Autoradiography and Phosphorimaging.

Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, ß-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.

NEUTRON DOSE ASSESSMENT USING SAMPLES OF HUMAN BLOOD AND HAIR.

The unique feature of nuclear accidents with neutron exposure is the induced radioactivity in body tissues. For dosimetry purposes, the most important stable isotopes occurring in human body, which can be activated by neutrons, are 23 Na and 32 S. The respective activation reactions are as follows:23Na(n,γ)24Na and32S(n,p)32P. While sodium occurs in human blood, sulfur is present in human hair. In order to verify the practical feasibility of this dosimetry technique in conditions of our laboratory, samples of human blood and hair were irradiated in a channel of a training reactor VR-1.24Na activity was measured by gamma-ray spectrometry.32P activity in hair was measured by means of a proportional counter. Based on neutron-spectrum calculation, relationships between neutron dose and induced activity were derived for both blood and hair.

Copepod nauplii use phosphorus from bacteria, creating a short circuit in the microbial loop.

In subtropical oceans phytoplankton carbon: phosphorus (C : P) ratios are high, and these ratios are predicted to increase further with rising ocean temperatures and stratification. Prey stoichiometry may pose a problem for copepod zooplankton nauplii, which have high phosphorus demands due to rapid growth. We hypothesised that nauplii meet this demand by consuming bacteria. Naupliar bacterial and phytoplankton carbon and phosphorus ingestion, assimilation and incorporation were traced using 33 P and 14 C radioisotopes. Bacterial carbon was incorporated four times less efficiently into biomass than phytoplankton carbon. In contrast, bacterial and phytoplankton phosphorus were incorporated at similar efficiencies, and bacteria could meet a substantial amount of naupliar phosphorus requirements. As parts of the ocean become more oligotrophic, bacteria could help sustain naupliar growth and survival under suboptimal stoichiometric conditions. Thus, nauplii may be a shortcut for phosphorus from the microbial loop to the classical food web.

Quantitative imaging of 33P in plant materials using 14C polymer references.

Phosphorus (P) research still lacks techniques for rapid imaging of P use and allocation in different soil, sediment, and biological systems in a quantitative manner. In this study, we describe a time-saving and cost-efficient digital autoradiographic method for in situ quantitative imaging of 33P radioisotopes in plant materials. Our method combines autoradiography of the radiotracer applications with additions of commercially available 14C polymer references to obtain 33P activities in a quantitative manner up to 2000 Bq cm-2. Our data show that linear standard regressions for both radioisotopes are obtained, allowing the establishment of photostimulated luminescence equivalence between both radioisotopes with a factor of 9.73. Validating experiments revealed a good agreement between the calculated and applied 33P activity (R2 = 0.96). This finding was also valid for the co-exposure of 14C polymer references and 33P radioisotope specific activities in excised plant leaves for both maize (R2 = 0.99) and wheat (R2 = 0.99). The outlined autoradiographic quantification procedure retrieved 100% ± 12% of the 33P activity in the plant leaves, irrespective of plant tissue density. The simplicity of this methodology opens up new perspectives for fast quantitative imaging of 33P in biological systems and likely, thus, also for other environmental compartments.

Verification of Safety Compliance of Delivering Radionuclides at Vanderbilt University.

Various radionuclides are transported at Vanderbilt University and Vanderbilt University Medical Center on a daily basis, to provide the necessities for diagnostic, therapeutic, and research applications. The delivery of the radionuclides takes various pathways where the general public may receive radiation doses. The Tennessee Department of Environment and Health Radiological Division regulates the dose limits for members of the public to be less than 1 mSv per year and 20 µSv in any hour. We designed a project to verify that potential doses received by the general public meet state regulations. Before the departure of the delivery, dose rates from three directions at a distance of 30 cm with respect to the transport vehicle, were measured using a tissue equivalent survey meter. During the shipment, times were recorded and the number of persons encountered along the path was estimated. Annual and hourly doses were calculated, conservatively assuming that a member of general public would follow the shipment at a distance of 30 cm, for the entire duration of the delivery. Calculated dose rates for each delivery and various combinations of radionuclides were found to be below state regulation limits.

