bluuebunny/crossref_metadata_2025 huggingface dataset:
A methodology to determine margins by EPID measurements of patient setup variation and motion as applied to immobilization devices
Authors: Prisciandaro Joann I. , Frechette Christina M. , Herman Michael G. , Brown Paul D. , Garces Yolanda I. , Foote Robert L.
Published: November 2004

Assessment of clinic and site specific margins are essential for the effective use of three‐dimensional and intensity modulated radiation therapy. An electronic portal imaging device (EPID) based methodology is introduced which allows individual and population based CTV‐to‐PTV margins to be determined and compared with traditional margins prescribed during treatment. This method was applied to a patient cohort receiving external beam head and neck radiotherapy under an IRB approved protocol. Although the full study involved the use of an EPID‐based method to assess the impact of (1) simulation technique, (2) immobilization, and (3) surgical intervention on inter‐ and intrafraction variations of individual and population‐based CTV‐to‐PTV margins, the focus of the paper is on the technique. As an illustration, the methodology is utilized to examine the influence of two immobilization devices, the UON™ thermoplastic mask and the Type‐S™ head/neck shoulder immobilization system on margins. Daily through port images were acquired for selected fields for each patient with an EPID. To analyze these images, simulation films or digitally reconstructed radiographs (DRR's) were imported into the EPID software. Up to five anatomical landmarks were identified and outlined by the clinician and up to three of these structures were matched for each reference image. Once the individual based errors were quantified, the patient results were grouped into populations by matched anatomical structures and immobilization device. The variation within the subgroup was quantified by calculating the systematic and random errors ( and ). Individual patient margins were approximated as 1.65 times the individual‐based random error and ranged from 1.1 to () and 1.1 to () for fields matched on skull and cervical structures, and 1.7 to () and 2.0 to () for supraclavicular fields. Population‐based margins ranging from 5.1 to () and 3.7 to () were calculated for the corresponding skull/cervical field and 9.3 to () and 6.3 to () for the supraclavicular fields, respectively. The reported CTV‐to‐PTV margins are comparable to a value based on traditional Mayo margins, but in some cases exceed the default values established in RTOG Head and Neck studies. The data suggests that the population‐based margins provide sufficient coverage for the majority of the patients. However, the population‐derived margins are excessive for some patients and insufficient for others, suggesting that a re‐evaluation of current treatment margins for individual patients is warranted. Finally, this methodology provides direct evidence of treatment variation and thus can demonstrate with confidence, the superiority of one technique over another.
X‐ray spectroscopy in mammography with a silicon PIN photodiode with application to the measurement of tube voltage
Authors: Künzel Roseli , Herdade Silvio Bruni , Terini Ricardo Andrade , Costa Paulo Roberto
Published: November 2004

In this work a silicon PIN photodiode was employed in mammographic x‐ray spectroscopy under clinical and nonclinical conditions. Measurements have been performed at a constant potential tungsten anode tube, adapted in this work with molybdenum filters to produce a beam like that used in mammography, and at a clinical equipment with a molybdenum anode tube by using an additional aluminum filtration. The corrected x‐ray spectra were in full agreement with those generated by theoretical models published in the literature and agree well with those measured with a CdZnTe detector for tube voltages less than 30 kV. The half value layer and the relative exposure values calculated from the corrected silicon PIN photodiode spectra were in agreement with those measured with an ionization chamber. These results indicate that a silicon PIN photodiode are very suitable for mammographic x‐ray spectroscopy. As an application, the voltage (kV) applied to mammographic x‐ray equipment has been measured through the evaluation of the spectra high energy cut off. Uncertainties evaluated for the voltage values calculated from the measured spectra are less than 0.13% for voltages in the range 20–35 kV. The low uncertainties associated with the obtained results in this work point out that the method employed can be accurately used for calibration of noninvasive mammographic meters.
Mutual information based CT registration of the lung at exhale and inhale breathing states using thin‐plate splines
Authors: Coselmon Martha M. , Balter James M. , McShan Daniel L. , Kessler Marc L.
Published: November 2004

