Design and construction of a 9 MeV γ-ray source based on capture of moderated plutonium-beryllium neutrons in nickel.

We have designed and constructed a high-energy γ-ray source for detector characterisation and calibration. The source is a composite type based on a plutonium-beryllium neutron emitter embedded in a paraffin moderator, which is homogeneously mixed with nickel powder. The 9 MeV γ-ray source produces approximately 450 photons per second in 4π when 2.2×105 neutrons per second are emitted, corresponding to a surface flux of 9 MeV γ-rays of approximately 2.5×10-6 cm-2 per emitted neutron. Here we discuss the properties and design of this source, including the characterisation of homogeneity and high-energy γ-ray emission spectra.

Metabolic engineering of Bacillus subtilis for high-level production of uridine from glucose.

As an intermediate in drug synthesis, uridine has practical applications in the pharmaceutical field. Bacillus subtilis is used as a host to boost uridine yield by manipulating its uridine biosynthesis pathway. In this study, we engineered a high-uridine-producing strain of B. subtilis by modifying its metabolic pathways in vivo. Overexpression of the aspartate ammonia-lyase (ansB) gene increased the relative transcriptional level of ansB in B. subtilis TD320 by 13·18 times and improved uridine production to 15·13 g l-1 after 72-h fermentation. Overexpression of the putative 6-phosphogluconolactonase (ykgB) gene increased uridine production by the derivative strain TD325 to 15·43 g l-1 . Reducing the translation of the amido phosphoribosyl transferase (purF) gene and inducing expression of the subtilisin E (aprE) gene resulted in a 1·99-fold increase in uridine production after 24 h shaking. Finally, uridine production in the optimal strain B. subtilis TD335, which exhibited reduced urease expression, reached 17·9 g l-1 with a yield of 314 mg of uridine g-1 glucose. To our knowledge, this is the first study to obtain high-yield uridine-producing B. subtilis in a medium containing only three components (80 g l-1 glucose, 20 g l-1 yeast powder, and 20 g l-1 urea).

Improving riboflavin production by modifying related metabolic pathways in Bacillus subtilis.

Riboflavin is a feed additive, food additive and clinical drug, with a significant annual demand of nearly 8000 t. Fermentation using recombinant Bacillus subtilis is currently one of the most important industrial production method for riboflavin. First, a suitable medium was selected and the expression of the ureABC operon was modified. The ykgB gene was overexpressed in B. subtilis RX10, the production of the derivative strain RX20 was increased to 4·61 g l-1 riboflavin, and the yield was increased to 52 mg riboflavin g-1 glucose. The relative transcription level of pyr operon in RX20 was reduced to 71%, the production of the derivative strain RX21 was increased to 5·82 g l-1 riboflavin, and the yield was 76 mg riboflavin g-1 glucose. The start codon of the pyrE gene in RX21 was modified to 'TTG', the production of the derivative strain RX22 was increased to 7·01 g l-1 riboflavin, and the yield was 89 mg riboflavin g-1 glucose. These results indicated that overexpression of the ykgB gene and reduction of the metabolic flux of de novo synthesis of pyrimidine nucleotides were beneficial to the synthesis of riboflavin.

First Report on the Occurrence of a New Pathotype, 714, of Plasmopara halstedii (Sunflower Downy Mildew) in Hungary.

