DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

Summary The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)₄, (GTG)₅, (CA)₈, and (TCC)₅, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.     

Sequence diversity of the nucleoprotein gene of iris yellow spot virus (genus Tospovirus, family Bunyaviridae) isolates from the western region of the United States

Summary. Iris yellow spot virus (IYSV), a tentative virus species in the genus Tospovirus and family Bunyaviridae, is considered a rapidly emerging threat to onion production in the western United States (US). The present study was undertaken to determine the sequence diversity of IYSV isolates from infected onion plants grown in California, Colorado, Idaho, Oregon, Utah and Washington. Using primers derived from the small RNA of IYSV, the complete sequence of the nucleoprotein (NP) gene of each isolate was determined and the sequences compared. In addition, a shallot isolate of IYSV from Washington was included in the study. The US isolates of IYSV shared a high degree of sequence identity (95 to 99%) with one another and to previously reported isolates. Phylogenetic analyses showed that with the exception of one isolate from central Oregon and one isolate from California, all the onion and shallot isolates from the western US clustered together. This cluster also included onion and lisianthus isolates from Japan. A second distinct cluster consisted of isolates from Australia (onion), Brazil (onion), Israel (lisianthus), Japan (alstroemeria), the Netherlands (iris) and Slovenia (leek). The IYSV isolates evaluated in this study appear to represent two distinct groups, one of which largely represents isolates from the western US. Understanding of the population structure of IYSV would potentially provide insights into the molecular epidemiology of this virus.     

Neuronal histamine in the gut wall releasable by gastrin and cholecystokinin

Histamine accumulated in the ligated vagus nerve of the rat, both above and below the ligature; maximum accumulation was after 4 h. The finding is suggestive of axonal flow. Further evidence for histamine in peripheral nerves was obtained in experiments showing that the guinea-pig gut wall could be labelled with [3H]histamine. The experiments were carried out with isolated strips of stomach wall and taenia coli. Electrical stimulation released [3H]histamine from these specimens. The release could be blocked by Ca2+-free medium or by tetrodotoxin. The release was unaffected by vagal denervation or chemical sympathectomy (6-hydroxydopamine) but prevented by reserpinization. Gastrin-17 and cholecystokinin-39 released radioactivity by a tetrodotoxin-sensitive mechanism. The possible existence of a gastrin/cholecystokinin-sensitive neuronal pool of histamine in the gut wall offers a new perspective on the postulated role of histamine as a physiological stimulant of gastric acid secretion and might explain why H2-receptor antagonists block gastrin-stimulated acid secretion.     

Risk factors for HIV infection in injection drug users and evidence for onward transmission of HIV to their sexual partners in Chennai, India

OBJECTIVES: Determining HIV prevalence in injection drug users (IDUs) and their regular sex partners in Chennai, India. METHODS: A total of 226 IDUs and their regular sex partners were enrolled during April-July 2003. After informed consent was obtained, a semistructured questionnaire was administered and serum was tested for HIV antibody. RESULTS: The HIV seroprevalence was 30% (68/226) in IDUs and 5% in their regular sex partners (11/226). While in 25% of couples only the male partner was HIV positive, 5% of the couples were concordant for HIV infection and 70% were HIV negative. Fifty-seven percent of the HIV-positive IDUs and 45% of the HIV-infected women thought that they had "no chance" or "very little chance" of getting HIV, reflecting low HIV risk perception. More than 20% IDUs reported borrowing or lending of injection equipment. In univariate analyses "sex" and "condom use" with sex workers had no bearing but "more than twice a day injecting frequency," "history of incarceration," "tattoos," "recruitment from northern part of the city," and ever-injecting drugs in drug-selling places had significant association with HIV infection in IDUs. In an adjusted model, the odds of HIV infection were 2 times higher among IDUs who had ever injected drugs in drug-selling places and 6 times higher in those who were recruited from the northern part of central Chennai. CONCLUSION: Reducing sharing of injection equipment and unsafe tattooing through targeted and environmental interventions, increasing HIV risk perception, and promoting safer sex practices among IDUs and their sex partners are urgent program needs.     

On the use of EPID-based implanted marker tracking for 4D radiotherapy

Four-dimensional (4D) radiotherapy delivery to dynamically moving tumors requires a real-time signal of the tumor position as a function of time so that the radiation beam can continuously track the tumor during the respiration cycle. The aim of this study was to develop and evaluate an electronic portal imaging device (EPID)-based marker-tracking system that can be used for real-time tumor targeting, or 4D radiotherapy. Three gold cylinders, 3 mm in length and 1 mm in diameter, were implanted in a dynamic lung phantom. The phantom range of motion was 4 cm with a 3-s "breathing" period. EPID image acquisition parameters were modified, allowing image acquisition in 0.1 s. Images of the stationary and moving phantom were acquired. Software was developed to segment automatically the marker positions from the EPID images. Images acquired in 0.1 s displayed higher noise and a lower signal-noise ratio than those obtained using regular (> 1 s) acquisition settings. However, the markers were still clearly visible on the 0.1-s images. The motion of the phantom blurred the images of the markers and further reduced the signal-noise ratio, though they could still be successfully segmented from the images in 10-30 ms of computation time. The positions of gold markers placed in the lung phantom were detected successfully, even for phantom velocities substantially higher than those observed for typical lung tumors. This study shows that using EPID-based marker tracking for 4D radiotherapy is feasible, however, changes in linear accelerator technology and EPID-based image acquisition as well as patient studies are required before this method can be implemented clinically.     