The advent of dynamic radiotherapy modeling and treatment techniques requires an infrastructure to weigh the merits of various interventions (breath holding, gating, tracking). The creation of treatment planning models that account for motion and deformation can allow the relative worth of such techniques to be evaluated. In order to develop a treatment planning model of a moving and deforming organ such as the lung, registration tools that account for deformation are required. We tested the accuracy of a mutual information based image registration tool using thin‐plate splines driven by the selection of control points and iterative alignment according to a simplex algorithm. Eleven patients each had sequential CT scans at breath‐held normal inhale and exhale states. The exhale right lung was segmented from CT and served as the reference model. For each patient, thirty control points were used to align the inhale CT right lung to the exhale CT right lung. Alignment accuracy (the standard deviation of the difference in the actual and predicted inhale position) was determined from locations of vascular and bronchial bifurcations, and found to be 1.7, 3.1, and about the RL, AP, and IS directions. The alignment accuracy was significantly different from the amount of measured movement during breathing only in the AP and IS directions. The accuracy of alignment including thin‐plate splines was more accurate than using affine transformations and the same iteration and scoring methodology. This technique shows promise for the future development of dynamic models of the lung for use in four‐dimensional (4‐D) treatment planning.
A study of the dosimetry of small field photon beams used in intensity modulated radiation therapy in inhomogeneous media: Monte Carlo simulations, and algorithm comparisons and corrections
Authors: Jones Andrew Osler
Published: November 2004

There is an increasing interest in the use of inhomogeneity corrections for lung, air, and bone in radiotherapy treatment planning. Traditionally, corrections based on physical density have been used. Modern algorithms use the electron density derived from CT images. Small fields are used in both conformal radiotherapy and IMRT, however, their beam characteristics in inhomogeneous media have not been extensively studied. This work compares traditional and modern treatment planning algorithms to Monte Carlo simulations in and near low‐density inhomogeneities. Field sizes ranging from in diameter are projected onto a phantom containing inhomogeneities and depth dose curves are compared. Comparisons of the Dose Perturbation Factors (DPF) are presented as functions of density and field size. Dose Correction Factors (DCF), which scale the algorithms to the Monte Carlo data, are compared for each algorithm. Physical scaling algorithms such as Batho and Equivalent Pathlength (EPL) predict an increase in dose for small fields passing through lung tissue, where Monte Carlo simulations show a sharp dose drop. The physical model‐based collapsed cone convolution (CCC) algorithm correctly predicts the dose drop, but does not accurately predict the magnitude. Because the model‐based algorithms do not correctly account for the change in backscatter, the dose drop predicted by CCC occurs farther downstream compared to that predicted by the Monte Carlo simulations. Beyond the tissue inhomogeneity all of the algorithms studied predict dose distributions in close agreement with Monte Carlo simulations. Dose–volume relationships are important in understanding the effects of radiation to the lung. The dose within the lung is affected by a complex function of beam energy, lung tissue density, and field size. Dose algorithms vary in their abilities to correctly predict the dose to the lung tissue. A thorough analysis of the effects of density, and field size on dose to the lung and how modern dose calculation algorithms compare to Monte Carlo data is presented in this research project. This work can be used as a basis to further refine an algorithm's accuracy in low‐density media or to correct prior dosimetric results.
Accuracy of the Burns equation for stopping‐power ratio as a function of depth and
Authors: Rogers D. W. O.
Published: November 2004

The accuracy of the Burns et al. equation [Med. Phys. 23, 489–501 (1996)] for the Spencer–Attix water to air stopping‐power ratio as a function of depth in a water phantom and electron beam quality in terms of is investigated by comparison to the original data on which this fit was based. It is shown that using this equation provides dose estimates on the central axis in a clinical electron beam that are accurate to within 1% of dose maximum for all 24 clinical beams investigated except very close to the surface in swept beams. In contrast, the error in the dose as a percentage of the local dose is much higher for values of the depth greater than 1.2.
Mapping of the prostate in endorectal coil‐based MRI/MRSI and CT: A deformable registration and validation study
Authors: Lian J. , Xing L. , Hunjan S. , Dumoulin C. , Levin J. , Lo A. , Watkins R. , Rohling K. , Giaquinto R. , Kim D. , Spielman D. , Daniel B.
Published: November 2004

