is the first study reporting the biological effect of carbon-ion radiation on E. multilocularis hydatid cysts.
doi:10.1371/journal.pntd.0002518
PMCID: PMC3812096  PMID: 24205427
20.  Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector 
Journal of Radiation Research  2014;56(2):360-365.
The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080–53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments.
doi:10.1093/jrr/rru091
PMCID: PMC4380042  PMID: 25324538
fluorescent nuclear track detector; ion beam tracks; DNA damage; localization
21.  Misrepair of DNA double-strand breaks after exposure to heavy-ion beams causes a peak in the LET–RBE relationship with respect to cell killing in DT40 cells 
Journal of Radiation Research  2013;54(6):1029-1035.
To determine the radiobiological mechanisms underlying relative biological effectiveness (RBE) and the repair efficiencies of DNA double-strand breaks (DSBs) as a function of linear energy transfer (LET), we exposed cells of the chicken B-lymphocyte cell line DT40 and its DSB repair pathway-deficient derivatives to heavy-ion beams produced at the Heavy-Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS), Chiba, Japan. The relationship between LET and cell lethality was investigated in the DNA DSB repair gene knockouts Ku70−/−, Rad54−/−, and Ku70−/−Rad54−/−, and in the wild-type cells. We found that cell-cycle stage and activity of the DNA DSB repair pathways influence LET-mediated biological effects. An expected LET–RBE relationship was observed in the cells capable of DNA repair, but no peak was found in the RBE with respect to cell survival in the Ku70−/−Rad54−/− cells or in Ku70−/− cells in the G1 and early S cell-cycle phases (when no sister chromatids were present and homologous recombination could not occur). These findings suggest that the peak in RBE is caused by deficient repair of the DNA DSBs.
doi:10.1093/jrr/rrt064
PMCID: PMC3823785  PMID: 23722078
linear energy transfer; relative biological effectiveness; sensitivity; misrepair; heavy ion
22.  Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation 
Current Genomics  2012;13(6):418-425.
The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.
doi:10.2174/138920212802510501
PMCID: PMC3426775  PMID: 23450137
DNA double strand breaks; Linear energy transfer; Radiation; γ-H2AX foci.
23.  Charge Transfer in Dynamical Biosystems, or The Treachery of (Static) Images 
Accounts of Chemical Research  2014;48(2):474-481.
Conspectus
The image is not the thing. Just as a pipe rendered in an oil painting cannot be smoked, quantum mechanical coupling pathways rendered on LCDs do not convey electrons. The aim of this Account is to examine some of our recent discoveries regarding biological electron transfer (ET) and transport mechanisms that emerge when one moves beyond treacherous static views to dynamical frameworks.
Studies over the last two decades introduced both atomistic detail and macromolecule dynamics to the description of biological ET. The first model to move beyond the structureless square-barrier tunneling description is the Pathway model, which predicts how protein secondary motifs and folding-induced through-bond and through-space tunneling gaps influence kinetics. Explicit electronic structure theory is applied routinely now to elucidate ET mechanisms, to capture pathway interferences, and to treat redox cofactor electronic structure effects. Importantly, structural sampling of proteins provides an understanding of how dynamics may change the mechanisms of biological ET, as ET rates are exponentially sensitive to structure. Does protein motion average out tunneling pathways? Do conformational fluctuations gate biological ET? Are transient multistate resonances produced by energy gap fluctuations? These questions are becoming accessible as the static view of biological ET recedes and dynamical viewpoints take center stage.
This Account introduces ET reactions at the core of bioenergetics, summarizes our team’s progress toward arriving at an atomistic-level description, examines how thermal fluctuations influence ET, presents metrics that characterize dynamical effects on ET, and discusses applications in very long (micrometer scale) bacterial nanowires. The persistence of structural effects on the ET rates in the face of thermal fluctuations is considered. Finally, the flickering resonance (FR) view of charge transfer is presented to examine how fluctuations control low-barrier transport among multiple groups in van der Waals contact. FR produces exponential distance dependence in the absence of tunneling; the exponential character emerges from the probability of matching multiple vibronically broadened electronic energies within a tolerance defined by the rms coupling among interacting groups. FR thus produces band like coherent transport on the nanometer length scale, enabled by conformational fluctuations. Taken as a whole, the emerging context for ET in dynamical biomolecules provides a robust framework to design and interpret the inner workings of bioenergetics from the molecular to the cellular scale and beyond, with applications in biomedicine, biocatalysis, and energy science.
doi:10.1021/ar500271d
PMCID: PMC4333612  PMID: 25307316
24.  Quantitative analysis of the ion-dependent folding stability of DNA triplexes 
Physical biology  2011;8(6):066006.
A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion-dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-bp DNA triplex and a pair of interacting 14-bp triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann Equation (PB). The improvement is more significant for a triplex, which has a higher charge density, than a duplex. This is possibly due to the higher ion concentration around the triplex and hence stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induced an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.
