Effect of inter-crystal scatter on estimation methods for random coincidences and subsequent correction.

Random coincidences can contribute substantially to the background in positron emission tomography (PET). Several estimation methods are being used for correcting them. The goal of this study was to investigate the validity of techniques for random coincidence estimation, with various low-energy thresholds (LETs). Simulated singles list-mode data of the MADPET-II small animal PET scanner were used as input. The simulations have been performed using the GATE simulation toolkit. Several sources with different geometries have been employed. We evaluated the number of random events using three methods: delayed window (DW), singles rate (SR) and time histogram fitting (TH). Since the GATE simulations allow random and true coincidences to be distinguished, a comparison between the number of random coincidences estimated using the standard methods and the number obtained using GATE was performed. An overestimation in the number of random events was observed using the DW and SR methods. This overestimation decreases for LETs higher than 255 keV. It is additionally reduced when the single events which have undergone a Compton interaction in crystals before being detected are removed from the data. These two observations lead us to infer that the overestimation is due to inter-crystal scatter. The effect of this mismatch in the reconstructed images is important for quantification because it leads to an underestimation of activity. This was shown using a hot-cold-background source with 3.7 MBq total activity in the background region and a 1.59 MBq total activity in the hot region. For both 200 keV and 400 keV LET, an overestimation of random coincidences for the DW and SR methods was observed, resulting in approximately 1.5% or more (at 200 keV LET: 1.7% for DW and 7% for SR) and less than 1% (at 400 keV LET: both methods) underestimation of activity within the background region. In almost all cases, images obtained by compensating for random events in the reconstruction algorithm were better in terms of quantification than the images made with precorrected data.

A true singles list-mode data acquisition system for a small animal PET scanner with independent crystal readout.

We present a unique data acquisition system designed to read out signals from the MADPET-II small animal LSO-APD PET tomograph. The scanner consists of 36 independent detector modules arranged in a dual-radial layer ring (phi 71 mm). Each module contains a 4 x 8 array of optically isolated, 2 x 2 mm LSO crystals, coupled one-to-one to a 32 channel APD. To take full advantage of the detector geometry, signals from each crystal are individually processed without any data reduction. This is realized using custom designed mixed-signal ASICs for analogue signal processing, and FPGAs to control the digitization of analogue signals and subsequent multiplexing. Analogue to digital converters (ADCs) digitize the signal peak height, time to digital converters (TDCs) time stamp each event relative to a system clock and two 32 bit words containing the energy, time and position information for each singles event are multiplexed through three FIFO stages before being written to disk via gigabit Ethernet. Every singles event is processed and stored in list-mode format, and coincidences are sorted post-acquisition in software. The 1152 channel data acquisition system was designed to be able to handle sustained data rates of up to 11 520 000 cps without loss (10 000 cps/channel). The timing resolution of the TDC was measured to be 1 ns FWHM. In addition to describing the data acquisition system, performance measurements made using a 128-channel detector prototype will be presented.

Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley.

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F(1) plants were self-pollinated and the F(2) generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F(2) plants with transposed Ds-bar was 100% stable, whereas only 23% of F(2) plants carrying Ds-bar at the original site expressed the transgene product stably. In F(3) and F(4) populations, transgene expression in 81.5% of plants from progeny of F(2) plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.

An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function.

To devise a method for function-based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted-repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds-interrupted uidA reporter gene, were used to demonstrate high-frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPase-expressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and first intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds-Ubi-bar. In F(1) plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F(2) progeny derived from individual selfed F(1) plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F(3) plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F(3) plants we observed two mutant phenotypes in which the transposed Ds elements co-segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes.

Validation and analysis of modeled predictions of growth of Bacillus cereus spores in boiled rice.

