Trap Mutation Research Articles

The thermodynamic and kinetic interactions of He interstitial clusters with bubbles in W

Due to its enviable properties, tungsten is a leading candidate plasma facing material in nuclear fusion reactors. However, like many other metals, tungsten is known to be affected by the high doses of helium atoms incoming from the plasma. Indeed, the implanted interstitial helium atoms cluster together and, upon reaching a critical cluster size, convert into substitutional nanoscale He bubbles. These bubbles then grow by absorbing further interstitial clusters from the matrix. This process can lead to deleterious changes in microstructure, degradation of mechanical properties, and contamination of the plasma. In order to better understand the growth process, we use traditional and accelerated molecular dynamics simulations to investigate the interactions between interstitial He clusters and pre-existing bubbles. These interactions are characterized in terms of thermodynamics and kinetics. We show that the proximity of the bubble leads to an enhancement of the trap mutation rate and, consequently, to the nucleation of satellite bubbles in the neighborhood of existing ones. We also uncover a number of mechanisms that can lead to the subsequent annihilation of such satellite nanobubbles.

A W[sbnd]Ne interatomic potential for simulation of neon implantation in tungsten

An interatomic pair potential for WNe is developed for atomistic molecular dynamics simulations of neon implantation in tungsten. The new potential predicts point defect energies and binding energies of small clusters that are in good agreement with electronic structure calculations. Molecular dynamics simulations of small neon clusters in tungsten show that trap mutation, in which an interstitial neon cluster displaces a tungsten atom from its lattice site, occurs for clusters of three or more neon atoms. However, near a free surface, trap mutation can occur at smaller sizes, including even a single neon interstitial in close proximity to a (100) or (110) surface.

Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.

In situ Observation of Microstructure Evolution in 4H–SiC under 3.5 keV He+ Irradiation

4H–SiC was irradiated with 3.5 keV He+ ions using the MIAMI facility at University of Huddersfield. The evolution of microstructure and gas bubbles during the irradiation at 700 °C, 800 °C and 900 °C was observed by in situ transmission electron microscopy. Under irradiation, isolated bubbles and bubble discs formed in the SiC matrix. Bubble discs lying on {0001} and {10-10} crystal planes were beginning to form at ion fluence above 2.3 × 1020 He+/m2 at 700 °C. The density of bubble discs increased with increasing irradiation fluence. However, growth rates were different at different of the implantation periods and temperature holding periods. The nucleation and growth of the bubble discs were attributed to be coalescence of the adjacent He vacancies and combination of loop punching and trap mutation, respectively.

Helium segregation on surfaces of plasma-exposed tungsten

We report a hierarchical multi-scale modeling study of implanted helium segregation on surfaces of tungsten, considered as a plasma facing component in nuclear fusion reactors. We employ a hierarchy of atomic-scale simulations based on a reliable interatomic interaction potential, including molecular-statics simulations to understand the origin of helium surface segregation, targeted molecular-dynamics (MD) simulations of near-surface cluster reactions, and large-scale MD simulations of implanted helium evolution in plasma-exposed tungsten. We find that small, mobile Hen (1 ⩽ n ⩽ 7) clusters in the near-surface region are attracted to the surface due to an elastic interaction force that provides the thermodynamic driving force for surface segregation. This elastic interaction force induces drift fluxes of these mobile Hen clusters, which increase substantially as the migrating clusters approach the surface, facilitating helium segregation on the surface. Moreover, the clusters’ drift toward the surface enables cluster reactions, most importantly trap mutation, in the near-surface region at rates much higher than in the bulk material. These near-surface cluster dynamics have significant effects on the surface morphology, near-surface defect structures, and the amount of helium retained in the material upon plasma exposure. We integrate the findings of such atomic-scale simulations into a properly parameterized and validated spatially dependent, continuum-scale reaction-diffusion cluster dynamics model, capable of predicting implanted helium evolution, surface segregation, and its near-surface effects in tungsten. This cluster-dynamics model sets the stage for development of fully atomistically informed coarse-grained models for computationally efficient simulation predictions of helium surface segregation, as well as helium retention and surface morphological evolution, toward optimal design of plasma facing components.

