bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostate brachytherapy seed localization by analysis of multiple projections: Identifying and addressing the seed overlap problem
Authors: Su Yi , Davis Brian J. , Herman Michael G. , Robb Richard A.
Published: May 2004

Intraoperative three‐dimensional reconstruction of seed locations during prostate brachytherapy for purposes of immediate computation of radiation dosimetry is an active area of current investigation, including methods which use multiple fluoroscopic projections. A simulation study using seed locations extracted from clinical CT data was performed; the result showed that on average one quarter of the seeds had a projection image overlapping with other seeds. The average percentage of non‐overlapping seeds for the prostate implants and seed types investigated was 74.5% with a range of 56.9%–92.9%. The distribution of seeds in different cluster sizes was analyzed as well as the distribution of pixel counts of connected components. A statistical classifier was developed to determine the number of seed images in a self‐connected component in the segmented images. The classifier was tested with simulation data, and the error rate was below 2%. A method to determine seed image position is also provided. A modified three‐film technique was used to reconstruct 3‐D seed locations. The algorithm allows unequal number of seed images for each projection as input while current methods require the same number of seed images detected in all projections. An accuracy analysis based on angular and positional uncertainty was performed. The reconstruction and seed localization algorithms were tested with simulation data, and the mean distance error of the reconstructed results was 0.61 mm. A phantom study was performed to validate the seed localization method. Three false positive seeds, 4.7% of the total, in the reconstruction result were observed in this study.
Evaluation of the imaging properties of an amorphous selenium‐based flat panel detector for digital fluoroscopy
Authors: Hunt D. C. , Tousignant O. , Rowlands J. A.
Published: May 2004

The imaging performance of an amorphous selenium flat‐panel detector for digital fluoroscopy was experimentally evaluated using the spatial frequency dependent modulation transfer function (MTF), noise power spectrum (NPS), and detective quantum efficiency (DQE). These parameters were investigated at beam qualities and exposures within the range typical of gastrointestinal fluoroscopic imaging 75 kV). The investigation does not take into consideration the detector cover, which in clinical use will lower the DQE measured here by its percent attenuation. The MTF was found to be less than the expected aperture response and the NPS was not white which together indicate presampling blurring. The cause of this blurring was attributed to charge trapping at the interface between two different layers of the The effect on the DQE was also consistent with presampling blur, which reduces the aliasing in the NPS and thereby reduces the spatial frequency dependence of the DQE. (The DQE was independent of spatial frequency from 0.12 to due to antialiasing of the NPS.) Moreover, the first zero of the measured MTF and the aperture response appeared at the same spatial frequency for a pixel of 150 μm). Hence, the geometric fill factor (77%) was increased to an effective fill factor of A large scale correlation in the noise due to the configuration of the readout electronics caused increased noise power in the gate line NPS at low spatial frequency The DQE was exposure independent over a large range of exposures but became exposure dependent at low exposures due to the electronic noise.
Classification of mammographic masses using generalized dynamic fuzzy neural networks
Authors: Lim Wei Keat , Er Meng Joo
Published: May 2004

In this article, computer‐aided classification of mammographic masses using generalized dynamic fuzzy neural networks (GDFNN) is presented. The texture parameters, derived from first‐order gradient distribution and gray‐level co‐occurrence matrices, were computed from the regions of interest. A total of 343 images containing 180 benign masses and 163 malignant masses from the Digital Database for Screening Mammography were analyzed. A fast approach of automatically generating fuzzy rules from training samples was implemented to classify tumors. This work is novel in that it alleviates the problem of requiring a designer to examine all the input‐output relationships of a training database in order to obtain the most appropriate structure for the classifier in a conventional computer‐aided diagnosis. In this approach, not only the connection weights can be adjusted, but also the structure can be self‐adaptive during the learning process. By virtue of the automatic generation of the classifier by the GDFNN learning algorithm, the area under the receiver‐operating characteristic curve, attains 0.868±0.020, which corresponds to a true‐positive fraction of 95.0% at a false positive fraction of 52.8%. The corresponding accuracy is 70.0%, the positive predictive value is 62.0%, and the negative predictive value is 91.4%.
Fractional scan algorithms for low‐dose perfusion CT
Authors: Hsieh Jiang , Wei Yuchuan , Wang Ge
Published: May 2004