NEUTRON FIELD MEASUREMENT OF P(35)+Be SOURCE USING THE MULTI-FOIL ACTIVATION METHOD.

Neutron field from the p+Be interaction was investigated at the NPI CAS for a proton beam energy of 35 MeV and thick beryllium target. Broad neutron spectra at close source-to-sample distances were determined using the multi-foil activation technique. Two large sets of dosimetry foils containing the Ni, Co, Au, In, Ti, Al, Y, Lu, Nb and Fe were irradiated at a distance of 74 mm at direct neutron beam axis and at a distance of 34 mm from beam axis. Supporting Monte-Carlo MCNPX calculations of the irradiation system were performed as well. From measured reaction rates, the neutron energy spectra at both positions were reconstructed employing the modified version of the SAND-II unfolding code and activation cross-section data from the EAF-2010 library. At the position of irradiated samples, the total fast neutron flux reaches the value up to 1010 cm-2 s-1, and the neutron field is utilizable for radiation hardness study and integral benchmark experiments within the International Fusion Material Irradiation Facility (IFMIF) program.

Assaying Protein Kinase Activity with Radiolabeled ATP.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Impact of raking and bioturbation-mediated ecological manipulation on sediment-water phosphorus diagenesis: a mesocosm study supported with radioactive signature.

The study examined the impact of raking and fish bioturbation on modulating phosphorus (P) concentrations in the water and sediment under different trophic conditions. An outdoor experiment was set to monitor physicochemical and microbiological parameters of water and sediment influencing P diagenesis. A pilot study with radioactive 32P was also performed under the agency of raking and bacteria (Bacillus sp.). Raking was more effective in release of P under unfertilized conditions by significantly enhancing orthophosphate (35%) and soluble reactive phosphate (31.8%) over respective controls. Bioturbation increased total and available P in sediments significantly as compared to control. The rates of increase were higher in the unfertilized conditions (17.6-28.4% for total P and 12.2 to 23.2% for available P) than the fertilized ones (6.5-12.4% for total P and 9.1 to 15% for available P). The combined effects of raking and bioturbation on orthophosphate and soluble reactive phosphate were also stronger under unfertilized state (54.5 and 81.8%) than fertilized ones (50 and 70%). The tracer signature showed that coupled action of introduced bacteria and repeated raking resulted in 59.2, 23 and 16% higher counts of radioactive P than the treatments receiving raking once, repeated raking and bacteria inoculation, respectively. Raking alone or in sync with bioturbation exerted pronounced impact on P diagenesis through induction of coupled mineralization and nutrient release. It has significant implication for performing regular raking of fish-farm sediments and manipulation of bottom-grazing fish to regulate mineralization of organic matter and release of obnoxious gases from the system. Further, they synergistically can enhance the buffering capacity against organic overload and help to maintain aquatic ecosystem health.

Monte Carlo dosimetric parameter study of a new (32)P brachytherapy source.

