Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0+/-0.5 degrees C during the 16h day with the light intensity of 7.5klx from white fluorescent tubes, and 20.0+/-0.5 degrees C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56+/-0.03 PMEs per 10(4) hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.     

Further yearly analyses of spontaneous pink mutant events in the stamen hairs of tradescantia clone BNL 4430 cultivated in the NSC growth chamber

In order to confirm the results obtained in the previous 1-year-term (December 12, 1998, through December 10, 1999) scorings and analyses of spontaneous pink mutant events (PMEs) in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating (NSC) growth chamber, similar scorings and analyses were continued for another 52-week period from December 11, 1999, through December 8, 2000. The environmental conditions were not changed, except for a minor modification in the method of supplying the nutrient solution used. During the scoring period, 732,128 stamen hairs with an average cell number of 24.90 cells were observed, and 2,368 PMEs were detected. The overall spontaneous somatic mutation frequency was 1.35 +/- 0.03 PMEs per 10(4) hair-cell divisions, which was significantly lower than the value of 1.56 +/- 0.03 determined in the previous 52-week period, and the frequencies were lower during April through September than in other months, the period showing lower frequencies lasting 1-month longer than in the previous year. The present results reconfirmed the occurrence of a clear seasonal variation in the spontaneous mutation frequency in the NSC growth chamber, and the lower overall frequency, probably related to the minor modification in supplying the nutrient solution, is helpful for conducting mutagenicity tests at low levels, offering a lower background level. The analyses of the sectoring patterns of all these PMEs showed that the most of the 203 cases of multiple (two to five) pink sectors observed in the same stamen hairs (scored as 253 PMEs for calculating mutation frequency) were the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells, agreeing with our previous study, and the significance of somatic recombinations in causing single PMEs was also reconfirmed.     

Responses of Tradescantia stamen hairs and pollen to UV-B irradiation

Clones 02 and 4430 of Tradescantia were tested in field, greenhouse and controlled environment chambers as monitors for the potentially hazardous UV-B irradiation increase that could result from stratospheric ozone decrease. In addition to about 16 hr of solar emissions at about 2100 micro-einsteins·m⁻²·s⁻¹ (400–700 nm) and 15 hr at about 1800 micro-einsteins·m⁻²·s⁻¹ in the field and greenhouse, respectively, plants were given 7 hr of supplemental UV-B irradiation per day for 27 days. After the first 7 days of UV-B irradiation exposure, cumulative data were recorded for 20 days. Cuttings of Tradescantia plants in controlled-environment, exposed to 16 hr of simulated solar emission of about 800 micro-einsteins·m⁻²·s⁻¹ (400–700 nm), were also exposed to 10 hr of supplemental UV-B irradiation per day for 1 or 2 days. All plants were checked for somatic aberrations (color changes in the flower petals and stamen hairs), number of hairs per stamen, and cells per hair. Pollen germination and pollen tube growth were noted after a 90-min UV-B irradiation period.Somatic aberrations occurred infrequently in the petals and were judged unreliable criteria for use in monitoring enhanced UV-B irradiation environments. The number of aberrant events within stamen hairs, however, was significantly increased by the UV-B irradiation treatments. while pollen germination and pollen tube growth were significantly reduced. These data indicate that color changes in stamen hairs and pollen viability are useful criteria for monitoring UV-B irradiation changes.     

Resolving the enigma of multiple mutant sectors in stamen hairs of tradescantia

Mutant sectors in stamen hairs of Clone 02 Tradescantia are designated as "multiple sectors" when two or more occupy the same hair, separated by non-mutant cells. Statistical analyses show that most multiple sectors do not arise as chance associations of independent events: when the frequency of stamens with two or more sectors is lowest, the probability that the sectors will be located in the same, rather than in different, hairs is highest. Ontogenetically, the ratio of sector pairs in different hairs to pairs in the same hair is highest in that period of response to acute irradiation prior to the appearance of entire-hair sectors; thereafter, the ratio subsides, approaching that of spontaneous mutation and indicating that the initiating event takes place early in hair development. Most mononemic chromosome models will not account for the production of multiple mutant and non-mutant sectors, dispersed along a linear structure such as a stamen hair, following a single mutational event. Consideration is given to two models (one mononemic, and the other dinemic) which will readily provide the possibilities for either the immediate segregation of mutant and non-mutant cells, or for the perpetuation in daughter nuclei of a "heterozygous" chromosome capable of segregation at some later mitosis. The dinemic model is preferred because it affords operation of the mutation mechanism (including breakage and deletion) at either the DNA molecule or subunit level.     