One of the key considerations in CT perfusion is the X‐ray dose received by the patient, since cine acquisition mode is typically used in which repeated x‐ray exposure occurs at the same anatomical location. In this technical note, we propose a new approach for dose reduction in which the X‐ray tube current is maintained and the number of projection views is reduced per gantry rotation. A projection interpolation algorithm and an image prediction algorithm are developed to address the incompleteness of such projection data. Both algorithms utilize a priori knowledge that the contrast‐uptake in perfusion images is gradual and predictable. As a result, excellent perfusion images can be reconstructed at a fraction of the nominal radiation dose.
Calibration of a scintillation dosemeter for beta rays using an extrapolation ionization chamber
Authors: Hakanen A. T. , Sipilä P. M. , Kosunen A.
Published: May 2004

A scintillation dosemeter is calibrated for beta rays from an ophthalmic applicator, using an extrapolation ionization chamber as a reference instrument. The calibration factor for the scintillation dosemeter agrees with that given by the manufacturer of the dosemeter within ca. 2%. The estimated overall uncertainty of the present calibration is ca. 6% (2 sd). A calibrated beta‐ray ophthalmic applicator can be used as a reference source for further calibrations performed in the laboratory or in the hospital.
Modeling scintillator‐photodiodes as detectors for megavoltage CT
Authors: Monajemi T. T. , Steciw S. , Fallone B. G. , Rathee S.
Published: May 2004

The use of cadmium tungstateand cesium iodide [CsI(Tl)] scintillation detectors is studied in megavoltage computed tomography (MVCT). A model describing the signal acquired from a scintillation detector has been developed which contains two steps: (1) the calculation of the energy deposited in the crystal due to MeV photons using theEGSnrcMonte Carlo code; and (2) the transport of the optical photons generated in the crystal voxels to photodiodes using the optical Monte Carlo codeDETECT2000. The measured detector signals in singleand CsI(Tl) scintillation crystals of baseand heights 0.4, 1, 1.2, 1.6 and 2 cm were, generally, in good agreement with the signals calculated with the model. A prototype detector array which contains 8crystals, eachin contact with a 16‐element array of photodiodes was built. The measured attenuation of a Cobalt‐60 beam as a function of solid water thickness behaves linearly. The frequency dependent modulation transfer functionnoise power spectrumand detective quantum efficiencywere measured for 1.25 MeV photons (in a Cobalt‐60 beam). For 6 MV photons, only thewas measured from a linear accelerator, where large pulse‐to‐pulse fluctuations in the output of the linear accelerator did not allow the measurement of theA two‐step Monte Carlo simulation was used to model the detector'sandThe DQE(0) of the detector array was found to be 26% and 19% for 1.25 MeV and 6 MV photons, respectively. For 1.25 MeV photons, the maximum discrepancies between the measured and modeledrelativeand thewere found to be 1.5%, 1.2%, and 1.9%, respectively. For the 6 MV beam, the maximum discrepancy between the modeled and the measuredwas found to be 2.5%. The modeling is sufficiently accurate for designing appropriate detectors for MVCT.
Efficient particle transport simulation through beam modulating devices for Monte Carlo treatment planning
Authors: Fippel Matthias
Published: May 2004

For Monte Carlo treatment planning it is essential to model efficiently patient dependent beam modifying devices, e.g., Multi‐Leaf Collimators (MLC). Therefore a Monte Carlo geometry tracking procedure is presented allowing the simulation of photon and electron transport through these devices within short calculation time. The tracking procedure is based on elemental regions, on surfaces (mainly planes) to separate the regions as well as on bit patterns and bit masks to identify the regions. Photon cross sections for photoelectric absorption, Compton scattering and pair production as well as electron stopping powers and ranges are provided by the Physical Reference Data of the National Institute of Standards and Technology (NIST). The tracking procedure is implemented in with object‐oriented design based on class hierarchies and inheritance. Using the geometry technique, several MLC models are constructed. Some of them take into account tongue‐and‐groove effects as well as curved leaf ends. The models are integrated into the Monte Carlo dose calculation engine XVMC for treatment planning. The system is tested by comparing different MLC implementations and by verification with measurement.
TCP isoeffect analysis using a heterogeneous distribution of radiosensitivity
Authors: Carlone Marco , Wilkins David , Nyiri Balazs , Raaphorst Peter
Published: May 2004