The endorectal coil is being increasingly used in magnetic resonance imaging (MRI) and MR spectroscopic imaging (MRSI) to obtain anatomic and metabolic images of the prostate with high signal‐to‐noise ratio (SNR). In practice, however, the use of endorectal probe inevitably distorts the prostate and other soft tissue organs, making the analysis and the use of the acquired image data in treatment planning difficult. The purpose of this work is to develop a deformable image registration algorithm to map the MRI/MRSI information obtained using an endorectal probe onto CT images and to verify the accuracy of the registration by phantom and patient studies. A mapping procedure involved using a thin plate spline (TPS) transformation was implemented to establish voxel‐to‐voxel correspondence between a reference image and a floating image with deformation. An elastic phantom with a number of implanted fiducial markers was designed for the validation of the quality of the registration. Radiographic images of the phantom were obtained before and after a series of intentionally introduced distortions. After mapping the distorted phantom to the original one, the displacements of the implanted markers were measured with respect to their ideal positions and the mean error was calculated. In patient studies, CT images of three prostate patients were acquired, followed by MR images with a rigid endorectal coil. Registration quality was estimated by the centroid position displacement and image coincidence index (CI). Phantom and patient studies show that TPS‐based registration has achieved significantly higher accuracy than the previously reported method based on a rigid‐body transformation and scaling. The technique should be useful to map the MR spectroscopic dataset acquired with ER probe onto the treatment planning CT dataset to guide radiotherapy planning.
An energy‐ and depth‐dependent model for x‐ray imaging
Authors: Gallas Brandon D. , Boswell Jonathan S. , Badano Aldo , Gagne Robert M. , Myers Kyle J.
Published: November 2004

In this paper, we model an x‐ray imaging system, paying special attention to the energy‐ and depth‐dependent characteristics of the inputs and interactions: x rays are polychromatic, interaction depth and conversion to optical photons is energy‐dependent, optical scattering and the collection efficiency depend on the depth of interaction. The model we construct is a random function of the point process that begins with the distribution of x rays incident on the phosphor and ends with optical photons being detected by the active area of detector pixels to form an image. We show how the point‐process representation can be used to calculate the characteristic statistics of the model. We then simulate a phosphor, estimate its characteristic statistics, and proceed with a signal‐detection experiment to investigate the impact of the pixel fill factor on detecting spherical calcifications (the signal). The two extremes possible from this experiment are that does not change with fill factor or changes in proportion to fill factor. In our results, the impact of fill factor is between these extremes, and depends on the diameter of the signal.
Dosimetric characteristics of the new RadioCoil™ wire line source for use in permanent brachytherapy implants
Authors: Meigooni A. S. , Zhang H. , Clark J. R. , Rachabatthula V. , Koona R. A.
Published: November 2004

Recently, a novel linear brachytherapy source in the form of a coiled wire has become available for use in interstitial implants of various treatment sites such as prostate gland. This source type employs a design completely different from that of most “seed” sources currently on the market, one which improves upon or eliminates several common problems with such sources. Dosimetric characteristics of these sources with active lengths were determined for clinical application. For and active length sources, the dose rate constant, radial dose function, and two‐dimensional (2D) anisotropy function were experimentally and theoretically determined following the updated AAPM Task Group 43 (TG‐43U1) recommendations. Radial dose functions and/or “along‐away” matrix functions were also obtained for sources with active lengths . Measurements were performed with LiF thermoluminescent dosimeters in Solid Water™ phantoms. Measured data was compared to Monte Carlo simulated data in Solid Water™ utilizing the PTRAN code, version 7.43. After finding the data to be in agreement, Monte Carlo calculations were performed in liquid water to obtain clinically applicable dosimetric data as per TG‐43U1 recommendations. The results indicated the dose rate constant of the long RadioCoil™ source in Solid Water™ to be when measured, and when simulated by Monte Carlo. The calculated dose rate constant in liquid water was found to be . These values are comparable to other commercially available sources. Complete dosimetric data and simulation results are described in this paper. Per TG‐43U1, clinical treatment planning systems should utilize the values reported for liquid water.
The effects of target size and tissue density on the minimum margin required for random errors
Authors: Witte Marnix G. , van der Geer Joris , Schneider Christoph , Lebesque Joos V. , van Herk Marcel
Published: November 2004