Reduction in the frequency of N-methyl-N-nitrosourea-induced somatic mutations in Tradescantia by pretreatment with low doses of alkylating agents

Treatment of Tradescantia cuttings with sub-mutagenic doses of N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea and methyl methanesulphonate before challenging doses of MNU reduced the frequency of somatic mutations in stamen hairs as compared with the effect of challenging dose alone. The highest response was about a 50% reduction in the mutagenic effect of the challenge dose.     

Mutagenicity of ground water (in the bore-holes) in Ararat Valley (Armenia) detected by Trad-SHM bioassay

The genotoxicity of ground water from four bore-holes of different depths (40-120m) in the Ararat valley (Armenia) used both for drinking and irrigation was investigated. The frequency of recessive somatic mutations was determined using the Tradescantia-stamen-hair-mutation (Trad-SHM) test. The Tradescantia clone 02 was used. The pink mutation events (PMEs) were increased by 3.18-6.81-fold in comparison with the control depending both on the depth of subterranean water location and the increase of Na(+) ion concentration in these water samples. The peak frequency was found in water from the 40-45m depth. The deeper the bore-holes, the lower the mutagenicity of water and the concentration of Na(+) ions. Different types of mutant sector arrangements and their frequencies changed depending on the subterranean water depth.     

Influence of X-ray dose fractionation on the frequency of somatic mutations induced in tradescantia stamen hairs

X-rays were used to investigate the influence of dose fractionation on the induction of pink and colorless somatic mutations in stamen hair cells of Tradescantia clone 02. Inflorescences were exposed to a single acute dose of 60 rad, two acute doses of 30 rad, or three acute doses of 20 rad. The dose rate in all cases was 30 rad/min. Intervals between dose fractions were varied from 35 sec to 48 h and the mutation frequency was compared with that resulting after the single dose of 60 rad. The data show a reduction in mutation frequency for fractionation intervals longer than 15 and 6 min for pink and colorless mutations, respectively, but not for shorter intervals.One interpretation of the data predicts that pink mutation frequencies are reduced by 11% for fraction intervals of from 30 min to 6 h, and that colorless mutation frequencies are reduced by 24% for intervals of from 15 min to 6 h. The corresponding sparing effect of dose fractionation is equal to 6 rad for pink mutations and 9 rad at the colorless mutation endpoint. A calculation has been made which indicates that the percentages of the total repairable (presumably two-hit) damage that is repaired during fraction intervals up to 6 h, are 16 and 35% for pink and colorless mutations respectively.     

Relative biological efficiency for the induction of various gene mutations in normal and enriched with 10B Tradescantia cells by neutrons from 252Cf source

The effectiveness of neutrons from a Californium-252 source in the induction of various abnormalities in the Tradescantia clone 4430 stamen hair cells (Trad-SH assay) were studied. A special attention was paid to check whether any enhancement in effects is visible in the cells enriched with boron ions. Inflorescences, normal or pretreated with chemicals containing boron, were irradiated in the air with neutrons from a 252Cf source at KAERI, Taejon, Korea. To estimate the relative biological effectiveness (RBE) of the beam under the study, numbers of Tradescantia inflorescence without chemical pretreatment were irradiated with various doses of X-rays. The ranges of radiation doses used for neutrons were 0-1.0Gy and for X-rays 0-0.5Gy. Following the culturing according to standard procedures screening of gene and lethal mutations in somatic cells of stamen hairs was done in the extended period, between days 7 and 19 after exposures. Maximal RBE values for the induction of pink, colorless and lethal mutations were evaluated from comparison of the slopes in linear parts of the dose response curves obtained after irradiation with X-rays and californium source. The RBE(max) value or the induction of gene mutation was estimated as 7.2 comparing the value 5.6 in the studies reported earlier. The comparison of dose-response curves and its alteration, due to changes in the cells and plants environment during and after irradiation, explains the observed differences. Inflorescence pretreated with borax responded to neutrons differently depending on the biological end points. Although, for the induction of pink mutations no significant difference was observed, though, in the case of cell lethality, pretreated with boron ion plants have shoved a statistically significant increase of the RBE value from 5.5 to 34.7, and in the case of colorless mutations from 1.6 to 5.6.     