A formula for the α/β ratio is derived using the heterogeneous (population averaged) tumor control model. This formula is nearly identical to the formula obtained using the homogeneous (individual) tumor control model, but the new formula includes extra terms showing that the α/β ratio, the ratio of the mean value of α divided by the mean value of β that would be observed in a patient population, explicitly depends on the survival level and heterogeneity. The magnitude of this correction is estimated for prostate cancer, and this appears to raise the mean value of the ratio estimate by about 20%. The method also allows investigation of confidence limits for α/β based on a population distribution of radiosensitivity. For a widely heterogeneous population, the upper 95% confidence interval for the α/β ratio can be as high as 7.3 Gy, even though the population mean is between 2.3 and 2.6 Gy.
Porous phantoms for PET and SPECT performance evaluation and quality assurance
Authors: DiFilippo Frank P. , Price James P. , Kelsch Daniel N. , Muzic Raymond F.
Published: May 2004

Characterization of PET and SPECT imaging performance often requires phantoms with complex radionuclide distributions. For example, lesion detection studies use multiple spherical regions of specific target‐to‐background ratios to simulate cancerous lesions. Such complex distributions are typically created using phantoms with multiple fillable chambers. However, such phantoms are typically difficult and time‐consuming to prepare accurately and reproducibly. A new approach using a single‐chamber phantom with a porous core can overcome these difficulties. Methods: Prototypes of two designs of porous core phantoms were produced and evaluated. The “hot spheres” phantom contained a multitude of simulated spherical lesions with diameters ranging from 6.35 to 25.4 mm (“multi‐resolution” slice) and with lesion‐to‐background ratios ranging from 1.6 to 4.4 (“multi‐contrast” slice). The “multi‐attenuation” phantom consisted of two halves. One half contained a porous core to produce regions of different attenuation but uniform activity. The other half mimicked the NEMA‐94 design with cold inserts of different attenuation. Results: Both phantoms produced the expected radionuclide distributions while requiring the preparation of only a single radionuclide solution and with much reduced preparation time. In images taken on clinical PET and SPECT scanners, the porous core structures were found to contribute negligible background noise or artifact. The measured lesion‐to‐background ratios from the hot spheres phantom differed slightly from calculated values, with the differences attributed mainly to uncertainty in pore diameter. The measured attenuation coefficients from the multi‐attenuation phantom agreed well with expected values. However, it was found that trapped air bubbles due to manufacturing defects in the porous core could potentially cause quantitative errors. Conclusion: The hot spheres and multi‐attenuation porous phantoms exhibited a wide range of imaging features providing thorough tests of lesion detection and of attenuation and scatter correction accuracy. Because the local activity concentration is set by the relative volume of radionuclide solution in the porous core, the quantitative accuracy is limited mainly by mechanical tolerance, and strict quality control during manufacturing is essential. Nonetheless, the single‐chamber design of the porous core phantoms is inherently more reproducible and more practical for routine use compared to conventional multi‐chamber phantoms.
X‐ray scatter correction algorithm for cone beam CT imaging
Authors: Ning Ruola , Tang Xiangyang , Conover David
Published: May 2004

Developing and optimizing an x‐ray scatter control and reduction technique is one of the major challenges for cone beam computed tomography (CBCT) because CBCT will be much less immune to scatter than fan‐beam CT. X‐ray scatter reduces image contrast, increases image noise and introduces reconstruction error into CBCT. To reduce scatter interference, a practical algorithm that is based upon the beam stop array technique and image sequence processing has been developed on a flat panel detector‐based CBCT prototype scanner. This paper presents a beam stop array‐based scatter correction algorithm and the evaluation results through phantom studies. The results indicate that the beam stop array‐based scatter correction algorithm is practical and effective to reduce and correct x‐ray scatter for a CBCT imaging task.
Simulation study for thermal dose optimization in ultrasound surgery of the breast
Authors: Malinen Matti , Huttunen Tomi , Hynynen Kullervo , Kaipio Jari P.
Published: May 2004