The minimum margins required to compensate for random geometric uncertainties in the delivery of radiotherapy treatment were determined for a spherical Clinical Target Volume, using an analytic model for the cumulative dose. Margins were calculated such that the minimum dose in the target would be no less than 95% of the prescribed dose for 90% of the patients. The dose distribution model incorporated two Gaussians, and could accurately represent realistic dose profiles for various target sizes in lung and water. It was found that variations in target size and tissue density lead to significant changes in the minimum margin required for random errors. The random error margin increased with tissue density, and decreased with target size. The required margins were similar for dose distributions of spherical and cylindrical symmetry. Significant dose outside the spherical high dose region, as could result from multiple incident beams, lead to an increased margin for the larger targets. We could confirm that the previously proposed margin of 0.7 times the standard deviation of the random errors is safe for standard deviations up to , except for very small targets in dense material.
A Monte Carlo dosimetry study of vaginal brachytherapy applications with a shielded cylindrical applicator set
Authors: Lymperopoulou G. , Pantelis E. , Papagiannis P. , Rozaki‐Mavrouli H. , Sakelliou L. , Baltas D. , Karaiskos P.
Published: November 2004

A durable recommendation for brachytherapy treatment planning systems to account for the effect of tissue, applicator and shielding material heterogeneities exists. As different proposed approaches have not been integrated in clinical treatment planning routine yet, currently utilized systems disregard or, most commonly, do not fully account for the aforementioned effects. Therefore, it is of interest to evaluate the efficacy of current treatment planning in clinical applications susceptible to errors due to heterogeneities. In this work the effect of the internal structure as well as the shielding used with a commercially available cylindrical shielded applicator set (Nucletron part # 084.320) for vaginal and rectum treatments is studied using three‐dimensional Monte Carlo simulation for a clinical treatment plan involving seven source dwell positions of the classic microSelectron HDR source. Results are compared to calculations of a treatment planning system (Plato BPS v.14.2.7), which assumes homogeneous water medium and applies a constant, multiplicative transmission factor only at points lying in the shadow of the shield. It is found that the internal structure of the applicator (which includes stainless steel, air and plastic materials) with no shield loaded does not affect the dose distribution relative to homogeneous water. In the unshielded side of the applicator with a 90°, 180°, or 270° tungsten alloy shield loaded, an overestimation of treatment planning system calculations relative to Monte Carlo results was observed which is both shield and position dependent. While significant (up to 15%) at increased distances, which are not of major clinical importance, this overestimation does not affect dose prescription distances by more than 3%. The inverse effect of approx. 3% dose increase at dose prescription distances is observed for stainless steel shields. Regarding the shielded side of the applicator, it is shown that the default treatment planning system transmission factors for tungsten alloy result in a consistent dose overestimation thus constituting a safe approach given the nature of associated clinical applications. Stainless steel is shown to be an ineffective shielding material with transmission factors reaching up to 0.68 at increased distances irrespective of shield geometry.
Nicotine Activation of α4* Receptors: Sufficient for Reward, Tolerance, and Sensitization
Authors: Tapper Andrew R. , McKinney Sheri L. , Nashmi Raad , Schwarz Johannes , Deshpande Purnima , Labarca Cesar , Whiteaker Paul , Marks Michael J. , Collins Allan C. , Lester Henry A.
Published: November 2004

The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with α4 nicotinic subunits containing a single point mutation, Leu
9′
→ Ala
9′
in the pore-forming M2 domain, rendering α4* receptors hypersensitive to nicotine. Selective activation of α4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of α4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.
A Link Between mRNA Turnover and RNA Interference in
Arabidopsis
Authors: Gazzani S. , Lawrenson T. , Woodward C. , Headon D. , Sablowski R.
Published: November 2004

