Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae.

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3-14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.

Intracellular inhibition of human neutrophil elastase by orally active pyrrolidine-trans-lactams.

Described are the acylation binding of trans-lactam 1 to porcine pancreatic elastase, the selection of the SO2Me activating group for the lactam N which also confers metabolic stability in hamster liver microsomes, the introduction of aqueous solubility through the piperidine salt 9, the in vivo oral activity of 9 and its bioavailability, and the introduction of 9 as an intracellular neutrophil elastase inhibitor.

Value of radiopaque markers in identifying partial small bowel obstruction.

Confirming partial small bowel obstruction is often a diagnostic challenge. In this case report, 4-mm solid radiopaque markers were used in 4 patients to show partial small bowel obstruction. Results of enteroclysis were normal in 2 of the 4 patients, and the markers were used to challenge suspected partial obstruction. The markers coalesced in the region of the partial obstruction, which was confirmed at surgery. Enteroclysis is the examination of choice in the diagnosis of partial small bowel obstruction. However, examinations with false-negative results can occur, particularly with adhesive and/or intermittent obstructions. The use of radiopaque markers in these cases proved an effective and useful method of establishing the diagnosis of partial small bowel obstruction, particularly in the 2 cases in which enteroclysis results were normal. Prospective studies are needed to establish the feasibility of this novel technique.

The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice.

The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed. CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers. Expression of this protein in plants imparts high levels of glyphosate tolerance. The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics. CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed, There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold tha anticipated consumption level of food products potentially containing CP4 EPSPS protein. CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins. These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybean are as safe and nutritious as traditional soybeans currently being marketed.

The effects of lossy compression on the detection of subtle pulmonary nodules.

We examined the ability of radiologists to detect pulmonary nodules in computed radiographic (CR) chest images subjected to lossy image compression. Low-contrast 1-cm diameter targets simulating noncalcified pulmonary nodules were introduced into clinical images and presented to ten radiologists in a series of two-alternative forced-choice (2AFC) observer experiments. The percentages of correct observer responses obtained while viewing noncompressed images (1:1) were compared with those obtained for the same images compressed 7:1, 16:1, 44:1, and 127:1. The images were compressed using a standard full-frame discrete cosine transform (DCT) technique. The degree of compression was determined by quantizing Fourier components in various frequency channels and then Huffman encoding the result. The data show a measurable decline in performance for each compression ratio. Through signal-to-noise ratio (SNR) analysis, we found that the reduction in performance was due primarily to the compression algorithm that increased image noise in the frequency channels of the signals to be detected.

Ultrasonic measurement of glomerular diameters in normal adult humans.

The average size of acoustic scatterers and the integrated backscatter coefficient of kidney cortex were measured in vivo from 2-4 MHz for a group of 50 normal adult volunteers. Our goal was to determine the sensitivity of the ultrasonic measurements under clinical conditions by identifying biologic sources of estimation uncertainty. Based on 10 measurements on each kidney of each volunteer, the average glomerular diameter for the group was found to be 216 +/- 27 microns (SD). Glomerular size was found to correlate with body surface area (r = 0.4), and the ratio of glomerular surface area to body surface area (GSA/BSA) was found to be constant throughout normal adult life with GSA/BSA = (8.24 +/- 1.35) x 10(-8) (SD). These results are consistent with histologic analyses found in the literature. Within an individual, 7% standard errors in GSA/BSA are typical. Biologic variability dominates the variance in scatterer size estimates in a group not matched for BSA, where it accounts for 47% of the variance. In a group of individuals matched for BSA, biologic variability accounts for only 21% of the variance; day-to-day variability accounts for 35% of the variance; and experimental parameters account for the remainder. If a deviation greater than 2 x SD is considered abnormal, then this technique can potentially detect changes in glomerular diameter as small as 30 microns within an individual. To detect abnormal GSA/BSA values for an individual, GSA/BSA would have to differ from the mean for that group by about 3.6 x 10(-8) or about 40%. Therefore, at this time scatterer size estimates appear most reliable for tracking the progression of disease and treatment for an individual over time.

Doppler US of helical flow in the portal vein.

In helical portal venous blood flow, the usual laminar flow in the portal vein is replaced by a spiral. This changes the color Doppler ultrasound (US) appearance to one of alternating or parallel red and blue bands. Duplex US may appear to show hepatopetal, hepatofugal, or simultaneous bidirectional flow depending on placement of the cursor within the helix. Helical portal venous flow is unusual in normal individuals (2.2% of 135 patients). Its presence should prompt further scrutiny for signs of liver disease, particularly portosystemic shunts, as in 20% of 41 patients who subsequently underwent liver transplantation. It is a normal finding immediately after liver transplantation (43% of 35 patients) and transjugular intrahepatic portosystemic shunt (TIPS) creation (28% of 36 patients). In both liver transplant and TIPS recipients, helical flow is usually transient. Its persistence long after transplantation in association with a prolonged increase in portal venous velocity is a useful sign of portal vein stenosis. Helical flow may also occur in cases of neoplastic invasion or displacement of the portal vein.

Ultrasound contrast-detail analysis: a comparison of low-contrast detectability among scanhead designs.

Contrast-detail (CD) analysis was used to compare the low-contrast detection capabilities of expert observers using different array-type scanhead technologies. Five expert observers viewed five different contrast targets to obtain CD curves for each scanhead. Differences in CD curves are interpreted in terms of the image contrast, resolution, and noise. It was found that differences in low-contrast detectability were due to differences in beam properties. Clinical images obtained during patient examinations are used to show how some clinically relevant tasks are distributed in their contrast and size.

Abdominal wall hernias: review of herniography and correlation with cross-sectional imaging.

