Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Survey of the potential environmental and health impacts in the immediate aftermath of the coal ash spill in Kingston, Tennessee.

An investigation of the potential environmental and health impacts in the immediate aftermath of one of the largest coal ash spills in U.S. history at the Tennessee Valley Authority (TVA) Kingston coal-burning power plant has revealed three major findings. First the surface release of coal ash with high levels of toxic elements (As = 75 mg/kg; Hg = 150 microg/kg) and radioactivity (226Ra + 228Ra = 8 pCi/g) to the environment has the potential to generate resuspended ambient fine particles (< 10 microm) containing these toxics into the atmosphere that may pose a health risk to local communities. Second, leaching of contaminants from the coal ash caused contamination of surface waters in areas of restricted water exchange, but only trace levels were found in the downstream Emory and Clinch Rivers due to river dilution. Third, the accumulation of Hg- and As-rich coal ash in river sediments has the potential to have an impact on the ecological system in the downstream rivers by fish poisoning and methylmercury formation in anaerobic river sediments.

Radium and barium removal through blending hydraulic fracturing fluids with acid mine drainage.

Wastewaters generated during hydraulic fracturing of the Marcellus Shale typically contain high concentrations of salts, naturally occurring radioactive material (NORM), and metals, such as barium, that pose environmental and public health risks upon inadequate treatment and disposal. In addition, fresh water scarcity in dry regions or during periods of drought could limit shale gas development. This paper explores the possibility of using alternative water sources and their impact on NORM levels through blending acid mine drainage (AMD) effluent with recycled hydraulic fracturing flowback fluids (HFFFs). We conducted a series of laboratory experiments in which the chemistry and NORM of different mix proportions of AMD and HFFF were examined after reacting for 48 h. The experimental data combined with geochemical modeling and X-ray diffraction analysis suggest that several ions, including sulfate, iron, barium, strontium, and a large portion of radium (60-100%), precipitated into newly formed solids composed mainly of Sr barite within the first ∼ 10 h of mixing. The results imply that blending AMD and HFFF could be an effective management practice for both remediation of the high NORM in the Marcellus HFFF wastewater and beneficial utilization of AMD that is currently contaminating waterways in northeastern U.S.A.

Impacts of shale gas wastewater disposal on water quality in western Pennsylvania.

The safe disposal of liquid wastes associated with oil and gas production in the United States is a major challenge given their large volumes and typically high levels of contaminants. In Pennsylvania, oil and gas wastewater is sometimes treated at brine treatment facilities and discharged to local streams. This study examined the water quality and isotopic compositions of discharged effluents, surface waters, and stream sediments associated with a treatment facility site in western Pennsylvania. The elevated levels of chloride and bromide, combined with the strontium, radium, oxygen, and hydrogen isotopic compositions of the effluents reflect the composition of Marcellus Shale produced waters. The discharge of the effluent from the treatment facility increased downstream concentrations of chloride and bromide above background levels. Barium and radium were substantially (>90%) reduced in the treated effluents compared to concentrations in Marcellus Shale produced waters. Nonetheless, (226)Ra levels in stream sediments (544-8759 Bq/kg) at the point of discharge were ~200 times greater than upstream and background sediments (22-44 Bq/kg) and above radioactive waste disposal threshold regulations, posing potential environmental risks of radium bioaccumulation in localized areas of shale gas wastewater disposal.

Do You Want to Hear the Bad News? The Value of Diagnostic Tests for Alzheimer's Disease.

OBJECTIVE: The diagnosis of Alzheimer's disease (AD) remains difficult. Lack of diagnostic certainty or possible distress related to a positive result from diagnostic testing could limit the application of new testing technologies. The objective of this paper is to quantify respondents' preferences for obtaining AD diagnostic tests and to estimate the perceived value of AD test information. METHODS: Discrete-choice experiment and contingent-valuation questions were administered to respondents in Germany and the United Kingdom. Choice data were analyzed by using random-parameters logit. A probit model characterized respondents who were not willing to take a test. RESULTS: Most respondents indicated a positive value for AD diagnostic test information. Respondents who indicated an interest in testing preferred brain imaging without the use of radioactive markers. German respondents had relatively lower money-equivalent values for test features compared with respondents in the United Kingdom. CONCLUSIONS: Respondents preferred less invasive diagnostic procedures and tests with higher accuracy and expressed a willingness to pay up to €700 to receive a less invasive test with the highest accuracy.

EAE cerebrospinal fluid augments in vitro phagocytosis and metabolism of CNS myelin by macrophages.

Previous studies from this laboratory have shown that CNS myelin is phagocytized and metabolized by cultured rat macrophages to a much larger extent when myelin is pretreated with serum containing antibodies to myelin constituents than when it is left untreated or pretreated with non-specific serum. In this study the effect of cerebrospinal fluid (CSF) from rabbits with experimental allergic encephalomyelitis (EAE) in promoting myelin phagocytosis was examined. Fourteen rabbits were immunized with purified myelin in Freund's complete adjuvant, seven of which developed clinical EAE symptoms. Serum and CSF were collected from EAE and control rabbits, and the CSF was centrifuged to remove cells. Sera and CSF from these rabbits and from Freund's adjuvant-immunized controls and untreated controls were measured for IgG content by radial diffusion assay, their myelin antibody characteristics were analyzed by immunoblots, and the ability of these serum and CSF samples to promote myelin phagocytosis when used for myelin opsonization was examined. The ability of a CSF sample to enhance radioactive myelin uptake and phagocytosis by cultured macrophages as measured by the appearance of radioactive cholesterol ester was linearly proportional to its total IgG titer, and correlated approximately both with clinical symptoms of the animal and the presence of antibody against the myelin constituents myelin basic protein, proteolipid protein, and galactocerebroside. The cholesterol esterification activities of EAE sera correlated to a lesser extent with IgG levels and clinical symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)