The growth of psychrotrophic Bacillus cereus 404 from spores in boiled rice was examined experimentally at 15, 20, and 30 degrees C. Using the Gompertz function, observed growth was modeled, and these kinetic values were compared with kinetic values for the growth of mesophilic vegetative cells as predicted by the U.S. Department of Agriculture's Pathogen Modeling Program, version 5.1. An analysis of variance indicated no statistically significant difference between observed and predicted values. A graphical comparison of kinetic values demonstrated that modeled predictions were "fail safe" for generation time and exponential growth rate at all temperatures. The model also was fail safe for lag-phase duration at 20 and 30 degrees C but not at 15 degrees C. Bias factors of 0.55, 0.82, and 1.82 for generation time, lag-phase duration, and exponential growth rate, respectively, indicated that the model generally was fail safe and hence provided a margin of safety in its growth predictions. Accuracy factors of 1.82, 1.60, and 1.82 for generation time, lag-phase duration, and exponential growth rate, respectively, quantitatively demonstrated the degree of difference between predicted and observed values. Although the Pathogen Modeling Program produced reasonably accurate predictions of the growth of psychrotrophic B. cereus from spores in boiled rice, the margin of safety provided by the model may be more conservative than desired for some applications. It is recommended that if microbial growth modeling is to be applied to any food safety or processing situation, it is best to validate the model before use. Once experimental data are gathered, graphical and quantitative methods of analysis can be useful tools for evaluating specific trends in model prediction and identifying important deviations between predicted and observed data.

Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA- and cytochrome P450 gene-mediated selection.

Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years various counter-selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non-toxic 5-fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU), was used for in vitro selection of germinating seedlings. Development of codA-expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5-FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono-oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T1 plants expressing the selectable marker gene showed striking morphological differences from the non-transgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large-scale screening under greenhouse or field conditions.

Effect of US contrast agents on spectral velocities: in vitro evaluation.

PURPOSE: To evaluate the effect of ultrasonographic (US) contrast agents on measurements of peak velocity with spectral Doppler US in stenotic and nonstenotic flow states. MATERIALS AND METHODS: Nonpulsatile flow was established in a flow phantom with 0%, 50%, 75%, and 90% stenoses. SH U 508A, perflenapent emulsion, and perfluorohexane emulsion were the contrast agents evaluated. Before and after administration of each contrast agent, two peak velocity measurements obtained proximal to, at the site of, and distal to the stenosis in each vessel model were averaged. The percentage difference in peak velocity after contrast agent administration was calculated for each site interrogated. The mean, SD, and coefficient of variation of the percentage difference in peak velocity were calculated. RESULTS: Percentage differences in peak velocity after contrast agent administration at different sample volume sites were not significantly different irrespective of the degree of stenosis or the contrast agent evaluated. CONCLUSION: The contrast agents evaluated do not produce a statistically significant increase in peak velocity. If this result is corroborated in clinical practice, contrast agents can be used without reevaluating existing Doppler US thresholds for stenosis.

Beneficial effect of selenium supplementation during murine infection with Trypanosoma cruzi.

Selenium (Se) has been shown to function as an antioxidant that may enhance immunity during microbial infection. To investigate the effect of elevated levels of Se on the course of experimental Chagas' disease, 5 groups of C3HeB/FeJ mice were infected with 10(3) bloodform trypomastigotes of a Brazil strain of Trypanosoma cruzi while receiving supplements of 0 ppm, 2 ppm, 4 ppm, 8 ppm, or 16 ppm Se as sodium selenate in drinking water. After 64 days of infection, survival ranged from 0 to 60%, with groups receiving 4 ppm and 8 ppm Se exhibiting 60% survival and the group without Se exhibiting 0% survival. In addition, parasitemia levels of mice supplemented with Se were significantly lower (P<0.01) than in nonsupplemented mice. The results of the present study suggest that Se supplementation does have a beneficial effect during murine infection with T. cruzi, resulting in decreased parasitemias and increased longevity.

Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue.

The development of a barley (Hordeum vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation (Ac/Ds) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co-transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uidA reporter gene (Gus), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.

Lateralization of pectoral stridulation sound production in the channel catfish.