Downy mildew of sunflower, caused by Plasmopara halstedii (Farlow) Berlese et de Toni, is an economically important disease in Hungary and much of Europe. The known pathotypes (races) of the pathogen influence the resistance genes (Pl genes) incorporated into new sunflower hybrids to manage the disease. There are at least 36 pathotypes of P. halstedii worldwide (3), but the number of races is increasing rapidly. In 2010, race 704 was identified in Hungary for the first time (2). Race 704 has been reported to confer virulence on Pl6, a broad spectrum resistance gene that is widely used in sunflower hybrids. This has coincided with a significant increase in disease severity since 2010 in the country. Our objectives are to continuously monitor this pathogen and identify pathotypes of P. halstedii. Because of the unfavorable weather conditions for downy mildew in 2013, samples were collected at a single site (Kunszentmárton, South Hungary) in the beginning of July from NK Neoma sunflower hybrids. Disease incidence (early and late primary infection) was as high as 40%. Systemically mildewed plants showed severe stunting and leaf chlorosis, signs and symptoms consistent with downy mildew. P. halstedii was identified microscopically. Examination of isolates was carried out using a set of sunflower differential lines based on the internationally standardized method for race identification of P. halstedii (1). Inoculum of the isolates was increased on a susceptible cultivar (cv. Iregi szürke csíkos) and tested by inoculating 3-day-old seedlings of sunflower differential lines. Inoculated seedlings were planted in trays in glasshouse. After 8 to 9 days, seedlings were sprayed with distilled water, covered with black plastic bags, and left overnight to induce sporulation. Disease incidence was determined by examining cotyledons at 9 days after inoculation for sporulation and true leaves on 12 to 13 days after inoculation for secondary symptoms, such as leaf chlorosis and stunting (1). While several differential lines showed no typical susceptible/resistant reactions, i.e., the infection was much lower than 100%, it was concluded that the isolates were mixtures of different P. halstedii pathotypes. To obtain single isolates, we collected zoosporangia from the differential lines in question separately, and then inoculated the seedlings of the same genotype and a uniformly susceptible line. A single isolate caused as high as 100% infection on HA-335, containing resistance gene Pl6. Subsequent evaluation of this isolate with the entire differential set resulted in an aggregate virulence phenotype of 714. As resistance gene Pl6 is incorporated to the majority of sunflower hybrids grown in Hungary, pathotypes virulent on this gene, such as 704 and 714, are likely to spread. This underscores the need to prove the resistance to these races in the newly registered hybrids and for further research to identify P. halstedii pathotypes. It is also important to establish the identity of this new pathotype by already discovered 714 pathotypes in other countries like France and Italy and to discover the real conditions of local evolving of new pathogens. To our knowledge, this is the first report of pathotype 714 of P. halstedii in both Hungary and Central Europe. References: (1) T. J. Gulya et al. Helia 14:11, 1991. (2) K. Rudolf et al. Növényvédelem 47:279, 2011. (3) F. Virányi and O. Spring. Eur. J. Plant Pathol. 129:207, 2011.

First Report on the Increased Distribution of Pathotype 704 of Plasmopara halstedii in Hungary.

Downy mildew of sunflower caused by Plasmopara halstedii (Farlow) Berlese et de Toni is a devastating disease worldwide. The treatment of seeds with fungicides and the use of resistant cultivars are widely employed control measures against this oomycete. Effective protection, however, may be hindered by the high genetic variability of the pathogen. There are 14 pathotypes of P. halstedii in Europe and as many as six of these were identified in Hungary before 2010 (1,4). In 2010, a new race, 704, was isolated in the eastern region of Hungary (3). Although the new pathotype was identified in two sunflower fields (near Vészto and Kondoros), it was expected to spread all over the country because of a lack of resistance against this race. The aim of our study, therefore, was to monitor the distribution of pathotype 704 in Hungary. Infected sunflower plants (2 to 5 samples/site) showing typical symptoms of downy mildew (leaf chlorosis, severe stunting) from four different sites (Árpádhalom, Rákóczifalva, Tiszasüly, and Újiráz) in the eastern region of the country were collected in mid-June 2012. Examination of isolates was carried out using a set of sunflower differential lines based on the internationally standardized method for race identification of P. halstedii (2). Inoculum of 17 isolates was increased on a susceptible cultivar (cv. Iregi Szürke Csíkos). Leaves containing zoosporangia were washed off in distilled water. The concentration of spore suspension for each isolate was adjusted to 20,000 to 30,000 viable zoosporangia per ml using a hemacytometer. Pre-germinated seeds of sunflower differential lines (20 seeds/line) with an optimal radicle length were selected and placed in separate petri dishes. They were filled with freshly prepared zoosporangial suspension of the isolates and incubated in the dark at 16°C for 6 h. Inoculated seeds were planted in trays. After 8 to 9 days when the first true leaves were ~0.5 to 1 cm long, the trays containing the plants were covered with transparent plastic bags overnight. Distilled water was sprayed into the bags to ensure a humid environment for stimulating sporulation. First disease assessment was performed immediately after incubation based on the appearance of characteristic white sporulation on cotyledons. A second evaluation was made of true leaves of 21-day-old plants. Twelve out of 17 isolates were pathotype 704, infecting either one of two commercial sunflower hybrids (NK Neoma and NK Brio) or volunteer sunflower plants. The remaining five isolates were races 700, 710, and 730, which are known to be widespread in Hungary (1). The presence of race 704 was proven in all sampling sites representing the eastern part of the country. This finding underscores the need to develop and grow improved sunflower hybrids with effective genes against this pathotype. To our knowledge, this is the first report of the wider distribution of pathotype 704 of P. halstedii in both Hungary and Central Europe. References: (1) T. J. Gulya. Adv. Downy Mildew Res. 3:121, 2007. (2) T. J. Gulya et al. Helia 14:11, 1991. (3) K. Rudolf et al. Növényvédelem 47:279, 2011. (4) F. Virányi and S. Masirevic. Helia 14:7, 1991.