OBJECTIVE: Recently, a new catheter-based (32)P brachytherapy source has been developed (College of Chemistry, Sichuan University) for use in high-dose-rate afterloader. This study presents the results of the dosimetric data of the Geant4 Monte Carlo (MC) simulation toolkit for this new (32)P brachytherapy source. METHODS: The new (32)P source had dimensions of 0.50-cm length and 0.08-cm diameter and was encapsulated in teflon. In this study, we attempted to obtain dosimetric data for this new source, as required by the formalism proposed by the American Association of Physicists in Medicine reports TG60 and TG149. The source was located in a 30-cm radius theoretical sphere water phantom, and the absorbed dose of the source was calculated using MC code. RESULTS: The dosimetric data included the reference absorbed dose rate, the radial dose function in the range of 0.10-0.50 cm at a longitudinal axis, the polynomial function for the radial dose function and the anisotropy function with a θ value of 0-90° in 5° intervals and an r of 0.10-0.35 cm in 0.01-cm intervals. The radial and axial dose profiles and away-along quality assurance table are also calculated for the unsheathed (32)P source. The dose rate D(r0,θ0) at the reference point for the unsheathed (32)P source is determined to be equal to 1.2660 ± 0.0006 cGy s(-1) mCi. The radial dose function of the new (32)P source shows good agreement with the other (32)P source presented in this work with an average difference of 1.78%. CONCLUSION: Dosimetric data are provided for the new (32)P source. These data could be used in treatment-planning systems in clinical practice. ADVANCES IN KNOWLEDGE: Provided a new beta-emitting brachytherapy source that is intended for treatment of liver cancer. A dosimetric study of the unsheathed (32)P source for which no published dosimetric data existed was performed.

Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening.

Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.

Determination of surface dose rate of indigenous (32)P patch brachytherapy source by experimental and Monte Carlo methods.

Isotope production and Application Division of Bhabha Atomic Research Center developed (32)P patch sources for treatment of superficial tumors. Surface dose rate of a newly developed (32)P patch source of nominal diameter 25 mm was measured experimentally using standard extrapolation ionization chamber and Gafchromic EBT film. Monte Carlo model of the (32)P patch source along with the extrapolation chamber was also developed to estimate the surface dose rates from these sources. The surface dose rates to tissue (cGy/min) measured using extrapolation chamber and radiochromic films are 82.03±4.18 (k=2) and 79.13±2.53 (k=2) respectively. The two values of the surface dose rates measured using the two independent experimental methods are in good agreement to each other within a variation of 3.5%. The surface dose rate to tissue (cGy/min) estimated using the MCNP Monte Carlo code works out to be 77.78±1.16 (k=2). The maximum deviation between the surface dose rates to tissue obtained by Monte Carlo and the extrapolation chamber method is 5.2% whereas the difference between the surface dose rates obtained by radiochromic film measurement and the Monte Carlo simulation is 1.7%. The three values of the surface dose rates of the (32)P patch source obtained by three independent methods are in good agreement to one another within the uncertainties associated with their measurements and calculation. This work has demonstrated that MCNP based electron transport simulations are accurate enough for determining the dosimetry parameters of the indigenously developed (32)P patch sources for contact brachytherapy applications.

Hair 32P measurement for body dose mapping in non-fatal exposures to fast neutrons.

Dosimetry bioassay methods are the backbone of a personal dosimetry in criticality accidents. Although methods like hair dosimetry and the use of activation foils (e.g., (32)S) have been employed for decades, capabilities of different techniques, effects of hair type and neutron spectrum on the dose response, sensitivity and uncertainties of different techniques, etc., need more investigations. For this reason, the use of the (32)S(n,p)(32)P reaction and hair samples for estimating non-fatal doses from fast neutrons was studied. The experiments were carried out with the hair samples attached on a RANDO phantom in a Cf-252 neutron field, in the dose range of about 0.05-1.15 Gy. In addition, the adequate post-accident preparation for hair samples including optimum conditioning and timing were investigated. Experimental results prove the good sensitivity and merit of the method for neutron quantification in the mentioned dose range for which other bioassay methods are of poor resolution and sensitivity. A rough estimation of the dose-response curve for Iranian hair was also derived.

Potential radiological impact of the phosphate industry on wildlife.