Molecular-dynamics analysis of mobile helium cluster reactions near surfaces of plasma-exposed tungsten

We report the results of a systematic atomic-scale analysis of the reactions of small mobile helium clusters (Hen, 4 ≤ n ≤ 7) near low-Miller-index tungsten (W) surfaces, aiming at a fundamental understanding of the near-surface dynamics of helium-carrying species in plasma-exposed tungsten. These small mobile helium clusters are attracted to the surface and migrate to the surface by Fickian diffusion and drift due to the thermodynamic driving force for surface segregation. As the clusters migrate toward the surface, trap mutation (TM) and cluster dissociation reactions are activated at rates higher than in the bulk. TM produces W adatoms and immobile complexes of helium clusters surrounding W vacancies located within the lattice planes at a short distance from the surface. These reactions are identified and characterized in detail based on the analysis of a large number of molecular-dynamics trajectories for each such mobile cluster near W(100), W(110), and W(111) surfaces. TM is found to be the dominant cluster reaction for all cluster and surface combinations, except for the He4 and He5 clusters near W(100) where cluster partial dissociation following TM dominates. We find that there exists a critical cluster size, n = 4 near W(100) and W(111) and n = 5 near W(110), beyond which the formation of multiple W adatoms and vacancies in the TM reactions is observed. The identified cluster reactions are responsible for important structural, morphological, and compositional features in the plasma-exposed tungsten, including surface adatom populations, near-surface immobile helium-vacancy complexes, and retained helium content, which are expected to influence the amount of hydrogen re-cycling and tritium retention in fusion tokamaks.

Energetics and kinetics unveiled on helium cluster growth in tungsten

The energetics and kinetics regarding helium (He) cluster growth in bcc tungsten (W) are unveiled using combined techniques of molecular statics and molecular dynamics. The principal mechanisms accounting for the decrease of system potential energy are identified to be trap mutation, → cluster transformation, loop punching, coalescence between 1/2[1 1-1] and 1/2[1-1-1] loops, and loop capturing. The kinetic barriers associated with these key atomistic events are estimated. This work provides new insights into the complex yet intriguing atomistic evolution sequence of the He cluster and interstitial loop in W-based nuclear fusion materials under irradiation.

Effects of temperature and surface orientation on migration behaviours of helium atoms near tungsten surfaces

Molecular dynamics simulations were performed to study the dependence of migration behaviours of single helium atoms near tungsten surfaces on the surface orientation and temperature. For W{100} and W{110} surfaces, He atoms can quickly escape out near the surface without accumulation even at a temperature of 400 K. The behaviours of helium atoms can be well-described by the theory of continuous diffusion of particles in a semi-infinite medium. For a W{111} surface, the situation is complex. Different types of trap mutations occur within the neighbouring region of the W{111} surface. The trap mutations hinder the escape of He atoms, resulting in their accumulation. The probability of a He atom escaping into vacuum from a trap mutation depends on the type of the trap mutation, and the occurrence probabilities of the different types of trap mutations are dependent on the temperature. This finding suggests that the escape rate of He atoms on the W{111} surface does not show a monotonic dependence on temperature. For instance, the escape rate at T = 1500 K is lower than the rate at T = 1100 K. Our results are useful for understanding the structural evolution and He release on tungsten surfaces and for designing models in other simulation methods beyond molecular dynamics.

Loss of preimplantation embryo resulting from a Pum1 gene trap mutation

Pumilio is a member of the highly conserved PUF family of RNA-binding proteins that function as a developmental regulator in diverse animal species. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals and are found to be involved in sperm development, neuron development as well as human diseases such as neurodegeneration. Generation of animal models disrupting different parts of Pum protein could help to further dissect their physiological function. Here we described characterization and analysis of a mouse line possessing a gene trap mutation of the Pumilio1 (Pum1) gene. Mice homozygous for the mutation (Pum1XE002) cannot be recovered in the adult offspring, at birth or at different time points of embryonic development (E18, E14, E12). Careful analysis of preimplantation embryos showed that no homozygous blastocysts could be detected on day 3.5 of gestation. 96-hr in vitro culture of 1-cell embryos either by natural mating or in vitro fertilization between heterozygotes failed to uncover any homozygous blastocysts, suggesting an early loss of homozygous preimplantation embryos. The lack of Pum1 gene trap homozygotes suggests a role of Pum1 in very early embryonic development or fertilization. This novel animal model affecting the beginning of embryonic development could help to understand not only the genetic mechanism underlying preimplantation embryonic development but also the translational regulation in development and diseases.