Frequency of micronuclei in root-tips of Tradescantia clone 02: Effects of diurnal temperature difference and Co gamma radiation

Frequency of micronuclei in root-tip cells was used to asses the influence and/or interaction of a large Diurnal Temperature Difference (DTD) on irradiated and non-irradiated Tradescantia Clone 02. Hydroponically grown plants were subjected to four different treatment combinations: no radiation/normal DTD (7.2°C), no radiation/large DTD (23.5°C), radiation (60R)/normal DTD, radiation/large DTD. An analysis of variance of Feulgen positive micronuclei arising from chromosome fragments in root-tip cells revealed that a 5-day chronic exposure to a large DTD without radiation does not significantly increase the frequency of cells with micronuclei (freq. = 0.53%). An accute 60 R radiation exposure, as expected, did significantly increase the frequency (freq. = 3.23%). None of the treatment interactions significantly altered micronuclei production. These data lend support to the contention that the mutation-induction mechanism of large DTD's, as expressed by variant color sectoring in stamen hair cells of this clone, is not mediated by chromosome breakage. Exposure to a large DTD, therefore, may be a method for inducing mutations in stamen hairs without concomitant chromosome breakage, and may also provide a good cytological means for analyzing somatic crossing over, which has been postulated as a possible mechanism for the expression of spontaneous mutations at the D⁺ locus. The frequency of micronuclei in irradiated tissue remained constant throughout the 5-day course of the experiments and several explanations for this occurrence are proposed.     

Mitotic crossing-over as an important mechanism of floral sectoring in tradescantia

A purplle flowered Tradescantia hirsuticaulis is described exhibiting three classes of somatic sectors: red-only, red/blue twin spots and blue-only. The twin spots are shown to be the results of a single event since the association of red and blue cells in the same stamenis non-random and intervening purple cells occur far too infrequently to permit hairs containing both red and blue sectors to arise by sequential, facilitated, red-only and blue-only events.As in T. clone 02, deletion, as evidenced by the presence of micronuclei (chromosome fragments), is indicated to be one mechanism producing some kinds of sectors (red-only) in response to ionizing radiation.The predominant, if not exclusive, mechanism of spontaneous sectoring and important mechanism of even 60 R-induced sectoring in this T. hirsuticaulis (and by inference, in other Tradescantia) is argued to be mitotic crossing-over, since other mechanisms operating on its genotype do not allow for twin spots arising as the result of one event. This is supported by the prediction of all three types of sectors in the proper order of frequency as well as by the calculation of a “coefficientof coincidence” and a radiation response that are similar to those found for mitotic crossing-over in Drosophila.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Comparative study of effect of infrared, submillimeter, and millimeter electromagnetic radiation on wing somatic mutations in Drosophila melanogaster induced by gamma-irradiation

It was shown that the number of spontaneous and gamma-radiation-induced somatic mutations in wing cells of fruit flies (third instar larvae) exposed to laser irradiation of submillimeter range (lambda = 81.5 microns) was significantly lower than in control. Laser irradiation did not affect the number of recombinations. Exposure to laser radiation in the infrared range and electromagnetic waves of the millimeter range (lambda = 3.8 mm) enhanced the effect of gamma-irradiation.     

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.     

Tradescantia bioassays for the determination of genotoxicity of water in the Panlong River, Kunming, People's Republic of China.

The Panlong river passes through Kunming City and receives a large quantity of municipal sewage and wastewater from industrial effluent. Along the river, 20 sites were selected to collect water samples to assess the genotoxicity using two Tradescantia assays, the micronucleus (Trad-MCN) and the stamen-hair-mutation (Trad-SHM) assays. The lowest frequencies in the Trad-MCN assay and the Trad-SHM assay are 3.19 MCN/100 tetrads and 1.32 M/1000 stamen hairs, respectively. In the water samples obtained from the Songhua Reservoir, the MCN frequencies and mutation rates are not statistically significantly different from the data found for the negative control (2.49 MCN/100 tetrads and 0.71 M/1000 stamen hairs). Among the other water samples, 19 in Trad-MCN assay and 17 in Trad-SHM assay show significantly higher genotoxicity than the control. The highest genotoxicity was in samples No. 19 for the MCN assay (8.73 MCN/100 cells), over three times higher than the negative control, and in sample No. 11 for the SHM assay (4.30 M/1000), six times higher than the negative control, and were about the same as for the positive control (10.0 mg/l NaN3, 9.28 MCN/100 tetrads and 7.44 SHM/1000 stamen hairs), respectively. The peak frequencies for the Trad-MCN assays were observed in the water samples obtained from the sites that were near industrial and municipal wastewater that ran into the river as effluent. In general, the frequency of MCN and SHM mutations increased where the river passed through Kunming. The Trad-MCN assay seemed more sensitive than that of the Trad-SHM assay in detecting genotoxicity of the river water pollution.     

Mutagenicity in lung of Big Blue mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO₂ reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC→TA, 29% GC→AT, 2% GC→CG, and 10% multiple mutations — primarily GG→TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO₂⁺ resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG→TT transversions. Nose-only exposure of Big Blue^® mice to PAN at 78 ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency ±S.E. of 6.16±0.58/10⁵ for controls versus 8.24±0.30/10⁵ for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with ³H-PAN showed that 24 h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG→TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.     