Oral administration of lactoferrin attenuates intestinal ischemia-reperfusion injury in rats.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) is a common and serious clinical condition. Lactoferrin (Lf) has displayed antioxidative and anti-inflammatory activities in protecting the intestinal mucosa. The objective of this study was to investigate whether oral administration of Lf could attenuate I/R-induced intestinal injury. METHODS: The experimental design consisted of three groups of Wistar rats (24 per group): sham operation, control (I/R, saline), Lf (I/R, Lf). Intestinal I/R was produced by occlusion of the superior mesenteric artery for 45 min. Eight rats from each group were randomly sacrificed 3, 12 or 36 h after reperfusion, and blood and intestinal samples were collected. RESULTS: Intestinal I/R resulted in gut damage evidenced by morphological alteration, reduction of γ-glutamyl transpeptidase (γ-GGT) activity and increased cell apoptosis. Daily administration of Lf (200 mg/kg) for 14 days before surgery significantly attenuated gut damage by reducing the histologic score and apoptosis index, and restoring intestinal γ-GGT activity. Lf reduced intestinal malondialdehyde and myeloperoxidase, restored glutathione and decreased serum levels of tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 compared with saline control in I/R rats. In addition, oral administration of Lf did not produce any significant effects in healthy rats; Lf at doses of 50 or 100 mg/kg also attenuated I/R-induced gut damage, but administration of Lf for 7 days did not exert a significant protective effect against I/R-induced gut damage. CONCLUSIONS: These results indicate that Lf may serve as a potent supplement in protecting the gut from intestinal I/R-induced injury by its antioxidative, anti-inflammatory and antiapoptotic activities.

Harmonization of individual dosimetry quality control within metrology system of a small country.

A programme of harmonization of individual dosimetry quality control organized in the framework of a distributed metrology system is presented as seen from the experiences gained in Slovenia. As a part of the programme intercomparison of dosimetry services was organized and basic characteristics of dosimetry systems compared. Results are discussed with suggestions for further improvements of quality assurance.

Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies.

Mammalian Aurora-A is related to a serine/threonine protein kinase that was originally identified by its close homology with Saccharomyces cerevisiae Ipl1p and Drosophila melanogaster aurora that are key regulators in the orchestration of mitotic events. The protein level of Aurora-A, its peak kinase activity during mitosis, and its activation have been attributed to phosphorylation. Here we show that this enzyme is an arginine-directed kinase and define its substrate specificity. We also found that Thr288 within the activation loop is a critical residue for activating phosphorylation events in vitro and that it is spatiotemporally restricted to a brief window at mitosis on duplicated centrosomes and on spindle microtubules proximal to the poles in vivo. Immunodepletion assays indicated that an upstream kinase(s) of Aurora-A might exist in mammalian cells in addition to autophosphorylation. Furthermore, human activated Aurora-A forms complexes with the negative regulator protein serine/threonine phosphatase type 1 (PP1) that was negatively phosphorylated on Thr320. Interestingly, phospho-specific Aurora-A monoclonal antibodies restrain Aurora-A kinase activity in vitro, providing further therapeutic avenues to explore.