In this paper the previously published optimization algorithm for thermal dose optimization is tested with numerical simulations. The simulations concern the thermal dose optimization in ultrasound surgery of the breast. The optimization algorithm is extended by setting the inequality constraint approximations to temperature in healthy tissue as well as in tumor region. In addition, the simulations are accomplished in realistic 3D geometry with varying thermal parameters. Another topic of the paper is to show the potential of the hemispherical phased array applicator for ultrasound surgery of the breast. With such an applicator larger tissue volumes can be treated with shorter time as compared to single element transducers. In simulations the geometrical focus of the applicator was placed mechanically in the middle of the treatable region. The whole tumor region was then scanned electrically by changing the phase of the emitted wave from individual elements. The simulations indicate that a feasible treatment plan can be achieved. In simulated cases the desired thermal dose was achieved for tumors with diameter from 1.5 to 2.4 cm, depending on the position of tumor. The maximum temperature limitations of 45 °C in healthy region and 80 °C in tumor region can be maintained in all simulations.
GAFChromic film dosimetry with a flatbed color scanner for Leksell Gamma Knife therapy
Authors: Yamauchi M. , Tominaga T. , Nakamura O. , Ueda R. , Hoshi M.
Published: May 2004

GAFChromic films MD‐55‐2 have recently been established widely in industrial, scientific, and medical applications as radiation dosimeters. We applied these films to the dosimetry for Leksell Gamma Knife therapy. We used a flatbed image scanner to take digital images of irradiated MD‐55‐2, and the data were converted to Red, Green and Blue pixel values. The absorbed dose, as derived from the response curve of the Red pixel value, was consistent with the Leksell Gamma Plan dose planning system, for exposures using collimator sizes, 4 mm, 8 mm, and 14 mm. However, the maximum dose in the exposure of the 18 mm collimator was measured to be about 5% smaller than Gamma Plan due to the density effect of the compound material in the head phantom.
A new smoothing procedure to reduce delivery segments for static MLC‐based IMRT planning
Authors: Sun Xuepeng , Xia Ping
Published: May 2004

In the application of pixel‐based intensity‐modulated radiation therapy (IMRT) using the step‐and‐shoot delivery method, one major difficulty is the prolonged delivery time. In this study, we present an integrated IMRT planning system that involves a simple smoothing method to reduce the complexity of the beam profiles. The system consists of three main steps: (a) an inverse planning process based on a least‐square dose‐based cost function; (b) smoothing of the intensity maps; (c) reoptimization of the segment weights. Step (a) obtains the best plan with the lowest cost value using a simulated annealing optimization algorithm with discrete intensity levels. Step (b) takes the intensity maps obtained from (a) and reduces the complexity of the maps by smoothing the adjacent beamlet intensities. During this process each beamlet is assigned a structure index based on anatomical information. A smoothing update is applied to average adjacent beamlets with the same index. To control the quality of the plan, a predefined clinical protocol is used as an acceptance criterion. The smoothing updates that violate the criterion are rejected. After the smoothing process, the segment weights are reoptimized in step (c) to further improve the plan quality. Three clinical cases were studied using this system: a medulloblastoma, a prostate cancer, and an oropharyngeal carcinoma. While the final plans demonstrate a degradation of the original plan quality, they still meet the plan acceptance criterion. On the other hand, the segment numbers or delivery times are reduced by 40%, 20%, and 20% for the three cases, respectively.
Lag measurement in an active matrix flat‐panel imager
Authors: Schroeder C. , Stanescu T. , Rathee S. , Fallone B. G.
Published: May 2004

Lag and residual contrast have been quantified in an amorphous selenium active‐matrix flat‐panel imager (AMFPI) as a function of frame time, kilovoltage (kV) and megavoltage (MV) x‐ray photon energies and amount of radiation incident on the detector. The AMFPI contains a 200 μm thick layer deposited on a thin film transistor (TFT) array of size with an 85‐μm pixel pitch. For all energies, the lag (signal normalized to the signal due to exposure) for the first and second frame after exposure ranges from 0.45% to 0.91% and from 0.29% to 0.51%, respectively. The amount of lag was determined to be a function of the time after the x‐ray exposure irrespective of frame time or the magnitude of exposure. The lag for MV photon energies was slightly less than that for kV photon energies. The residual contrast for all energies studied ranges from 0.41% to 0.75% and from 0.219% to 0.41% for the and frames, respectively. These results show that lag and residual contrast in kV and MV radiographic applications are always less than 1% for the detection system used and only depend on the time after x‐ray exposure.
Origins of Bilateral Symmetry:
Hox
and
Dpp
Expression in a Sea Anemone
Authors: Finnerty John R. , Pang Kevin , Burton Pat , Paulson Dave , Martindale Mark Q.
Published: May 2004

