OptiPhase HiSafe Research Articles

Application of portable liquid scintillation counter for on-field measurement of tritium in aqueous samples during radiation emergency

Screening of aqueous samples for 3H contamination is required to decide suitability of water intended for human consumption during radiation emergency. BARC, Trombay has recently procured Portable Triathler liquid scintillation counter as a screening tool for on-site response to radiation emergency. For this purpose, parameters like, different available scintillators, scintillator to sample ratio and influence of different capacity scintillation vials were optimized for 3H concentration measurement. The study indicated that for 7 mL vials, the optimized scintillator to sample ratio was 4:1 for Optiphase Hisafe II, Ultima Gold Ultra Low Level Tritium, Aqualight AB and Dioxane based scintillators whereas for Ultima Gold AB and Optiphase Hisafe III scintillators it was 3:1. In case of 20 mL vials, the ratio was optimized to 8:1 for all the above mentioned six scintillators. Additionally, the effect of applying counting efficiency using calibration curves generated using distilled water and that obtained using optimized scintillator to sample ratio in various spiked water samples was also studied. Results in 3H concentration (2000–5700 Bq/mL) indicate about ±10% deviation using both the methods for commercially available scintillators. However, in case of Dioxane based scintillator, the deviation was higher (20%). For analysis of 3H concentration ∼200 Bq/mL using commercially available scintillators results in higher deviation of about 21% due to unreliable quench indicating parameter for count rate less than 4000. The study indicated that calibration curves constructed using optimized parameters are universally applicable for determination of 3H concentration in wide variety of water samples.

Разработка и верификация метода разложения b-спектра смеси радионуклидов на основе кусочно-комбинированных функций с полиномиальной или кусочно- линейной аппроксимацией их параметров

Development and verification of own method of b-spectrum processing of ultra-low background radiometer-spectrometer Quantulus-1220 with minimal or no sample preparation, with H-3, K-40 and Sr-90+Y-90 presence in liquid sample at various ratio by activity considering quenching. Development of software Aspect-SUBI which implements the method.
 Materials and Methods. The method is based on deconvolution of the measured b-spectrum in the form of a superposition of model spectra of several radionuclides. b-spectra, obtained from standard radioactive solutions H-3, K-40 and Sr-90+Y-90 mixed with liquid scintillator Optiphase Hisafe 3 in volume ratio 8 ml : 12 ml were used. Methyl orange was used as a quencher. A sum of piecewisecombined functions was used for radionuclide libraries building. The lest squares method was used to determine radionuclide activities in the sample. Computer software Microsoft Excel, Sigma Plot 2000 and R version 4.0.2 was used for statistical processing of findings, their visualization as well as verification of the developed method.
 Results. Libraries of model b-spectra were built as a sum of piecewise-combined functions with polynomial approximation (library «P») or approximation by piecewise linear functions of their parameters (library «L») depending on quench parameter SQP(E). Verification of interpretation method on paired samples proved its capacity for simultaneous measurement of activity: 1) H-3 (library «P», with relative error ±9%) and K-40 (library «L», with relative error ±20%); 2) H-3 (library «P», with relative error ±11%) and Sr-90+Y-90 (library «L», with relative error ±21%) with limitations due to the levels of activities ratio and strong quenching. At simultaneous measurement of activity K-40 (library «L») and Sr-90+Y-90 (library «L») only correct measurement of activity Sr-90+Y-90 is possible with the error level ±10% taken quench parameter SQP(E) is in the range from 600 to 706. 
 Conclusion. The developed method can be used for simultaneous measurement of volume activity of H-3 and K-40 in urine samples of Ozyorsk population and Mayak PA workers, as well as volume activity of H-3 and Sr-90+Y-90 in samples from Lake Kyzyltash located in the Mayak PA affected area. The developed software Aspect-SUBI can be used not only with Quantulus-1220 but also with any other liquid scintillation spectrometer with logarithmic type of intensification and automatic quench detection.

