Microdosimetry and chromosome aberrations: Effects of 230 keV neutrons on Vicia faba chromosomes

Physical energy deposition events have been related to sub-nuclear cytological events (chromosomal changes) in metaphases sequentially accumulated from the latter part of the cell cycle of Vicia faba. 230 keV neutrons produce about 0.4 recoil protons per late interphase nucleus per rad with the majority of protons travelling 1 to 2 microns from their origin, depositing energy at around 90 keV per micron. The frequency of induced aberrations is basically linear with dose, though varying through consecutive cell sampling periods because of differential induced mitotic delay. Distributions of chromosomal aberrations and total cytological events are overdispersed in relation to the Poisson distribuyion indicating that some proton recoils produce multiple events. When gaps and aberrations within chromosomes and multiple aberrations between chromosomes, are considered as discrete events, distributions follow Poisson expectations. About 40% of proton recoils result in observable cytological change. The highly energetic proton recoils (～90 keV per micron) which can induce multiple events are the ones most likely to produce effects which result in cell death. The sphere of influence of the proton recoils is probably adequately estimated from their range (～1 to 2 μm) since it seems compatible with the spatial proximity of the initial components of the resultant chromosome aberrations.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Single-cell responses to ionizing radiation

While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.     

The efficacy of sampling strategies for estimating scabies prevalence

BackgroundEstimating community level scabies prevalence is crucial for targeting interventions to areas of greatest need. The World Health Organisation recommends sampling at the unit of households or schools, but there is presently no standardised approach to scabies prevalence assessment. Consequently, a wide range of sampling sizes and methods have been used. As both prevalence and drivers of transmission vary across populations, there is a need to understand how sampling strategies for estimating scabies prevalence interact with local epidemiology to affect the accuracy of prevalence estimates.MethodsWe used a simulation-based approach to compare the efficacy of different scabies sampling strategies. First, we generated synthetic populations broadly representative of remote Australian Indigenous communities and assigned a scabies status to individuals to achieve a specified prevalence using different assumptions about scabies epidemiology. Second, we calculated an observed prevalence for different sampling methods and sizes.ResultsThe distribution of prevalence in subpopulation groups can vary substantially when the underlying scabies assignment method changes. Across all of the scabies assignment methods combined, the simple random sampling method produces the narrowest 95% confidence interval for all sample sizes. The household sampling method introduces higher variance compared to simple random sampling when the assignment of scabies includes a household-specific component. The school sampling method overestimates community prevalence when the assignment of scabies includes an age-specific component.DiscussionOur results indicate that there are interactions between transmission assumptions and surveillance strategies, emphasizing the need for understanding scabies transmission dynamics. We suggest using the simple random sampling method for estimating scabies prevalence. Our approach can be adapted to various populations and diseases.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Intrachromosomal changes and genomic instability in site-specific microbeam-irradiated and bystander human-hamster hybrid cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.     

Oncogenic transformation by fractionated doses of neutrons

Oncogenic transformation was assayed after C3H 10T1/2 cells were irradiated with monoenergetic neutrons; cells were exposed to 0.23-, 0.35-, 0.45-, 5.9-, and 13.7-MeV neutrons given singly or in five equal fractions over 8 h. At the biologically effective neutron energy of 0.45 MeV, enhancement of transformation was evident with some small fractionated doses (below 1 Gy). When transformation was examined as a function of neutron energy at 0.5 Gy, enhancement was seen for cells exposed to three of the five energies (0.35, 0.45, and 5.9 MeV). Enhancement was greatest for cells irradiated with 5.9-MeV neutrons. Of the neutron energies examined, 5.9-MeV neutrons had the lowest dose-averaged lineal energy and linear energy transfer. This suggests that enhancement of transformation by fractionated low doses of neutrons may be radiation-quality dependent.     

Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome

Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.     

The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells

It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation. Recently, evidence has accumulated that in cell populations exposed to low doses of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. The end points observed include chromosome aberrations, mutations and gene expression. The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of alpha particles, and to assay for oncogenic transformation. The single-cell microbeam delivered no, one, two, four or eight alpha particles through the nuclei of all or just 10% of C3H 10T1/2 cells. We show that (a) more cells can be inactivated than were actually traversed by alpha particles and (b) when 10% of the cells on a dish are exposed to alpha particles, the resulting frequency of induced transformation is not less than that observed when every cell on the dish is exposed to the same number of alpha particles. These observations constitute evidence suggesting a bystander effect, i.e., that unirradiated cells are responding to damage induced in irradiated cells. This bystander effect in a biological system of relevance to carcinogenesis could have significant implications for risk estimation for low-dose radiation.