Sounds of the channel catfish Ictalurus punctatus were found to consist of a rapid series of pulses produced by rubbing a ridged process on the first pectoral spine against the rough surface of a groove in the pectoral girdle during fin abduction. Although sounds can be made with either fin, approximately half of the individuals exhibited a fin preference, and 90% of these preferred the right fin. Unlike examples of handedness in other invertebrates and fishes, this preference is not simply a matter of anatomical asymmetry, but as in humans, reflects a preference between two equally developed limbs.

Influence of arterial coronary bypass grafts on the mortality in coronary reoperations.

From 1988 through 1991, 1663 patients underwent a first reoperation for isolated coronary bypass grafting with 62 (3.7%) in-hospital deaths. At the primary operation, 575 patients had received at least one internal thoracic artery graft and 489 patients had at least one patent internal thoracic artery graft present at the time of reoperation. At reoperation, 1014 patients received at least one internal thoracic artery graft, 10 received an inferior epigastric graft, and 37 received a gastroepiploic graft. Of 489 patients with patent internal thoracic artery grafts at reoperation, the internal thoracic artery was damaged in 17 (3.5%); of 428 patients with a patent internal thoracic artery graft to the left anterior descending coronary artery, 14 (3.3%) had graft damage necessitating regrafting. All patients with damaged internal thoracic arteries survived. Multivariate testing of variables for their association with in-hospital mortality identified no internal thoracic artery graft at either primary surgery or reoperation (p < 0.0001), a history of congestive heart failure (p < 0.0001), advancing age (p = 0.018), female gender (p = 0.029), and emergency operation (p = 0.01) as factors linked to increased risk. Left ventricular function, left main stenosis, extent of native coronary atherosclerosis, and the interval between operations did not influence mortality. Furthermore, the presence of an atherosclerotic vein graft to the left anterior descending coronary artery a factor shown to increase in-hospital risk in previous studies did not increase risk during these years. We attribute the observation that patent internal thoracic artery and atherosclerotic vein grafts do not appear to be factors specifically increasing the risk of reoperation to the use of retrograde cardioplegic solution and increased surgical experience. The use of internal thoracic artery grafts at a primary operation does not increase the risk of a reoperation, and the use of internal thoracic artery grafts at reoperation does not increase in-hospital morbidity or mortality.

Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice.

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5' regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the beta-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.

Systemic induction of a potato pin2 promoter by wounding, methyl jasmonate, and abscisic acid in transgenic rice plants.

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial beta-glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5'-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.

Characterization of cis-acting elements regulating transcription from the promoter of a constitutively active rice actin gene.

The promoter of the constitutively expressed rice (Oryza sativa) actin 1 gene (Act1) is highly active in transformed rice plants (W. Zhang, D. McElroy, and R. Wu, Plant Cell 3:1150-1160, 1991). A region 834 bp upstream of the Act1 transcription initiation site contains all the regulatory elements necessary for maximal gene expression in transformed rice protoplasts (D. McElroy, W. Zhang, J. Cao, and R. Wu, Plant Cell 2:163-171, 1990). We have constructed a series of Act1 promoter deletions fused to a bacterial beta-glucuronidase reporter sequence (Gus). Transient expression assays in transformed rice protoplasts, as well as transformed maize cells and tissues, identified two distinct cis-acting regulatory elements in the Act1 promoter. A 38-bp poly(dA-dT) region was found to be a positive regulator of Act1 promoter activity. Deletion of the poly(dA-dT) element lowered Gus expression by at least threefold compared with expression produced by the full-length Act1 promoter. By gel retardation and footprinting, we identified a ubiquitous rice protein which specifically recognizes this poly(dA-dT) element in the constitutively active Act1 promoter. A CCCAA pentamer repeat-containing region was found to be a negative regulator of the Act1 promoter in transformed rice protoplasts. Transient expression assays in different maize cells and tissues with use of the Act1 deletion constructs suggested that the CCCAA pentamer repeat region functions in a complex tissue-specific manner. A CCCAA-binding protein was detected only in root extracts.