Efficiency calibration and optimization of a liquid scintillation spectrometer for low level 55Fe and 63Ni measurements

An experimental study on the counting efficiency, background, and Lower Limit of Detection (LLD) of a liquid scintillation spectrometer was carried out for establishing a measurement technique suitable for low level activity measurements of 55Fe and 63Ni in environmental samples. The spectrometer's response, using two scintillators (PerkinElmer Optiphase Hisafe 3 and Meridian Prosafe HC+) and glass and plastic vials, was studied. Colour and chemical quenching effects were also investigated for both scintillators and vials. Finally, a spectrum integration window selection method able to reduce the LLD through a Figure of Merit (FOM) optimization, was tested. Efficiency and LLD curves were obtained for both scintillators and types of vials with this technique.

The effect of sample stability on the determination of radioactivity for various radionuclides by liquid scintillation counting

For measuring a sample stored for a long period of time using liquid scintillation counting (LSC), it is necessary to study the long-term stability of the sample. The effect of sample stability on the determination of radioactivity for 241Am, 90Sr/90Y, 137Cs, 147Pm, 237Np/233Pa, and 3H by LSC has been investigated. The variation of quench level over time can be an indication of sample stability. If the variation in a sample is little, the effect of sample stability on the determination of the above radionuclides can be neglected. Otherwise, the sample stability will have impact not only on the counting efficiency (especially for low energy β emitters), but also on the results of α/β discrimination. For studying the stability of a sample, special attention should be paid to the radionuclides with chemical form apt to be adsorbed, because the quench level of a sample cannot be reflected by the quench index SQP(E) alone when significant physical quench exists. Shaking a sample stored for a long period of time and checking the LSC spectra can give the information on physical quench in the sample. In the range of this study, OptiPhase Hisafe 3 has much better quench resistance than Ultima Gold AB.

Radionuclide, scintillation cocktail and chemical/color quench influence on discriminator setting in gross alpha/beta measurements by LSC

Gross alpha/beta measurements in drinking waters enable radiochemical composition analysis in environmental studies providing efficient screening method that can indicate whether water contains elevated levels of any radionuclide. Routine gross alpha/beta activity monitoring in drinking waters has been carried out for a few years in laboratory for low-level radioactivity measurements in Novi Sad according to ASTM method, performing measurements on liquid scintillation counter Quantulus 1220 which can simultaneously generate alpha/beta spectra of samples by Pulse Shape Analysis (PSA circuit). In this paper, PSA discriminator impact was investigated to ensure obtaining of accurate and reliable alpha/beta activities. One novelty of presented work is PSA parameter setup with two combinations of radionuclides (241Am, 226Ra and 90Sr/90Y) with varying activity concentrations. Performed experiments also make contribution to investigations on the manner in which chemical and color quench affect optimal PSA parameter setting and further on, their altogether influence on gross alpha/beta activity measurements. Nitromethane, 15.8 M nitric acid and water, as well as yellow and yellow-orange dye, were used as quenching agents in order to test PSA/interference factor behavior in the presence of quenchers with different quenching strengths. Variation of PSA setting in quenched samples with two different commercially available cocktails (Ultima Gold LLT and OptiPhase HiSafe 3) was also tested. Lastly, application i.e. assessment of obtained PSA-SQP(E) correlation on the obtained results of activity concentrations of few artesian well water samples and colored spiked samples, based on the measured SQP(E) value of samples, has been demonstrated.