The activities of the phosphate industry may lead to enhanced levels of naturally occurring radioactivity in terrestrial and aquatic ecosystems. We performed a preliminary environmental risk assessment (ERA) of environmental contamination resulting from the activities of 5 phosphate fertiliser plants (located in Belgium, Spain, Syria, Egypt, Brazil), a phosphate-mine and a phosphate-export platform in a harbour (both located in Syria). These sites were selected because of the availability of information on concentrations of naturally occurring radionuclides in the surrounding environments. Assessments were generally performed considering highest environmental concentrations reported in the studies. The ERICA Tool, operating in a Tier 2 assessment mode, was used to predict radiation dose rates and associated risk to the selected reference organisms using the ERICA default parameter setting. Reference organisms were those assigned as default by the ERICA Tool. Potential impact is expressed as a best estimate risk quotient (RQ) based on a radiation screening value of 10 µGy h(-1). If RQ ≤ 1, the environment is considered unlikely to be at risk and further radiological assessment is not deemed necessary. Except for one of the cases assessed, the best estimate RQ exceeded 1 for at least one of the reference organisms. Internal exposure covered for 90-100 % of the total dose. (226)Ra or (210)Po were generally the highest contributors to the dose. The aquatic ecosystems in the vicinity of the phosphate fertiliser plants in Tessenderlo (Belgium), Huelva (Spain), Goiás (Brazil) and the terrestrial environment around the phosphate mine in Palmyra (Syria) are the ecosystems predicted to be potentially most at risk.

Measure post-bloodmeal dispersal of mosquitoes and duration of radioactivity by using the isotope ³²P.

The radioactive isotope (32)P-labeled disodium phosphate (Na2H(32)PO4) was injected via the jugular vein into a cow kept in a shed in Maozhuang Village, Cao Township of Shanxian County, China. Over the following 5 d, mosquitoes feeding on the cow were captured at distances up to 400 m to determine dispersal distance. The duration of radioactivity in the cow and marked mosquitoes was 10 d. The results showed that after blood feeding, Anopheles sinensis and Culex tritaeniorhynchus temporarily rested in the cattle shed and then flew outdoors. In contrast, Culex pipiens pallens remained in the cattle shed after feeding. These findings confirmed that local An. sinensis and Cx. tritaeniorhynchus were partially endophilic and tended to rest out of doors, whereas Cx. pipiens pallens was endophilic. For marked An. sinensis and Cx. tritaeniorhynchus, there was a significant tendency for dispersal to be in a northeast and east direction, probably because of the presence of heavy shading by an agricultural field, a small river for mosquito oviposition sites, and locations downwind from the blood source. The furthest flight distances for An. sinensis and Cx. tritaeniorhynchus were 210 and 240 m; therefore, control of these mosquitoes should include resting places indoors and outdoors within a radius of 250 m from confirmed cases.

(32)P measurment of urine samples and internal dose assessment for radiation workers in life science laboratories.

(32)P measurements of urine samples and internal dose assessments were conducted for workers in life science laboratories. A procedure for sample pre-treatment was established and validation was performed to exclude interference and to detect (32)P levels accurately. The detection conditions for Cherenkov radiation were evaluated and the accuracy of Cherenkov radiation measurements validated. The analytical and measurement procedures were applied to urine samples collected from 11 workers from life sciences laboratories. The results of the measurements generally indicated very low background radiation levels, but daily urine samples from two workers were above the minimum detectable activity. The (32)P concentrations for two of the workers were 29.3 ± 10.4 Bq•d(-1) and 24.1 ± 11.8 Bq•d(-1), respectively, at intake levels of 4.12 kBq and 2.61 kBq. The effective doses for these two workers were 4.6 µSv and 2.9 µSv. Overall, the results indicate very low levels of radioactivity, except for cases related to specific working conditions.

A microautoradiographic method for fresh-frozen sections to reveal the distribution of radionuclides at the cellular level in plants.