Over 99% of modern animals are members of the evolutionary lineage Bilateria. The evolutionary success of Bilateria is credited partly to the origin of bilateral symmetry. Although animals of the phylum Cnidaria are not within the Bilateria, some representatives, such as the sea anemone
Nematostella vectensis
, exhibit bilateral symmetry. We show that
Nematostella
uses homologous genes to achieve bilateral symmetry: Multiple
Hox
genes are expressed in a staggered fashion along its primary body axis, and the transforming growth factor–β gene
decapentaplegic
(
dpp
) is expressed in an asymmetric fashion about its secondary body axis. These data suggest that bilateral symmetry arose before the evolutionary split of Cnidaria and Bilateria.
Regulation of an
ATG7
-
beclin 1
Program of Autophagic Cell Death by Caspase-8
Authors: Yu Li , Alva Ajjai , Su Helen , Dutt Parmesh , Freundt Eric , Welsh Sarah , Baehrecke Eric H. , Lenardo Michael J.
Published: May 2004

Caspases play a central role in apoptosis, a well-studied pathway of programmed cell death. Other programs of death potentially involving necrosis and autophagy may exist, but their relation to apoptosis and mechanisms of regulation remains unclear. We define a new molecular pathway in which activation of the receptor-interacting protein (a serine-threonine kinase) and Jun amino-terminal kinase induced cell death with the morphology of autophagy. Autophagic death required the genes
ATG7
and
beclin 1
and was induced by caspase-8 inhibition. Clinical therapies involving caspase inhibitors may arrest apoptosis but also have the unanticipated effect of promoting autophagic cell death.
Characterization of Humanized Antibodies Secreted byAspergillus niger
Authors: Ward Michael , Lin Cherry , Victoria Doreen C. , Fox Bryan P. , Fox Judith A. , Wong David L. , Meerman Hendrik J. , Pucci Jeff P. , Fong Robin B. , Heng Meng H. , Tsurushita Naoya , Gieswein Christine , Park Minha , Wang Huaming
Published: May 2004

Two different humanized immunoglobulin G1(κ) antibodies and an Fab′ fragment were produced byAspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage byA. nigerKexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex6GlcNAc2to Hex15GlcNAc2. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced byAspergillusand that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.
Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products
Authors: Pesaro Manuel , Nicollier Gilles , Zeyer Josef , Widmer Franco
Published: May 2004

Prior to registration of crop protection products (CPPs) their persistence in soil has to be determined under defined conditions. For this purpose, soils are collected in the field and stored for up to 3 months prior to the tests. During storage, stresses like drying may induce changes in microbiological soil characteristics (MSCs) and thus may influence CPP degradation rates. We investigated the influence of soil storage-related stress on the resistance and resilience of different MSCs by assessing the impact of a single severe drying-rewetting cycle and by monitoring recovery from this event for 34 days. The degradation and mineralization of the fungicide metalaxyl-M and the insecticide lufenuron were delayed by factors of 1.5 to 5.4 in the dried and rewetted soil compared to the degradation and mineralization in an undisturbed reference. The microbial biomass, as estimated by direct cell counting and from the soil DNA content, decreased on average by 51 and 24%, respectively. The bulk microbial activities, as determined by measuring substrate-induced respiration and fluorescein diacetate hydrolysis, increased after rewetting and recovered completely within 6 days after reequilibration. The effects onBacteria,Archaea, andPseudomonaswere investigated by performing PCR amplification of 16S rRNA genes and reverse-transcribed 16S rRNA, followed by restriction fragment length polymorphism (RFLP) and terminal RFLP (T-RFLP) fingerprinting. Statistical analyses of RFLP and T-RFLP profiles indicated that specific groups in the microbial community were sensitive to the stress. In addition, evaluation of rRNA genes and rRNA as markers for monitoring the stress responses of microbial communities revealed overall similar sensitivities. We concluded that various structural and functional MSCs were not resistant to drying-rewetting stress and that resilience depended strongly on the parameter investigated.
Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells
Authors: Muñoz Rosario , García José L. , Carrascosa Alfonso V. , Gonzalez Ramon
Published: May 2004