Cytogenetic effects of separate and combined effect of thorium-232 and potassium nitrates on Tradescantia (clone 02)

Under both separate and combined influence of 232nd-nitrate and potassium nitrate a study was made of the yield of somatic mutation frequencies, the level of morphologically abnormal cells and the loss of reproduction integrity in the stamen hairs of Tradescantia (clone 02). All investigated concentrations of 232nd-nitrate and potassium nitrate exerted statistically significant genotoxic effects and increased the level of morphological cell abnormalities in the stamen hairs of Tradescantia. Also, 0.36 mg/l 232nd and 0.51 mg/l KNO3 concentrations cause a statistically significant loss of reproduction integrity in the stamen hair. The combined action of 232nd-nitrate and potassium nitrate in all investigated concentrations and parameters causes a statistically significant effect.     

Genotoxicity of 5-azacytidine in somatic cells of Drosophila

The newly developed somatic mutation and recombination test, utilizing the wing-hair mutations mwh and flr3, was used to evaluate the genotoxicity of the base analog 5-azacytidine in larvae of Drosophila melanogaster. Third instar larvae were fed media wetted with various concentrations of the compound, and wings of surviving adults were removed and scored for the presence of clones of cells possessing malformed hairs. Wings of exposed flies trans-dihybrid for mwh and flr3 had significantly increased frequencies of twin spots, small single spots and large single spots. Significant linear regression of twin-spot frequencies upon concentration was also obtained. Induction of twin spots by 5-azacytidine unambiguously demonstrates its recombinogenic activity in somatic cells of Drosophila. Significantly increased frequencies of large single spots on wings of inversion-heterozygous flies were also observed and suggest that 5-azacytidine may also be inducing somatic gene mutations (or deletions).     

Detection of the loss of 1 chromosome from pair III in Saccharomyces cerevisiae yeasts

A diploid strain of Saccharomyces cerevisiae, T6 is described which monitors both mitotic crossing over and induction of aneuploidy. The chromosome III carries recessive markers: rgh12 of "rough colony" phenotype closely linked to centromere, the left arm is marked with his4, the right arm is marked both with thr4 and the locus of mating type alpha. Expression of all the markers on chromosome III leads to formation of colonies which are rough, require histidine and threonine, and are of alpha mating type. These colonies arise as a result of the loss of a chromosome during mitosis, which makes the strain allow detection of monosomic cells formation. Chromosome XV carries two phenotypically distinguishable and recessive alleles of the gene ade2: ade2-192 (causes red coloration of colonies) and ade2-G45 (causes pink coloration of colonies). Mitotic crossing over generates two reciprocal products which can be revealed together in colonies as pink and red sectors in double-spotted colonies. Both double-spotted and monosomic colonies have been obtained after treatment with gamma-rays. The frequency of mitotic crossing over after irradiation by 1000-3000 Gray increased up to 2-3.2% (the spontaneous level was 0.006%), the frequency of aneuploidy was 0.12 to 0.57% at plating immediately after irradiation (the spontaneous monosomics were not observed among 1.5 X 10(5) cells scored). Induction of mitotic crossing over and aneuploidy by benomyl was rather slight (up to 0.05 and 0.006%, respectively).     

Life-history styles and somatic allocation in iteroporous arctic charr and semelparous pink salmon

Synopsis RNA/DNA ratios were used to estimate recent somatic allocation of spawning arctic charr,Salvelinus alpinus, and pink salmon,Oncorhynchus gorbuscha. Smaller arctic charr morphs had lower ratios than larger morphs. Larger male pink salmon had lower ratios than smaller males. Thus recent somatic allocation (anabolic metabolic activity and growth) is coupled with cumulative lifetime growth in iteroparous arctic charr, but is uncoupled in semelparous pink salmon. These data suggest that somatic investment in these species is being differentially effected by the energetic costs of reproduction.     

Comparison of X-ray and gamma-ray dose-response curves for pink somatic mutations in Tradescantia clone 02

Summary Microdosimetric data indicate that the mean specific energy,, produced by individual charged particles from X rays and gamma rays is different for the two radiation qualities by nearly a factor of two. In order to test whether this influences the initial, linear component in the dose-effect relations, a comparison was made between dose-response curves for pink somatic mutations inTradescantia clone 02 stamen hairs following X and gamma irradiations. Absorbed doses ranged from 2.66 to 300 rad. The results are in agreement with predictions made on the basis of microdosimetric data. At low doses gamma rays are substantially less effective than X rays. The RBE of gamma rays vs. X rays at low doses was approximately 0.6, a value lower than those usually reported in other experimental systems.Research supported by Public Health Service Research Grant No. CA-12536 from the National Cancer Institute and the U.S. Energy Research and Development Administration at Brookhaven National Laboratory. By acceptance of the article, the publisher and/or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright concerning this paper