Microautoradiography (MAR) is a conventional imaging method based on the daguerreotype. The technique is used to visualize the distribution of radionuclide-labeled compounds within a tissue section. However, application of the classical MAR method to plant tissue sections is associated with several difficulties. In this study, we report an MAR method applicable to fresh-frozen plant sections. Our method had two features: (i) the sample was kept frozen from plant tissue collection to radioisotope detection, making it possible to fix solutes without solvent exchange; and (ii) 1.2 µm thick polyphenylene sulfide film was inserted between the fresh-frozen plant section and the photosensitive nuclear emulsion to separate the section from the emulsion before autoradiography was conducted, which significantly improved the quality of the section until microscopic detection, the quality of the MAR image and the success rate. Then, the passage of cadmium (Cd) through vegetative rice stem tissue after 24 h of (109)Cd absorption was described for the first time using the MAR method. MAR clearly revealed the distribution of (109)Cd at the tissue level with high resolution. The (109)Cd concentration in phloem cells was found to be particularly high, whereas the xylem cells contained only small amounts of (109)Cd. The MAR method was also applicable for detecting (109)Cd and [(33)P]phosphate in roots. The MAR method developed here is expected to provide distribution images for a variety of compounds and ions in plant tissue.

Clinical observation of 32P metabolic mode at the level of sub-target nuclides.

AIM: The aim of this paper was to observe the metabolic mode of 32P at the level of sub-target nuclides. METHODS: Twenty-one cancer patients were locally injected with 32P-labelled glass microspheres and then observed to determine the equalization of 32P radionuclide metabolism in the tumor target. We imaged 3 sub-target regions of interest (ROI) 1/3 the size in both the anterior and posterior directions by bremsstrahlung single-photon emission computed tomography (SPECT) X-ray imaging. The radiation dose parameters of the beta rays including the initial dose rate, the effective half-life, and the effective half-life of the cumulative radiation dose were then calculated. RESULTS: The radionuclide metabolism of the 21 complete tumor targets complied with the mono-compartmental model of index metabolism, but the level of tumor control did not correlate with radiation dose parameters. In contrast, the radionuclide metabolism of the 63 sub-targets did not comply with the mono-compartmental model. Instead, 32 sub-targets were better represented by bi-compartmental or tri-compartmental metabolic models. None of the remaining 31 sub-targets complied with index metabolism. CONCLUSION: The complexity of the radiation dose at the sub-target level partially explains poor local tumor control. Future studies will be required to improve the expression of internal exposure to radiation dose parameters.

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

BACKGROUND AND AIMS: Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor. METHODS: (32)P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare 'Fleet') to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used. KEY RESULTS: The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants. CONCLUSIONS: Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.

Electrophoretic mobility-shift assays.

In an electrophoretic mobility-shift assay (EMSA, or simply "gel shift"), a (32)P-labeled DNA fragment containing a specific DNA site is incubated with a cognate DNA-binding protein. The protein-DNA complexes are separated from free (unbound) DNA by electrophoresis through a nondenaturing polyacrylamide gel. The protein retards the mobility of the DNA fragments to which it binds. Thus, the free DNA will migrate faster than the DNA-protein complex. An image of the gel is used to reveal the positions of the free and bound radiolabeled DNAs.

Measurement of phosphoinositides in the zebrafish Danio rerio.

Phosphoinositides represent a minor fraction of the total glycerolipids in cells. Despite the fact that phosphoinositides are present in small quantities, they have crucial roles during cell signaling and in regulating numerous intracellular processes. Measuring changes in the levels of different phosphoinositides in animals is difficult, but it is essential in order to define the important functions of specific members of the phosphoinositide family. Here we detail procedures for measuring phosphoinositides in 2-days-postfertilization (2-d.p.f.) embryos in zebrafish (Danio rerio). Both in vivo radiolabeling (using [(32)P]orthophosphate) followed by thin-layer or high-performance liquid chromatography (TLC or HPLC) analysis and specific in vitro phosphorylation assays (using [(32)P]γATP) permit the quantitative measurement of phosphoinositides. Normalization of both measurements can be achieved by the determination of total lipid phosphate in embryos. All the techniques described are relatively inexpensive and accessible to most laboratories with an interest in studying the effect of gene manipulation on phosphoinositide metabolism in zebrafish. All the procedures described herein will take up to 10 working days.