Blood transfusion increases radical promoting non-transferrin bound iron in preterm infants.

BACKGROUND: Blood transfusion has been recognised as a risk factor for the development of retinopathy of prematurity (ROP) or chronic lung disease (CLD) in preterm infants, but the precise mechanism involved is not understood. AIM: To investigate the level of non-transferrin bound "free" iron, which has the potential to promote the generation of reactive oxygen species, and its redox status in the plasma of preterm infants immediately before and after blood transfusion. METHODS: Twenty one preterm infants with a median gestational age and birth weight of 27 weeks and 1021 g respectively were prospectively enrolled in the study. Sixteen of the 21 infants developed ROP and/or CLD. The infants were transfused with concentrated red blood cells at a median age of 32 days. The plasma concentration of total bleomycin detectable iron (BDI) was measured and also the ferrous iron (Fe(2+)) activity by bleomycin-iron complex dependent degradation of DNA. RESULTS: Even before blood transfusion, BDI was detectable in one third of the blood samples, and all but one sample had ferrous iron activity. After transfusion, both BDI and ferrous iron activity were significantly increased, in contrast with the situation in full term infants. Plasma ascorbic acid (AA) concentration was significantly decreased after blood transfusion, whereas the level of its oxidation product, dehydroascorbic acid (DHAA), and the DHAA/AA ratio were significantly increased compared with before the transfusion. The activity of plasma ferroxidase, which converts iron from the ferrous to the ferric state, was appreciably decreased in preterm infants, as expected from their very low plasma caeruloplasmin concentration. CONCLUSIONS: Plasma non-transferrin bound iron was significantly increased in preterm infants after blood transfusion and existed partly in the ferrous form, because of the low ferroxidase activity and the reduction of ferric iron (Fe(3+)) by ascorbic acid. This finding was specific to preterm infants and was not observed in full term infants after blood transfusion. Non-transferrin bound "free" iron may catalyse the generation of reactive oxygen species, which may be responsible for the clinical association of blood transfusion with ROP and CLD.

KL-6, a mucinous glycoprotein, as an indicator of chronic lung disease of the newborn.

KL-6 is a mucinous glycoprotein that is preferentially expressed by alveolar type 2 cells. Plasma KL-6 was higher in infants with chronic lung disease (n = 12) than in infants without chronic lung disease (n = 14) on day 0-1, 10, and 30 (P =.04). KL-6 correlated with the alveolar-arterial oxygen tension difference on day 10 and day 30. Plasma KL-6 may be useful as an early marker of chronic lung disease and an indicator of severity.

[Determination of lactic acid in fermentation broth of Rhizopus oryzae by reversed-phase high performance liquid chromatography (RP-HPLC)].

A method for determining lactic acid in fermentation broth of Rhizopus oryzae by RP-HPLC is described. The operating conditions were Wakosil-II 5 C18 RS column(4.6 mm i.d. x 150 mm, 5 microns) at room temperature, 0.01 mol/L phosphoric acid solution (pH 2.5) as mobile phase with a flow rate of 1.0 mL/min and UV detection at 210 nm. The retention time of lactic acid was 3.820 min. This method is simple, rapid and accurate. The results will not be affected by other components in the broth. The relative standard deviation was 0.22% (n = 5), and the recovery was over 99%.

[Development of a high resolution beta camera].

We have developed and tested a high resolution beta camera. The beta camera consists of thin CaF2(Eu) scintillator, tapered fiber optics plate, position sensitive photomultiplier tube (PSPMT). The output of the PSPMT is fed to position calculation circuit and accumulated in the memory. The data in the memory is fed to personal computer for display and analysis. We have developed two types of beta cameras. One is 20 mm diameter field of view (FOV) camera, and the other is 10 mm diameter camera. Intrinsic spatial resolutions were 0.8 mm FWHM and 0.5 mm FWHM for 20 mm and 10 mm FOV camera, respectively. We confirmed that developed beta cameras may overcome the limitation of the resolution of the PET camera.