Modeling of helium bubble nucleation and growth in austenitic stainless steels using an Object Kinetic Monte Carlo method

Implantation of 10keV helium in 316L steel thin foils was performed in JANNuS-Orsay facility and modeled using a multiscale approach. Density Functional Theory (DFT) atomistic calculations [1] were used to obtain the properties of He and He-vacancy clusters, and the Binary Collision Approximation based code MARLOWE was applied to determine the damage and He-ion depth profiles as in [2,3]. The processes involved in the homogeneous He bubble nucleation and growth were defined and implemented in the Object Kinetic Monte Carlo code LAKIMOCA [4]. In particular as the He to dpa ratio was high, self-trapping of He clusters and the trap mutation of He-vacancy clusters had to be taken into account. With this multiscale approach, the formation of bubbles was modeled up to nanometer-scale size, where bubbles can be observed by Transmission Electron Microscopy. Their densities and sizes were studied as functions of fluence (up to 5×1019He/m2) at two temperatures (473 and 723K) and for different sample thicknesses (25–250nm). It appears that the damage is not only due to the collision cascades but is also strongly controlled by the He accumulation in pressurized bubbles. Comparison with experimental data is discussed and sensible agreement is achieved.

Challenges and opportunities of modeling plasma–surface interactions in tungsten using high-performance computing

The performance of plasma facing components (PFCs) is critical for ITER and future magnetic fusion reactors. The ITER divertor will be tungsten, which is the primary candidate material for future reactors. Recent experiments involving tungsten exposure to low-energy helium plasmas reveal significant surface modification, including the growth of nanometer-scale tendrils of “fuzz” and formation of nanometer-sized bubbles in the near-surface region. The large span of spatial and temporal scales governing plasma surface interactions are among the challenges to modeling divertor performance. Fortunately, recent innovations in computational modeling, increasingly powerful high-performance computers, and improved experimental characterization tools provide a path toward self-consistent, experimentally validated models of PFC and divertor performance. Recent advances in understanding tungsten–helium interactions are reviewed, including such processes as helium clustering, which serve as nuclei for gas bubbles; and trap mutation, dislocation loop punching and bubble bursting; which together initiate surface morphological modification.

Modeling of the self trapping of helium and the trap mutation in tungsten using DFT and empirical potentials based on DFT

Abstract

TRAP mutations and immunology

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Modelling self trapping and trap mutation in tungsten using DFT and Molecular Dynamics with an empirical potential based on DFT

Density Functional Theory calculations and Molecular Dynamics with a recently developed potential for W–He were used to evaluate the thermal stability of helium–vacancy clusters (nHe.mv) as well as pure interstitial helium clusters in tungsten. The stability of such objects results from a competitive process between thermal emission of vacancies, self interstitial atoms (SIAs) and helium, depending on the helium-to-vacancy ratio in mixed clusters or helium number in pure interstitial helium clusters. We investigated in particular the thermodynamics and kinetics of self trapping and trap mutation, i.e. the emission of one SIA along with the creation of one vacancy from a vacancy–helium or pure helium object.

P53-Independent Cell Cycle and Erythroid Differentiation Defects in Murine Embryonic Stem Cells Haploinsufficient for Diamond Blackfan Anemia-Proteins: RPS19 versus RPL5

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression; however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.

Enhanced modification of tungsten surface by nanostructure formation during high flux deuterium plasma exposure

The morphology changes induced by low energy deuterium ions on tungsten surfaces is studied for particle fluxes similar to those expected in the ITER divertor, as high as 1024m−2s−1. A new type of surface modifications by formation of nanostructures is observed to occur under those conditions. The nanostructures formation is critically dependent on the particle flux and ion energy, and affected by surface temperature and particle fluence. It correlates with the formation of nano-cavities at a depth of about 40nm below the surface. In addition, the positron annihilation Doppler broadening (PADB) measurements reveal a significant increase of defect concentration (mostly vacancy-type) after plasma exposure, and the evolution of defects is in line with the surface morphology changes with fluence and surface temperature. The possible mechanism of cyclic trap mutation and dislocation loop punching due to high flux D exposure is discussed.

Primitive Erythropoiesis and Osteogenesis Are Differentially Impaired In Rpl5 and Rps19 Mutant Murine Embryonic Stem Cell Models Of Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

TDAG8 activation inhibits osteoclastic bone resorption

Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis.

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.