Calculations and measurements of the scintillator-to-water stopping power ratio of liquid scintillators for use in proton radiotherapy

Liquid scintillators are a promising detector for high-resolution three-dimensional proton therapy dosimetry. Because the scintillator comprises both the active volume of the detector and the phantom material, an ideal scintillator will exhibit water equivalence in its radiological properties. One of the most fundamental of these is the scintillator’s stopping power. The objective of this study was to compare calculations and measurements of scintillator-to-water stopping power ratios to evaluate the suitability of the liquid scintillators BC-531 and OptiPhase HiSafe 3 for proton dosimetry. We also measured the relative scintillation output of the two scintillators. Both calculations and measurements show that the linear stopping power of OptiPhase is significantly closer to water than that of BC-531. BC-531 has a somewhat higher scintillation output. OptiPhase can be mixed with water at high concentrations, which further improves its scintillator-to-water stopping power ratio. However, this causes the solution to become cloudy, which has a negative impact on the scintillation output and spatial resolution of the detector. OptiPhase is preferred over BC-531 for proton dosimetry because its density and scintillator-to-water stopping power ratio are more water equivalent.

Study on quench effects in liquid scintillation counting during tritium measurements

Quench effects can cause a serious reduction in counting efficiency for a given sample/cocktail mixture in liquid scintillation counting (LSC) experiments. This paper presents a simple experiment performed in order to test the influence of quenching on the LSC efficiency of 3H. The aim of this study was to investigate the behavior of several quench agents with different quench strengths (nitromethane, nitric acid, acetone, dimethyl-sulfoxide) added in different amounts to tritiated water in order to obtain standard sets for quench calibration curves. The OptiPhase HiSafe 2 and OptiPhase HiSafe 3 scintillation cocktails were used in this study in order to compare their quench resistance. Measurements were performed using a low-level LS counter (Wallac, Quantulus 1220).

Measurement of wavelength-dependent refractive indices of liquid scintillation cocktails

Refractive indices of several commercial liquid scintillation cocktails were measured by means of an automatic critical-angle dispersion refractometer in the wavelength range from 404.7nm to 706.5nm. The results are needed for various applications. In particular, detailed Monte Carlo simulations of liquid scintillation counters that include the computation of optical light require these data. In addition, the refractive index is an important parameter for studies of micelle sizes by means of dynamic light scattering. In this work, the refractive indices were determined for Ultima Gold™, Ultima Gold™ F, Ultima Gold™ LLT, Ultima Gold™ AB, Hionic Fluor™, Permafluor®E+, Mineral Oil Scintillator, Insta-Gel Plus, OptiPhase HiSafe 2, OptiPhase HiSafe 3, Ultima Gold™ XR, Insta-Gel Plus, AquaLight, MaxiLight and Ultima Gold™ MV at 16°C, 18°C, 20°C and 22°C. The carbon dioxide absorber Carbo-Sorb®E was also analyzed.For some scintillators, various batches were compared and mixtures with water or nitromethane were studied.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

SU-E-T-105: An Evaluation of Two Organic Liquid Scintillators for Use in Real Time 3D Proton Dosimetry

Purpose: To evaluate the suitability of the organic liquid scintillators BC‐531 and OptiPhase HiSafe 3 for real‐time 3D proton dosimetry by measuring their scintillator‐to‐water stopping power ratios, density, and relative scintillation output. Methods: Stopping power ratios were measured for BC‐531 (not miscible with water) and OptiPhase at 0%, 25%, and 50% water concentrations. The ratios were derived from the water equivalent thickness of various scintillator volumes, which was quantified by the shift the scintillator introduces to the pristine Bragg peak of a 200 MeV proton beam. These depth‐dose distributions were measured with the IBA Zebra detector.Relative scintillation output was measured for a small volume of scintillator in the flat region of a 10 cm spread‐out Bragg peak. Output was quantified for the OptiPhase solutions by integrating over an image taken with the Andor Technology Luca S electron‐multiplying CCD camera. Results: BC‐531 showed the largest deviation from water in stopping power and density, with a scintillator‐to‐water stopping power ratio of 0.876 and density of 0.869 g/mL. For pure OptiPhase, that ratio was 0.990, and it increased to 0.997 and 0.996 for OptiPhase at 25% and 50% water concentrations. The density of the OptiPhase solutions was 0.963, 0.969, and 0.986 g/mL, respectively.The scintillation output of OptiPhase at 25% and 50% water concentrations was 72% and 59% of that for the pure scintillator. Conclusion: OptiPhase HiSafe 3 is a promising scintillator for real‐time 3D dosimetry for proton therapy. Its ability to mix with water improves its water equivalence without an unacceptable loss of scintillation output. However, the stopping power of the pure scintillator (0% water) is already within 1% of that of water, so dilution is unnecessary to achieve water equivalence. The addition of water also causes the solution to become cloudy, which would limit the spatial accuracy of the dosimeter.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Maternal corticosterone regulates nutrient allocation to fetal growth in mice