[Evaluation of non-uniform attenuation correction using simultaneous transmission and emission computed tomography--basic analysis with myocardial phantom].

Simultaneous transmission emission protocol (STEP), developed for the non-uniform attenuation correction of single photon emission computed tomography (SPECT) was evaluated using the cardiac phantom prepared with and without a myocardial wall defect. Emission computed tomography (ECT) of the cardiac phantom using 201Tl was acquired. Transmission data (TCT) were taken using a line source of 99mTc. Myocardial images with STEP method were superior in the homogeneity of intramyocardial radioactivity and spatial resolution to the conventional SPECT images. This is an excellent method because of the accurate matching position between TCT and ECT images and shortening the examination time by simultaneous data acquisition. It would be clinically useful for diagnosing various myocardial diseases.

Fractional mean transit time in transplanted kidneys studied by technetium-99m-DTPA: comparison of clinical and biopsy findings.

UNLABELLED: To determine the usefulness of fractional mean transit time (MTT) in the differential diagnosis of postrenal transplant complications, 99mTc-DTPA was used to evaluate differences in MTT between the outer zone (cortical nephron) and middle zone (juxtamedullary nephron, calcyces and cortical nephron) of the kidney. It is well known that acute rejection is characterized by delayed cortical transit time, whereas cortical nephron function is well maintained and juxtamedullary function is impaired after renal ischemia. METHODS: Technetium-99m-DTPA fractional MTT was determined by deconvolution analysis of 89 renograms obtained within 5 days of the date of kidney graft biopsy and evaluation. RESULTS: Outer zone MTT was significantly shorter than middle zone MTT in normals (2.7 +/- 0.4 versus 3.0 +/- 0.6 min, n = 22, p < 0.001), acute tubular necrosis (3.4 +/- 1.1 versus 3.6 +/- 1.4 min, n = 19, p < 0.01), chronic rejection (3.9 +/- 1.5 versus 5.0 +/- 2.3 min, n = 14, p < 0.001) and obstruction (4.1 +/- 0.6 versus 8.9 +/- 3.4 min, n = 13, p < 0.001). In contrast, outer zone MTT was significantly longer than middle zone MTT in acute rejection (4.8 +/- 3.2 versus 4.2 +/- 2.5 min, n = 21, p < 0.05). CONCLUSION: Fractional MTT was demonstrated to be useful in differentiating acute rejection and ATN in transplanted kidneys.

[Dual-energy data acquisition for simultaneous 81mKr ventilation/99mTc-MAA perfusion SPECT: ventilation/perfusion ratio mapping in cross section].

Methods of simultaneous pulmonary ventilation/perfusion (V/Q) tomogram (SPECT) for V/Q ratio mapping were reported. Crosstalk was corrected by subtraction assuming crosstalk/signal ratio is constant for an individual examination. Crosstalk was reduced by 85% for that of 99mTc-to-81mKr window (R12), and by 83% for that of 81mKr-to-99mTc window (R21) in a phantom experiment. The R12 and R21 were 1.0-1.9% (mean 1.3%) and 34.6-55.3% (mean 41.9%), respectively in 9 patients (mean age, 56.1 years; range, 38-82 years). V/Q SPECT was undertaken in 7 of the 9 patients. 99mTc-macroaggregated albumin (185 MBq) was injected intravenously in supine position, and 81mKr gas was administered continuously during the SPECT data acquisition. Sixty-four 64 x 64-matrix (size = 8 mm/pixel) images, 20-second acquisition time for each, were collected by a rotating gamma camera in a step-and-shoot fashion during a 360-degree rotation with the two photopeaks, one at 140 keV for 99mTc and another at 190 keV for 81mKr. Examination time was about 40 minutes, and the V/Q ratio maps in cross section were obtained in all of the examinations.

[A new polar map to quantify reversible area by myocardial 201Tl single photon emission computed tomography and clinical application to patients before and after percutaneous transluminal coronary angioplasty].