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in
Escherichia coli
and
Saccharomyces cerevisiae
. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.
A Novel Alpha-Proteobacterium Resides in the Mitochondria of Ovarian Cells of the TickIxodes ricinus
Authors: Beninati Tiziana , Lo Nathan , Sacchi Luciano , Genchi Claudio , Noda Hiroaki , Bandi Claudio
Published: May 2004

An intracellular bacterium fromIxodes ricinusticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of theProteobacteria. ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.
Bacterial Community Dynamics and Hydrocarbon Degradation during a Field-Scale Evaluation of Bioremediation on a Mudflat Beach Contaminated with Buried Oil
Authors: Röling Wilfred F. M. , Milner Michael G. , Jones D. Martin , Fratepietro Francesco , Swannell Richard P. J. , Daniel Fabien , Head Ian M.
Published: May 2004

A field-scale experiment with a complete randomized block design was performed to study the degradation of buried oil on a shoreline over a period of almost 1 year. The following four treatments were examined in three replicate blocks: two levels of fertilizer treatment of oil-treated plots, one receiving a weekly application of liquid fertilizer and the other treated with a slow-release fertilizer; and two controls, one not treated with oil and the other treated with oil but not with fertilizer. Oil degradation was monitored by measuring carbon dioxide evolution and by chemical analysis of the oil. Buried oil was degraded to a significantly greater extent in fertilized plots, but no differences in oil chemistry were observed between the two different fertilizer treatments, although carbon dioxide production was significantly higher in the oil-treated plots that were treated with slow-release fertilizer during the first 14 days of the experiment. Bacterial communities present in the beach sediments were profiled by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments and 16S rRNA amplified by reverse transcriptase PCR. Similarities between the DGGE profiles were calculated, and similarity matrices were subjected to statistical analysis. These analyses showed that although significant hydrocarbon degradation occurred both in plots treated with oil alone and in the plots treated with oil and liquid fertilizer, the bacterial community structure in these plots was, in general, not significantly different from that in the control plots that were not treated with oil and did not change over time. In contrast, the bacterial community structure in the plots treated with oil and slow-release fertilizer changed rapidly, and there were significant differences over time, as well as between blocks and even within plots. The differences were probably related to the higher concentrations of nutrients measured in interstitial water from the plots treated with slow-release fertilizer. Bacteria with 16S rRNA sequences closely related (>99.7% identity) to
Alcanivorax borkumensis
and
Pseudomonas stutzeri
sequences dominated during the initial phase of oil degradation in the plots treated with slow-release fertilizer. Field data were compared to the results of previous laboratory microcosm experiments, which revealed significant differences.
Response of Archaeal Communities in Beach Sediments to Spilled Oil and Bioremediation
Authors: Röling Wilfred F. M. , Couto de Brito Ivana R. , Swannell Richard P. J. , Head Ian M.
Published: May 2004

While the contribution of
Bacteria
to bioremediation of oil-contaminated shorelines is well established, the response of
Archaea
to spilled oil and bioremediation treatments is unknown. The relationship between archaeal community structure and oil spill bioremediation was examined in laboratory microcosms and in a bioremediation field trial. 16S rRNA gene-based PCR and denaturing gradient gel analysis revealed that the archaeal community in oil-free laboratory microcosms was stable for 26 days. In contrast, in oil-polluted microcosms a dramatic decrease in the ability to detect
Archaea
was observed, and it was not possible to amplify fragments of archaeal 16S rRNA genes from samples taken from microcosms treated with oil. This was the case irrespective of whether a bioremediation treatment (addition of inorganic nutrients) was applied. Since rapid oil biodegradation occurred in nutrient-treated microcosms, we concluded that
Archaea
are unlikely to play a role in oil degradation in beach ecosystems. A clear-cut relationship between the presence of oil and the absence of
Archaea
was not apparent in the field experiment. This may have been related to continuous inoculation of beach sediments in the field with
Archaea
from seawater or invertebrates and shows that the reestablishment of
Archaea
following bioremediation cannot be used as a determinant of ecosystem recovery following bioremediation. Comparative 16S rRNA sequence analysis showed that the majority of the
Archaea
detected (94%) belonged to a novel, distinct cluster of group II uncultured
Euryarchaeota
, which exhibited less than 87% identity to previously described sequences. A minor contribution of group I uncultured
Crenarchaeota
was observed.
Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts by Using Tunable Surface Microparticles
Authors: Chandler Darrell P. , Jarrell Ann E.
Published: May 2004