In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in
Arabidopsis
was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5′-3′ exonucleolysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.
Biodiversity Effects on Soil Processes Explained by Interspecific Functional Dissimilarity
Authors: Heemsbergen D. A. , Berg M. P. , Loreau M. , van Hal J. R. , Faber J. H. , Verhoef H. A.
Published: November 2004

The loss of biodiversity can have significant impacts on ecosystem functioning, but the mechanisms involved lack empirical confirmation. Using soil microcosms, we show experimentally that functional dissimilarity among detritivorous species, not species number, drives community compositional effects on leaf litter mass loss and soil respiration, two key soil ecosystem processes. These experiments confirm theoretical predictions that biodiversity effects on ecosystem functioning can be predicted by the degree of functional differences among species.
Tracking SNARE Complex Formation in Live Endocrine Cells
Authors: An Seong J. , Almers Wolfhard
Published: November 2004

Syntaxin, synaptosome-associated protein of 25 kD (SNAP25), and vesicle-associated membrane protein/synaptobrevin are collectively called SNAP receptor (SNARE) proteins, and they catalyze neuronal exocytosis by forming a “core complex.” The steps in core complex formation are unknown. Here, we monitored SNARE complex formation in vivo with the use of a fluorescent version of SNAP25. In PC12 cells, we found evidence for a syntaxin-SNAP25 complex that formed with high affinity, required only the amino-terminal SNARE motif of SNAP25, tolerated a mutation that blocks formation of other syntaxin-SNAP25 complexes, and assembled reversibly when Ca
2+
entered cells during depolarization. The complex may represent a precursor to the core complex formed during a Ca
2+
-dependent priming step of exocytosis.
MHC Class I Peptides as Chemosensory Signals in the Vomeronasal Organ
Authors: Leinders-Zufall Trese , Brennan Peter , Widmayer Patricia , S. Prashanth Chandramani , Maul-Pavicic Andrea , Jäger Martina , Li Xiao-Hong , Breer Heinz , Zufall Frank , Boehm Thomas
Published: November 2004

The mammalian vomeronasal organ detects social information about gender, status, and individuality. The molecular cues carrying this information remain largely unknown. Here, we show that small peptides that serve as ligands for major histocompatibility complex (MHC) class I molecules function also as sensory stimuli for a subset of vomeronasal sensory neurons located in the basal Gao- and V2R receptor–expressing zone of the vomeronasal epithelium. In behaving mice, the same peptides function as individuality signals underlying mate recognition in the context of pregnancy block. MHC peptides constitute a previously unknown family of chemosensory stimuli by which MHC genotypic diversity can influence social behavior.
Enhancement of Ferroelectricity in Strained BaTiO
3
Thin Films
Authors: Choi K. J. , Biegalski M. , Li Y. L. , Sharan A. , Schubert J. , Uecker R. , Reiche P. , Chen Y. B. , Pan X. Q. , Gopalan V. , Chen L.-Q. , Schlom D. G. , Eom C. B.
Published: November 2004

Biaxial compressive strain has been used to markedly enhance the ferroelectric properties of BaTiO
3
thin films. This strain, imposed by coherent epitaxy, can result in a ferroelectric transition temperature nearly 500°C higher and a remanent polarization at least 250% higher than bulk BaTiO
3
single crystals. This work demonstrates a route to a lead-free ferroelectric for nonvolatile memories and electro-optic devices.
Highly Efficient Production of Laccase by the Basidiomycete
Pycnoporus cinnabarinus
Authors: Alves Alexandra M. C. R. , Record Eric , Lomascolo Anne , Scholtmeijer Karin , Asther Marcel , Wessels Joseph G. H. , Wösten Han A. B.
Published: November 2004