Stresses during pregnancy that increase maternal glucocorticoids reduce birth weight in several species. However, the role of natural glucocorticoids in the mother in fetal acquisition of nutrients for growth remains unknown. This study aimed to determine whether fetal growth was reduced as a consequence of altered amino acid supply when mice were given corticosterone in their drinking water for 5 day periods in mid to late pregnancy (day, D, 11-16 or D14-19). Compared to controls drinking tap water, fetal weight was always reduced by corticosterone. At D16, corticosterone had no effect on materno-fetal transfer of [(14)C]methylaminoisobutyric acid (MeAIB), although placental MeAIB accumulation and expression of the Slc38a1 and Slc38a2 transporters were increased. However, at D19, 3 days after treatment ended, materno-fetal transfer of MeAIB was increased by 37% (P < 0.04). During treatment at D19, placental accumulation and materno-fetal transfer of MeAIB were reduced by 40% (P < 0.01), although expression of Slc38a1 was again elevated. Permanent reductions in placental vascularity occurred during the earlier but not the later period of treatment. Placental Hsd11b2 expression, which regulates feto-placental glucocorticoid bioavailability, was also affected by treatment at D19 only. Maternal corticosterone concentrations inversely correlated with materno-fetal MeAIB clearance and fetal weight at D19 but not D16. On D19, weight gain of the maternal carcass was normal during corticosterone treatment but reduced in those mice treated from D11 to D16, in which corticosterone levels were lowest. Maternal corticosterone is, therefore, a physiological regulator of the amino acid supply for fetal growth via actions on placental phenotype.

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

A selective separation method for 93Zr in radiochemical analysis of low and intermediate level wastes from nuclear power plants

The zirconium isotope 93Zr is a long-lived pure β-particle-emitting radionuclide produced from 235U fission and from neutron activation of the stable isotope 92Zr and thus occurring as one of the radionuclides found in nuclear reactors. Due to its long half life, 93Zr is one of the radionuclides of interest for the performance of assessment studies of waste storage or disposal. Measurement of 93Zr is difficult owing to its trace level concentration and its low activity in nuclear wastes and further because its certified standards are not frequently available. A radiochemical procedure based on liquid–liquid extraction with 1-(2-thenoyl)-3,3,3-trifluoroacetone in xylene, ion exchange with Dowex resin and selective extraction using TRU resin has to be carried out in order to separate zirconium from the matrix and to analyze it by liquid scintillation spectrometry technique (LSC). To set up the radiochemical separation procedure for 93Zr, a tracer solution of 95Zr was used in order to follow the behavior of zirconium during the process by γ-ray spectrometry through measurement of the 95Zr. Then, the protocol was applied to low level waste (LLW) and intermediate level waste (ILW) from nuclear power plants. The efficiency detection for 63Ni was used to determination of 93Zr activity in the matrices analyzed. The limit of detection of the 0.05 Bq l−1 was obtained for 63Ni standard solutions by using a sample:cocktail ratio of 3:17 mL for OptiPhase HiSafe 3 cocktail.