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA (∼10
4
cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA (∼10
5
cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.
Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of
Kluyveromyces lactis
Authors: Donnini Claudia , Farina Francesca , Neglia Barbara , Compagno Maria Concetta , Uccelletti Daniela , Goffrini Paola , Palleschi Claudio
Published: May 2004

The secreted production of heterologous proteins in
Kluyveromyces lactis
was studied. A glucoamylase (GAA) from the yeast
Arxula adeninivorans
was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of
K. lactis.
The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of
Saccharomyces cerevisiae
. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a
dgr151
-
1
mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain.
dgr151
-
1
is an allele of
RAG5
, the gene encoding the only hexokinase present in
K. lactis
(a homologue of
S. cerevisiae HXK2
). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the
dgr151
-
1
allele also showed a reduction in N- and O-glycosylation. Therefore, the
dgr151
strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.
In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris
Authors: Vervecken Wouter , Kaigorodov Vladimir , Callewaert Nico , Geysens Steven , De Vusser Kristof , Contreras Roland
Published: May 2004

ThePichia pastorisN-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereasPichiacells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of theP. pastorisN-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation ofOCH1and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum andN-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of anyP. pastorisexpression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.
Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins
Authors: Johnsen Line , Fimland Gunnar , Mantzilas Dimitris , Nissen-Meyer Jon
Published: May 2004

The immunity proteins of pediocin-like bacteriocins show a high degree of specificity with respect to the pediocin-like bacteriocin they recognize and confer immunity to. The aim of this study was to identify regions of the immunity proteins that are involved in this specific recognition. Six different hybrid immunity proteins were constructed from three different pediocin-like bacteriocin immunity proteins that have similar sequences but confer resistance to different bacteriocins. These hybrid immunity proteins were then tested for their ability to confer immunity to various pediocin-like bacteriocins. The specificities of the hybrid immunity proteins proved to be similar to those of the immunity proteins from which the C-terminal halves were derived, thus revealing that the C-terminal half of immunity proteins for pediocin-like bacteriocins contains a domain that is involved in specific recognition of the bacteriocins they confer immunity to. Moreover, the results also revealed that the effectiveness of an immunity protein is strain dependent and that its functionality thus depends in part on interplay with strain-dependent factors. To further investigate the structure-function relationship of these immunity proteins, the enterocin A and leucocin A immunity proteins (EntA-im and LeuA-im) were purified to homogeneity and structurally analyzed under various conditions by Circular dichroism (CD) spectroscopy. The results revealed that both immunity proteins are α-helical and well structured in an aqueous environment, the denaturing temperature being 78.5°C for EntA-im and 58.0°C for LeuA-im. The CD spectra also revealed that there was no further increase in the structuring or α-helical content when the immunity proteins were exposed to dodecylphosphocholine micelles or dioleoyl-
l
-α-phosphatidyl-
dl
-glycerol (DOPG) liposomes, indicating that the immunity proteins, in contrast to the bacteriocins, do not interact extensively with membranes. They may nevertheless be loosely associated with the membrane, possibly as peripheral membrane proteins, thus enabling them to interact with their cognate bacteriocin.
Regulation of Fumonisin Biosynthesis inFusarium verticillioidesby a Zinc Binuclear Cluster-Type Gene,ZFR1
Authors: Flaherty Joseph E. , Woloshuk Charles P.
Published: May 2004