An efficient transformation and expression system was developed for the industrially relevant basidiomycete
Pycnoporus cinnabarinus
. This was used to transform a laccase-deficient monokaryotic strain with the homologous
lac1
laccase gene placed under the regulation of its own promoter or that of the
SC3
hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (
GPD
) gene of
Schizophyllum commune. SC3
-driven expression resulted in a maximal laccase activity of 107 nkat ml
−1
in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the
GPD
and
lac1
promoters, respectively.
lac1
-driven expression strongly increased when 25 g of ethanol liter
−1
was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml
−1
. These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably,
lac1
mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter
−1
when
lac1
was expressed behind the
SC3
or
GPD
promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml
−1
(i.e., 360 mg liter
−1
) was obtained. Laccase production was further increased in transformants expressing
lac1
behind its own promoter or that of
GPD
by growth in the presence of 40 g of ethanol liter
−1
. In this case, maximal activities were 3,900 and 4,660 nkat ml
−1
, respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that
P. cinnabarinus
may be a host of choice for the production of other proteins as well.
Redox Transformations of Arsenic Oxyanions in Periphyton Communities
Authors: Kulp Thomas R. , Hoeft Shelley E. , Oremland Ronald S.
Published: November 2004

Periphyton (
Cladophora
sp.) samples from a suburban stream lacking detectable dissolved As were able to reduce added As(V) to As(III) when incubated under anoxic conditions and, conversely, oxidized added As(III) to As(V) with aerobic incubation. Both types of activity were abolished in autoclaved controls, thereby demonstrating its biological nature. The reduction of As(V) was inhibited by chloramphenicol, indicating that it required the synthesis of new protein. Nitrate also inhibited As(V) reduction, primarily because it served as a preferred electron acceptor to which the periphyton community was already adapted. However, part of the inhibition was also caused by microbial reoxidation of As(III) linked to nitrate. Addition of [
14
C]glucose to anoxic samples resulted in the production of
14
CO
2
, suggesting that the observed As(V) reduction was a respiratory process coupled to the oxidation of organic matter. The population density of As(V)-reducing bacteria within the periphyton increased with time and with the amount of As(V) added, reaching values as high as ∼10
6
cells ml
−1
at the end of the incubation. This indicated that dissimilatory As(V) reduction in these populations was linked to growth. However, As(V)-respiring bacteria were found to be present, albeit at lower numbers (∼10
2
ml
−1
), in freshly sampled periphyton. These results demonstrate the presence of a bacterial population within the periphyton communities that is capable of two key arsenic redox transformations that were previously studied in As-contaminated environments, which suggests that these processes are widely distributed in nature. This assumption was reinforced by experiments with estuarine samples of
Cladophora sericea
in which we detected a similar capacity for anaerobic As(V) reduction and aerobic As(III) oxidation.
Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children
Authors: Song Yuli , Liu Chengxu , Finegold Sydney M.
Published: November 2004

Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate threeClostridiumclusters and oneClostridiumspecies,C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell ofC. bolteaecould be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children forC. bolteaeand the followingClostridiumgroups were statistically significant: mean counts ofC. bolteaeand clusters I and XI in autistic children were 46-fold (P= 0.01), 9.0-fold (P= 0.014), and 3.5-fold (P= 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 × 108CFU/g in autistic children and 4.8 × 108CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.
Measurements of Fitness and Competition in CommensalEscherichia coliandE. coliO157:H7 Strains
Authors: Durso Lisa M. , Smith David , Hutkins Robert W.
Published: November 2004

Although the main reservoirs for pathogenicEscherichia coliO157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensalE. coliandE. coliO157:H7 strains. Indirect antagonism assays revealed thatE. coliO157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensalE. colistrains but not by theE. coliO157:H7 strains. Despite the observed physiological and biochemical differences between these two groups ofE. colistrains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.
Frequency and Spatial Distribution of EnvironmentalCampylobacterspp
Authors: Brown P. E. , Christensen O. F. , Clough H. E. , Diggle P. J. , Hart C. A. , Hazel S. , Kemp R. , Leatherbarrow A. J. H. , Moore A. , Sutherst J. , Turner J. , Williams N. J. , Wright E. J. , French N. P.
Published: November 2004