Herniography is an accurate means of identifying groin hernias when the clinical diagnosis is uncertain. Its role in evaluation of other types of ventral hernias is less clear; however, with minor modifications in technique, herniography is also useful in these cases. This article reviews the technique of herniography, normal variations in anatomy, and interpretation of herniograms of the groin and anterior abdominal wall on the basis of 72 patients studied over 3 1/2 years. Herniography is also compared with cross-sectional imaging techniques. Inguinal and femoral hernias are best shown and classified with herniography. Ventral hernias are well demonstrated by both herniography and cross-sectional imaging; however, herniation of only fat and supine imaging may lead to misdiagnosis with computed tomography or ultrasound. Spigelian hernias are probably best depicted with cross-sectional imaging. Diagnostic pitfalls of herniography are reviewed, including the need for obtaining postexercise radiographs, oblique radiographs, and tangential radiographs.

Effects of endothelin-1 on renal microvasculature measured using quantitative ultrasound.

Renal vascular resistance is an important feature of kidney function and disease. To maintain adequate blood flow, renal vascular resistance varies in response to changes in systemic pressure. Vascular resistance is largely determined by arteriolar diameter, which is regulated by local and systemic factors. We used quantitative ultrasound techniques to follow renal vascular changes in anesthetized dogs during local intraarterial infusion of a potent vasoconstrictor, endothelin-1 (ET-1). Average arteriolar diameters were estimated by analyzing echo-signal spectra (5-15 MHz) obtained from renal cortex in vivo before, during, and after ET-1 infusion. At calculated arterial concentrations of 0.01 nM, 0.1 nM, and 1.0 nM, ET-1 reduced the average arteriolar diameter of 38 +/- 2 microns by 2%, 63%, and 91%, respectively, without producing a significant change in systemic blood pressure. Changes in scatterer size were consistent with the observed changes in renal hemodynamics detected using Doppler techniques. In addition, acoustic attenuation was found to increase with ET-1 concentration. These data suggest that quantitative ultrasound methods are sensitive to changes in renal arteriolar diameter, and may be a new noninvasive method for continuously monitoring changes in vascular resistance.

Ultrasound contrast-detail analysis: a preliminary study in human observer performance.

Results of a human observer performance study using a new ultrasound contrast-detail (CD) phantom are reported. The new phantom allows estimates of the fundamental statistical uncertainties involved in CD analysis. Results of these experiments show that observers generally considered to be "trained" require experience at the specific task of low-contrast target detection under signal known exactly (SKE) conditions, and that observers require an orientation period at the beginning of each observation session to obtain stable responses. The results obtained in this study can be used to estimate the number of independent images, observers, and repeat observation sessions required for a desired uncertainty in CD curves, and show that 10% fractional standard deviations are obtainable with modest observer effort.

Abnormal spinal curvature in the fetus.

We reviewed 20 cases of sonographically detected abnormal fetal spinal curvature to help determine the significance of this finding. Both marked (13 cases) and mild (7 cases) curves were diagnosed sonographically. Sonography showed associated anomalies in 19 fetuses, including neural tube and ventral wall defects. Outcomes were poor, with only three infants surviving. Isolated scoliosis was identified prenatally in one neurologically normal infant with hemivertebrae. We conclude that abnormal spinal curvature in the fetus is a significant finding, whether mild or severe. Even as an isolated finding, it is significant, as prenatal detection assists postnatal management.

Image calibration of laser digitizers, printers, and gray-scale displays.

Laser film digitizers, interactive gray-scale monitors, and laser film printers are necessary to transmit digital image information. These devices must be standardized so that hard- and soft-copy images are as similar as possible. Standardization of appropriate calibration procedures is necessary to attain this goal. Radiographs are converted into digital data representations by a laser film digitizer. These representations (and those obtained with other modalities) are transferred to a laser film printer or to an interactive monitor with gray-scale display. To obtain the best gray-level fidelity, printer output optical densities should be identical to those of the input film. Laser printers should be calibrated regularly to ensure uniform results. A gray-scale controller functions as an adjunct to the host computer and can automate the calibration process. Gray-scale controller functions may someday be incorporated into an accelerator or array-processor board.

Enhancement of storage phosphor plate images: a C-language program.

A C-language software program has been developed for emulating the image enhancement processing of a storage phosphor plate system. This software has been implemented on a VAX 3400 computer. There are 2,100 lines of C-language code in the program. There are seven parameters used to specify the degree of enhancement. The software is being implemented on a single accelerator board.

Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain.

Pseudomonas aureofaciens Q2-87 produces the antibiotic 2,4-diacetophloroglucinol (Phl), which inhibits Gaeumannomyces graminis var. tritici and other fungi in vitro. Strain Q2-87 also provides biological control of take-all, a root disease of wheat caused by this fungus. To assess the role of Phl in the antifungal activity of strain Q2-87, a genetic analysis of antibiotic production was conducted. Two mutants of Q2-87 with altered antifungal activity were isolated by site-directed mutagenesis with Tn5. One mutant, Q2-87::Tn5-1, did not inhibit G. graminis var. tritici in vitro and did not produce Phl. Two cosmids were isolated from a genomic library of the wild-type strain by probing with the mutant genomic fragment. Antifungal activity and Phl production were coordinately restored in Q2-87::Tn5-1 by complementation with either cosmid. Mobilization of one of these cosmids into two heterologous Pseudomonas strains conferred the ability to synthesize Phl and increased their activity against G. graminis var. tritici, Pythium ultimum, and Rhizoctonia solani in vitro. Subcloning and deletion analysis of these cosmids identified a 4.8-kb region which was necessary for Phl synthesis and antifungal activity.