In the studies of stress 201Tl single photon emission computed tomography, we have developed a computer method to discriminate reversible from irreversible defect, then quantify each area and display the area on the polar map. Initial percent uptake (%Ui) and delayed percent uptake (%Ud) were expressed as percent of the maximum uptake area and were defined as regional myocardial 201Tl uptake in initial and delayed image, respectively. The %Ud/%Ui ratio was defined as redistribution ratio (RD-ratio). The values of %Ui obtained from each pixel of short axis slices were plotted against the values of RD-ratio on XY co-ordinates. In this graphic display, normal area, ischemic viable area and non viable area were separated by the following four lines. A; The straight line (Y = 2.0-0.012X) indicating the relationship between %Ui and RD-ratio for the group with ischemic viable myocardium. B; The parallel line to A and shifted to -1.5 SD from A. C; The vertical line at 67.0% level (ischemic upper level). D; The vertical line at 27.6% level (viable lower level). Each area was discriminated by color display and calculated relative area values were displayed on the polar map. Criteria for discriminating each area were derived from the results of ischemic pre-transluminal coronary angioplasty (PTCA) lesions (n = 21) in which viability was confirmed by successful PTCA and previous results of 66 cases with single vessel disease and 16 cases of control group. This new computerized technique was applied for evaluation of another group with successful PTCA (n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)

[Three-dimensional display of 99mTc-MIBI myocardial scintigraphy].

One of 99mTc-hexakis, 99mTc-methoxyisobutyl isonitrile (MIBI), has been demonstrated to have a myocardial uptake proportional to regional coronary blood flow. In this study, 99mTc-MIBI myocardial scintigraphy were performed for 16 patients with ischemic heart disease. After injection of 740 MBq of 99mTc-MIBI, 64 projection images were collected during a 360-degree rotation. Three-dimensional (3D) display of the left ventricle was reconstructed with depth-shading method from 99mTc-MIBI SPECT images, which were reconstructed by filtered back projection method. In 9 of the patients, left ventricular cineangiography were performed as diagnostic gold standard. Four physicians blinded to patients' clinical informations interpreted 3D images and SPECT images on separate occasions. Diagnosis of hypoperfusion by 3D displays agreed with those of SPECT in 92.9% (104/112 segments), and disagreed in 7.1% (8 segments). Sensitivity and specificity of 3D images were 87.0 and 93.9%, which were not statistically different (p less than 0.05) from that of SPECT images (91.3, 97.0%). Receiver operating characteristic (ROC) analysis revealed nearly identical curves for the two. Although 3D display had nearly identical diagnostic ability with SPECT, observers reported that 3D images were easier to diagnose than SPECT images. An advantage of the 3D display is that the display gives a more realistic impression of the left ventricle to an observer than tomography or planar imaging. Another advantage is that 3D display can reduce the amount of data storage compared with that of SPECT. In conclusion, 3D images may be useful for diagnosis of hypoperfusion of left ventricle.

Value of three-dimensional surface display of brain perfusion imaging. Comparison with tomographic imaging.

The diagnostic ability of a three-dimensional surface display of I-123 IMP brain scintigrams and tomographic images was compared in 12 patients who underwent I-123 brain tomography for cerebral cortical hypoperfusion. Foci of decreased accumulation were independently diagnosed by four radiologists, first with transaxial images, second with three-dimensional images, and finally with both images subjectively compared for the purpose of diagnosing the existence, localization, and extent of lesions. Six different lesions (0 to 3 lesions/radiologist) were found only on the transaxial images, but 30 different lesions (1 to 14 lesions/radiologist) were found on the three-dimensional images. In 9 out of 12 cases, the three-dimensional images were regarded as superior for evaluating the extension of the lesion and for lesion existence and lesion localization in 5 out of 12 (P less than 0.05). In conclusion, a three-dimensional display of an I-123 IMP brain tomogram is superior to tomographic images in diagnosing the extent of cerebral cortical hypoperfusion. However, the display is not superior to tomographic images for diagnosing the existence and localization of hypoperfusion.