Humans are exposed toCampylobacterspp. in a range of sources via both food and environmental pathways. For this study, we explored the frequency and distribution of thermophilicCampylobacterspp. in a 10- by 10-km square rural area of Cheshire, United Kingdom. The area contains approximately 70, mainly dairy, farms and is used extensively for outdoor recreational activities.Campylobacterspp. were isolated from a range of environmental samples by use of a systematic sampling grid. Livestock (mainly cattle) and wildlife feces and environmental water and soil samples were cultured, and isolates were presumptively identified by standard techniques. These isolates were further characterized by PCR.Campylobacter jejuniwas the most prevalent species in all animal samples, ranging from 11% in samples from nonavian wildlife to 36% in cattle feces, and was isolated from 15% of water samples.Campylobacter coliwas commonly found in water (17%) and sheep (21%) samples, but rarely in other samples.Campylobacter lariwas recovered from all sample types, with the exception of sheep feces, and was found in moderate numbers in birds (7%) and water (5%).Campylobacter hyointestinaliswas only recovered from cattle (7%) and birds (1%). The spatial distribution and determinants ofC. jejuniin cattle feces were examined by the use of model-based spatial statistics. The distribution was consistent with very localized within-farm or within-field transmission and showed little evidence of any larger-scale spatial dependence. We concluded that there is a potentially high risk of human exposure toCampylobacterspp., particularlyC. jejuni, in the environment of our study area. The prevalence and likely risk posed byC. jejuni-positive cattle feces in the environment diminished as the fecal material aged. After we took into account the age of the fecal material, the absence or presence of rain, and the presence of bird feces, there was evidence of significant variation in the prevalence ofC. jejuni-positive cattle feces between grazing fields but no evidence of spatial clustering beyond this resolution. The spatial pattern ofC. jejuniis therefore consistent with that for an organism that is ubiquitous in areas contaminated with cattle feces, with a short-scale variation in infection intensity that cannot be explained solely by variations in the age of the fecal material. The observed pattern is not consistent with large-scale transmission attributable to watercourses, wildlife territories, or other geographical features that transcend field and farm boundaries.
Aflatoxin Biosynthesis Cluster GenecypAIs Required for G Aflatoxin Formation
Authors: Ehrlich Kenneth C. , Chang Perng-Kuang , Yu Jiujiang , Cotty Peter J.
Published: November 2004

Aspergillus flavusisolates produce only aflatoxins B1 and B2, whileAspergillus parasiticusandAspergillus nomiusproduce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene,pksA, revealed thatA. flavusisolates are missing portions of genes (cypAandnorB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption ofcypAinA. parasiticusyielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded bycypAhas highest amino acid identity toGibberella zeaeTri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring ofO-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.
Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades
Authors: Castro Hector , Ogram Andrew , Reddy K. R.
Published: November 2004

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order
Methanomicrobiales
was observed in
mcrA
clone libraries, suggesting primer bias for this group. A greater diversity within the
Methanomicrobiales
was observed in
mcrA
clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to
Methanosaeta
spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to
Methanomicrobiales
, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders
Methanobacteriales
and
Methanococcales
were found in any 16S rRNA clone library, although some
Methanobacteriales
were found in
mcrA
libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.
Novel Endophytic Nitrogen-Fixing Clostridia from the Grass
Miscanthus sinensis
as Revealed by Terminal Restriction Fragment Length Polymorphism Analysis
Authors: Miyamoto Takuya , Kawahara Makoto , Minamisawa Kiwamu
Published: November 2004

Anaerobic nitrogen-fixing consortia consisting of N
2
-fixing clostridia and diverse nondiazotrophic bacteria were previously isolated from various gramineous plants (K. Minamisawa, K. Nishioka, T. Miyaki, B. Ye, T. Miyamoto, M. You, A. Saito, M. Saito, W. Barraquio, N. Teaumroong, T. Sein, and T. Tadashi, Appl. Environ. Microbiol.
70:
3096-3102, 2004). For this work, clostridial populations and their phylogenetic structures in a stand of the grass
Miscanthus sinensis
in Japan were assessed by a 16S rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable-number (MPN) counts. PCR primers and restriction enzymes were optimized for analyses of the plant clostridia. Clostridia were detected in strongly surface-sterilized leaves, stems, and roots of the plants at approximately 10
4
to 10
5
cells/g of fresh weight; they made up a large proportion of N
2
-fixing bacterial populations, as determined by MPN counts associated with an acetylene reduction assay. Phylogenetic grouping by MPN-TRFLP analysis revealed that the clostridial populations belonged to group II of cluster XIVa and groups IV and V of cluster I; this result was supported by a culture-independent TRFLP analysis using direct DNA extraction from plants. When phylogenetic populations from
M. sinensis
and the soil around the plants were compared, group II clostridia were found to exist exclusively in
M. sinensis
.
Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods
Authors: Jean Julie , D'Souza Doris H. , Jaykus Lee-Ann
Published: November 2004