Development and validation of a technique for the determination of 226Ra and 228Ra by liquid scintillation in liquid samples

Radium isotopes are dispersed in the environment according to their physicochemical characteristics. Considering their long half-lives and radiological effects, (226)Ra and (228)Ra are very important issues in radiological protection. In Brazil, radium isotopes represent an exposure problem both in nuclear fuel cycle installations and in high natural radiation background areas. The experimental part of this work includes the development of a technique for the determination of (226)Ra and (228)Ra by liquid scintillation with potential application in biological samples. Radium was concentrated and then separated from other constituents of the sample by co-precipitation/precipitation with Ba(Ra)SO(4). The precipitate was filtered and weighted to calculate the chemical yield. The filter containing the precipitate of Ba(Ra)SO(4) was transferred to a scintillation vial. Two methods were used to prepare the sources. The first one consisted of the addition of water (8 ml), Instagel XF (8 ml) and UltimaGold (4 ml) in the vial containing the filter and the precipitate, forming a gel suspension. In the second method, the precipitate was dissolved with 0.2 M ethylene-diamine-tetra-acetic-acid solution (9 ml) and 11 ml of scintillation solution (Optiphase Hisafe 3) was added to the vial, forming an aqueous and an organic phase. The solutions obtained were counted in a low background scintillation spectrometry system (Quantulus) suitable for the detection and identification of both alpha and beta particles for the determination of (226)Ra and (228)Ra. The activity values of (226)Ra and (228)Ra calculated by the two methods are in good agreement with the reference values, indicating that both methods are suitable for the determination of (226)Ra and (228)Ra.

Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

A performance comparison of nine selected liquid scintillation cocktails

Sample load capacity, sample compatibility, influence of sample load on 3H/ 14C counting efficiency, background count rate, figure of merit, quench resistance, and sample stability for nine selected, commercially available, liquid scintillation cocktails are presented in this study. The cocktails tested are Ecoscint A, Insta-Gel Plus, OptiPhase Hisafe 3, OptiPhase Trisafe, Ready Gel, SafeScint 1:1, Ultima Gold, Ultima Gold LLT, and Ultima Gold XR. For the data acquisition a Packard TriCarb Model 1900CA and a Quantulus 1220 liquid scintillation counter is used. All samples are prepared in either 20 mL low potassium, borosilicate glass vials or 20 mL high density, polyethylene vials. The aim of the full comparison study is to determine a single cocktail that best suits all measurement requirements of the liquid scintillation laboratory at SCK·CEN for the determination of low levels of radioactivity in biological and environmental samples. The results published in this report are intended to help other laboratories in selecting a cocktail fit for their purposes or to set up a similar study.

The Yersinia Protein Kinase A Is a Host Factor Inducible RhoA/Rac-binding Virulence Factor

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

Development of a homogeneous pulse shape discriminating flow-cell radiation detection system

A homogeneous flow-cell radiation detection system which utilizes coincidence counting and pulse shape discrimination circuitry was assembled and tested with five commercially available liquid scintillation cocktails. Two of the cocktails, Ultima Flo (Packard) and Mono Flow 5 (National Diagnostics) have low viscosities and are intended for flow applications; and three of the cocktails, Optiphase HiSafe 3 (Wallac), Ultima Gold AB (Packard), and Ready Safe (Beckman), have higher viscosities and are intended for static applications. The low viscosity cocktails were modified with 1-methylnaphthalene to increase their capability for alpha/beta pulse shape discrimination. The sample loading and pulse shape discriminator setting were optimized to give the lowest minimum detectable concentration for alpha radiation in a 30 s count time. Of the higher viscosity cocktails, Optiphase HiSafe 3 had the lowest minimum detectable activities for alpha and beta radiation, 0.2 and 0.4 Bq/ml for 233 U and 90 Sr/ 90 Y , respectively, for a 30 s count time. The sample loading was 70% and the corresponding alpha/beta spillover was 5.5%. Of the low viscosity cocktails, Mono Flow 5 modified with 2.5% (by volume) 1-methylnaphthalene resulted in the lowest minimum detectable activities for alpha and beta radiation; 0.3 and 0.5 Bq/ml for 233 U and 90 Sr/ 90 Y , respectively, for a 30 s count time. The sample loading was 50%, and the corresponding alpha/beta spillover was 16.6%. HiSafe 3 at a 10% sample loading was used to evaluate the system under simulated flow conditions.