The effect of dose calculation uncertainty on the evaluation of radiotherapy plans

Monte Carlo dose calculations will potentially reduce systematic errors that may be present in currently used dose calculation algorithms. However, Monte Carlo calculations inherently contain random errors, or statistical uncertainty, the level of which decreases inversely with the square root of computation time. Our purpose in this study was to determine the level of uncertainty at which a lung treatment plan is clinically acceptable. The evaluation methods to decide acceptability were visual examination of both isodose lines on CT scans and dose volume histograms (DVHs), and reviewing calculated biological indices. To study the effect of systematic and/or random errors on treatment plan evaluation, a simulated "error-free" reference plan was used as a benchmark. The relationship between Monte Carlo statistical uncertainty and dose was found to be approximately proportional to the square root of the dose. Random and systematic errors were applied to a calculated lung plan, creating dose distributions with statistical uncertainties of between 0% and 16% (1 s.d.) at the maximum dose point and also distributions with systematic errors of -16% to 16% at the maximum dose point. Critical structure DVHs and biological indices are less sensitive to calculation uncertainty than those of the target. Systematic errors affect plan evaluation accuracy significantly more than random errors, suggesting that Monte Carlo dose calculation will improve outcomes in radiotherapy. A statistical uncertainty of 2% or less does not significantly affect isodose lines, DVHs, or biological indices.     

Chaetogaster limnaei (Annelida: Oligochaeta) as a parasite of the zebra mussel Dreissena polymorpha,and the quagga mussel Dreissena bugensis (Mollusca: Bivalvia)

Dreissenid mussels, Dreissena polymorpha and D. bugensis, were found to be infected by the naidid oligochaete Chaetogaster limnaei at four sites in the St. Lawrence River. This is the first report of this species infecting dreissenids anywhere in the world. Most worms inhabited the mantle cavity, where they caused erosion of the mantle and gill epithelia as determined by histopathological examination. Others penetrated various tissues; one had invaded the ovary and was feeding on oocytes and ovarian tissues. Of 606 mussels examined, 166 (27.4%) harbored at least 1 C. limnaei. The prevalence varied between 1% and 80%, depending on the collection site and date. The worms were slightly but significantly more prevalent in D. bugensis than in D. polymorpha. The intensity ranged from 1 to 18 worms per infected host. Variations in prevalence and intensity were not related to the size or sex of the host, but the data did suggest some seasonality.     

Mutations selected in rotavirus enterotoxin NSP4 depend on the context of its expression

The rotavirus NSP4 protein is cytotoxic when transiently expressed in cells and is capable of inducing secretory diarrhea in neonatal mice. NSP4 consists of 175 amino acids, and sequences important for its toxic effects have been mapped to the carboxy-terminal half of the protein. In this report, we compared NSP4-encoding nucleotide sequences recovered from cell lines engineered to express NSP4 from human rotavirus strain Wa with NSP4 sequences recovered from cells persistently infected with either Wa or simian rotavirus strain SA11. In cells stably transfected with Wa NSP4, we found that proline(138) was changed to either serine or threonine. However, in cells persistently infected with SA11, we found that phenylalanine(33) was changed to leucine, and in cells persistently infected with Wa, no changes were observed in NSP4. Expression of Wa NSP4 in Caco-2 cells resulted in increased cell-doubling times and decreased cell viability in comparison to cells expressing NSP4-serine(138) or NSP4-threonine(138). This result suggests that sequence polymorphism at residue 138 in Wa NSP4 influences the cytotoxicity of the protein. Therefore, mutations in the carboxy-terminal half of NSP4 are selected when NSP4 is expressed in cells in the absence of other viral proteins, but not in the context of viral replication. These findings suggest that cytotoxic functions of NSP4 are not operant during natural rotavirus infection.     

A method for photon beam Monte Carlo multileaf collimator particle transport

Monte Carlo (MC) algorithms are recognized as the most accurate methodology for patient dose assessment. For intensity-modulated radiation therapy (IMRT) delivered with dynamic multileaf collimators (DMLCs), accurate dose calculation, even with MC, is challenging. Accurate IMRT MC dose calculations require inclusion of the moving MLC in the MC simulation. Due to its complex geometry, full transport through the MLC can be time consuming. The aim of this work was to develop an MLC model for photon beam MC IMRT dose computations. The basis of the MC MLC model is that the complex MLC geometry can be separated into simple geometric regions, each of which readily lends itself to simplified radiation transport. For photons, only attenuation and first Compton scatter interactions are considered. The amount of attenuation material an individual particle encounters while traversing the entire MLC is determined by adding the individual amounts from each of the simplified geometric regions. Compton scatter is sampled based upon the total thickness traversed. Pair production and electron interactions (scattering and bremsstrahlung) within the MLC are ignored. The MLC model was tested for 6 MV and 18 MV photon beams by comparing it with measurements and MC simulations that incorporate the full physics and geometry for fields blocked by the MLC and with measurements for fields with the maximum possible tongue-and-groove and tongue-or-groove effects, for static test cases and for sliding windows of various widths. The MLC model predicts the field size dependence of the MLC leakage radiation within 0.1% of the open-field dose. The entrance dose and beam hardening behind a closed MLC are predicted within +/- 1% or 1 mm. Dose undulations due to differences in inter- and intra-leaf leakage are also correctly predicted. The MC MLC model predicts leaf-edge tongue-and-groove dose effect within +/- 1% or 1 mm for 95% of the points compared at 6 MV and 88% of the points compared at 18 MV. The dose through a static leaf tip is also predicted generally within +/- 1% or 1 mm. Tests with sliding windows of various widths confirm the accuracy of the MLC model for dynamic delivery and indicate that accounting for a slight leaf position error (0.008 cm for our MLC) will improve the accuracy of the model. The MLC model developed is applicable to both dynamic MLC and segmental MLC IMRT beam delivery and will be useful for patient IMRT dose calculations, pre-treatment verification of IMRT delivery and IMRT portal dose transmission dosimetry.