Fusarium verticillioides, a pathogen of maize, produces a class of mycotoxins called fumonisins in infected kernels. In this study, a candidate regulatory gene,ZFR1, was identified in an expressed sequence tag library enriched for transcripts expressed byF. verticillioidesduring fumonisin B1(FB1) biosynthesis.ZFR1deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain.ZFR1encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression ofZFR1in colonized germ and degermed kernel tissues correlated with FB1levels. Overexpression ofZFR1inzfr1mutants restored FB1production to wild-type levels; however, FB1was not restored in anfcc1(FusariumC-type cyclin) mutant by overexpression ofZFR1. The results of this study indicate thatZFR1is a positive regulator of FB1biosynthesis inF. verticillioidesand suggest thatFCC1is required forZFR1function.
Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in
Escherichia coli
Authors: Aertsen Abram , Vanoirbeek Kristof , De Spiegeleer Philipp , Sermon Jan , Hauben Kristel , Farewell Anne , Nyström Thomas , Michiels Chris W.
Published: May 2004

A random library of
Escherichia coli
MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (
dnaK
,
lon
,
clpPX
), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of
rpoH
, encoding the heat shock-specific sigma factor σ
32
, was also induced by high pressure; (ii) heat shock rendered
E. coli
significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of
E. coli
compared to wild-type levels.
Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator
Thlaspi goesingense
Authors: Idris Rughia , Trifonova Radoslava , Puschenreiter Markus , Wenzel Walter W. , Sessitsch Angela
Published: May 2004

Thlaspi goesingense
is able to hyperaccumulate extremely high concentrations of Ni when grown in ultramafic soils. Recently it has been shown that rhizosphere bacteria may increase the heavy metal concentrations in hyperaccumulator plants significantly, whereas the role of endophytes has not been investigated yet. In this study the rhizosphere and shoot-associated (endophytic) bacteria colonizing
T. goesingense
were characterized in detail by using both cultivation and cultivation-independent techniques. Bacteria were identified by 16S rRNA sequence analysis, and isolates were further characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction—Ni tolerance, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore production. In the rhizosphere a high percentage of bacteria belonging to the
Holophaga/Acidobacterium
division and
α-Proteobacteria
were found. In addition, high-G+C gram-positive bacteria,
Verrucomicrobia
, and microbes of the
Cytophaga/Flexibacter/Bacteroides
division colonized the rhizosphere. The community structure of shoot-associated bacteria was highly different. The majority of clones affiliated with the
Proteobacteria
, but also bacteria belonging to the
Cytophaga/Flexibacter/Bacteroides
division, the
Holophaga/Acidobacterium
division, and the low-G+C gram-positive bacteria, were frequently found. A high number of highly related
Sphingomonas
16S rRNA gene sequences were detected, which were also obtained by the cultivation of endophytes. Rhizosphere isolates belonged mainly to the genera
Methylobacterium, Rhodococcus
, and
Okibacterium
, whereas the majority of endophytes showed high levels of similarity to
Methylobacterium mesophilicum
. Additionally,
Sphingomonas
spp. were abundant. Isolates were resistant to Ni concentrations between 5 and 12 mM; however, endophytes generally tolerated higher Ni levels than rhizosphere bacteria. Almost all bacteria were able to produce siderophores. Various strains, particularly endophytes, were able to grow on ACC as the sole nitrogen source.
Detection of Enteric Viruses in Shellfish from the Norwegian Coast
Authors: Myrmel M. , Berg E. M. M. , Rimstad E. , Grinde B.
Published: May 2004

Common blue mussels (
Mytilus edulis
), horse mussels (
Modiolus modiolus
), and flat oysters (
Ostrea edulis
) obtained from various harvesting and commercial production sites along the Norwegian coast were screened for the presence of norovirus by a real-time reverse transcription (RT)-nested PCR assay and for possible indicators of fecal contamination, i.e., for F-specific RNA bacteriophages (F-RNA phages) by plaque assay and for human adenoviruses and human circoviruses by nested PCR assay. The aims were to obtain relevant information for assessing the risk of transmission of enteric viruses by shellfish and to investigate the potential of various indicator viruses in routine screening. Noroviruses were detected in 6.8% of the samples, and the indicators were detected in 23.8% (F-RNA phages), 18.6% (adenoviruses), and 8.0% (circoviruses) of the samples. A seasonal variation was observed, with the exception of circoviruses, with more positive samples in the winter. A positive correlation was found between F-RNA phages and noroviruses. However, F-RNA phages were present in only 43% of the norovirus-positive samples. The results show that mussels from the Norwegian coast can constitute a risk of infection with enteric viruses and that routine testing of samples may be justified. Advantages and disadvantages of various options for screening are discussed.