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10
−1
reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10
0
to 10
2
reverse transcription-PCR-detectable units (or PFU)/9 cm
2
in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.
Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi
Authors: Pawlowska Teresa E. , Charvat Iris
Published: November 2004

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species,
Glomus etunicatum
and
Glomus intraradices
, had different levels of sensitivity to metal stress.
G. etunicatum
was more sensitive to all three metals than was
G. intraradices
. A unique response of increased presymbiotic hyphal extension occurred in
G. intraradices
exposed to Cd and Pb. Presymbiotic hyphae of
G. intraradices
formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In
G. intraradices
grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by
G. intraradices
spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.
Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a
Sphingomonas
Strain That Degrades Various Polycyclic Aromatic Hydrocarbons
Authors: Demanèche Sandrine , Meyer Christine , Micoud Julien , Louwagie Mathilde , Willison John C. , Jouanneau Yves
Published: November 2004

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism
Sphingomonas
sp. strain CHY-1 were investigated. [
14
C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in
Novosphingobium aromaticivorans
F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (
phnB
). The second locus contained five genes encoding an extradiol dioxygenase (
phnC
), a ferredoxin (
phnA3
), another oxygenase component (PhnII), and an isomerase (
phnD
). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (
phnA3
) and a reductase (
phnA4
). Disruption of the
phnA1
a
gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in
Escherichia coli
functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.
DNA Macroarray Profiling of
Lactococcus lactis
subsp.
lactis
IL1403 Gene Expression during Environmental Stresses
Authors: Xie Yi , Chou Lan-szu , Cutler Adele , Weimer Bart
Published: November 2004

This report describes the use of an oligonucleotide macroarray to profile the expression of 375 genes in
Lactococcus lactis
subsp.
lactis
IL1403 during heat, acid, and osmotic stress. A set of known stress-associated genes in IL1403 was used as the internal control on the array. Every stress response was accurately detected using the macroarray, compared to data from previous reports. As a group, the expression patterns of the investigated metabolic genes were significantly altered by heat, acid, and osmotic stresses. Specifically, 13 to 18% of the investigated genes were differentially expressed in each of the environmental stress treatments. Interestingly, the methionine biosynthesis pathway genes (
metA-metB1
and
metB2-cysK
) were induced during heat shock, but methionine utilization genes, such as
metK
, were induced during acid stress. These data provide a possible explanation for the differences between acid tolerance mechanisms of
L. lactis
strains IL1403 and MG1363 reported previously. Several groups of transcriptional responses were common among the stress treatments, such as repression of peptide transporter genes, including the
opt
operon (also known as
dpp
) and
dtpT
. Reduction of peptide transport due to environmental stress will have important implications in the cheese ripening process. Although stress responses in lactococci were extensively studied during the last decade, additional information about this bacterium was gained from the use of this metabolic array.
Seasonal Shedding of MultipleCryptosporidiumGenotypes in California Ground Squirrels (Spermophilus beecheyi)
Authors: Atwill Edward R. , Phillips Ralph , Pereira Maria Das Graças C. , Li Xunde , McCowan Brenda
Published: November 2004

Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces−1. The mean annual environmental loading rate ofCryptosporidiumoocysts was 57,882 oocysts squirrel−1day−1, with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel−1day−1in fall, winter, and spring to levels of 2 × 105oocysts squirrel−1day−1in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes ofCryptosporidiumspecies in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbersAY462231toAY462233, respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcineC. parvum(AF115377) andC. wrairi(AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species ofCryptosporidium.