doi:10.1088/1478-3975/8/6/066006
PMCID: PMC3427753  PMID: 22067830
folding stability; energy landscape; Triplex; TBI model
25.  Clustering phenotype populations by genome-wide RNAi and multiparametric imaging 
How to predict gene function from phenotypic cues is a longstanding question in biology.Using quantitative multiparametric imaging, RNAi-mediated cell phenotypes were measured on a genome-wide scale.On the basis of phenotypic ‘neighbourhoods', we identified previously uncharacterized human genes as mediators of the DNA damage response pathway and the maintenance of genomic integrity.The phenotypic map is provided as an online resource at http://www.cellmorph.org for discovering further functional relationships for a broad spectrum of biological module
Genetic screens for phenotypic similarity have made key contributions for associating genes with biological processes. Aggregating genes by similarity of their loss-of-function phenotype has provided insights into signalling pathways that have a conserved function from Drosophila to human (Nusslein-Volhard and Wieschaus, 1980; Bier, 2005). Complex visual phenotypes, such as defects in pattern formation during development, greatly facilitated the classification of genes into pathways, and phenotypic similarities in many cases predicted molecular relationships. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cultured cells has become feasible in many organisms whose genome have been sequenced (Boutros and Ahringer, 2008). One of the current challenges is the computational categorization of visual phenotypes and the prediction of gene function and associated biological processes. With large parts of the genome still being in unchartered territory, deriving functional information from large-scale phenotype analysis promises to uncover novel gene–gene relationships and to generate functional maps to explore cellular processes.
In this study, we developed an automated approach using RNAi-mediated cell phenotypes, multiparametric imaging and computational modelling to obtain functional information on previously uncharacterized genes. To generate broad, computer-readable phenotypic signatures, we measured the effect of RNAi-mediated knockdowns on changes of cell morphology in human cells on a genome-wide scale. First, the several million cells were stained for nuclear and cytoskeletal markers and then imaged using automated microscopy. On the basis of fluorescent markers, we established an automated image analysis to classify individual cells (Figure 1A). After cell segmentation for determining nuclei and cell boundaries (Figure 1C), we computed 51 cell descriptors that quantified intensities, shape characteristics and texture (Figure 1F). Individual cells were categorized into 1 of 10 classes, which included cells showing protrusion/elongation, cells in metaphase, large cells, condensed cells, cells with lamellipodia and cellular debris (Figure 1D and E). Each siRNA knockdown was summarized by a phenotypic profile and differences between RNAi knockdowns were quantified by the similarity between phenotypic profiles. We termed the vector of scores a phenoprint (Figure 3C) and defined the phenotypic distance between a pair of perturbations as the distance between their corresponding phenoprints.
To visualize the distribution of all phenoprints, we plotted them in a genome-wide map as a two-dimensional representation of the phenotypic similarity relationships (Figure 3A). The complete data set and an interactive version of the phenotypic map are available at http://www.cellmorph.org. The map identified phenotypic ‘neighbourhoods', which are characterized by cells with lamellipodia (WNK3, ANXA4), cells with prominent actin fibres (ODF2, SOD3), abundance of large cells (CA14), many elongated cells (SH2B2, ELMO2), decrease in cell number (TPX2, COPB1, COPA), increase in number of cells in metaphase (BLR1, CIB2) and combinations of phenotypes such as presence of large cells with protrusions and bright nuclei (PTPRZ1, RRM1; Figure 3B).
To test whether phenotypic similarity might serve as a predictor of gene function, we focused our further analysis on two clusters that contained genes associated with the DNA damage response (DDR) and genomic integrity (Figure 3A and C). The first phenotypic cluster included proteins with kinetochore-associated functions such as NUF2 (Figure 3B) and SGOL1. It also contained the centrosomal protein CEP164 that has been described as an important mediator of the DNA damage-activated signalling cascade (Sivasubramaniam et al, 2008) and the largely uncharacterized genes DONSON and SON. A second phenotypically distinct cluster included previously described components of the DDR pathway such as RRM1 (Figure 3A–C), CLSPN, PRIM2 and SETD8. Furthermore, this cluster contained the poorly characterized genes CADM1 and CD3EAP.
Cells activate a signalling cascade in response to DNA damage induced by exogenous and endogenous factors. Central are the kinases ATM and ATR as they serve as sensors of DNA damage and activators of further downstream kinases (Harper and Elledge, 2007; Cimprich and Cortez, 2008). To investigate whether DONSON, SON, CADM1 and CD3EAP, which were found in phenotypic ‘neighbourhoods' to known DDR components, have a role in the DNA damage signalling pathway, we tested the effect of their depletion on the DDR on γ irradiation. As indicated by reduced CHEK1 phosphorylation, siRNA knock down of DONSON, SON, CD3EAP or CADM1 resulted in impaired DDR signalling on γ irradiation. Furthermore, knock down of DONSON or SON reduced phosphorylation of downstream effectors such as NBS1, CHEK1 and the histone variant H2AX on UVC irradiation. DONSON depletion also impaired recruitment of RPA2 onto chromatin and SON knockdown reduced RPA2 phosphorylation indicating that DONSON and SON presumably act downstream of the activation of ATM. In agreement to their phenotypic profile, these results suggest that DONSON, SON, CADM1 and CD3EAP are important mediators of the DDR. Further experiments demonstrated that they are also required for the maintenance of genomic integrity.
In summary, we show that genes with similar phenotypic profiles tend to share similar functions. The power of our computational and experimental approach is demonstrated by the identification of novel signalling regulators whose phenotypic profiles were found in proximity to known biological modules. Therefore, we believe that such phenotypic maps can serve as a resource for functional discovery and characterization of unknown genes. Furthermore, such approaches are also applicable for other perturbation reagents, such as small molecules in drug discovery and development. One could also envision combined maps that contain both siRNAs and small molecules to predict target–small molecule relationships and potential side effects.
Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.
doi:10.1038/msb.2010.25
PMCID: PMC2913390  PMID: 20531400
DNA damage response signalling; massively parallel phenotyping; phenotype networks; RNAi screening

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