bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermodynamic modeling of ammoniacal nickel sulphate solutions
Authors: Kolhinen Tero , Jalkanen Heikki
Published: December 2005

The aim of this study was to build a thermodynamic model to extrapolate the system NiSO4–(NH4)2SO4–NH3–H2O which exists in aqueous hydrogen reduction processes of nickel. These solutions are relatively concentrated, and the temperature for solution preparation and reduction stages might range from room temperature up to 180–200°C. Temperature has a great effect on solution equilibria and the equilibrium solubility of simple and complex crystalline salts such as sulphates and hydroxides known to exist in the system. In this work, the role of kinetics is recognized, but only equilibrium phenomena reconsidered. The main focus of the study was on the modeling of salt solubility. In cases of a process solution where the precipitation of solid crystalline salts is highly undesirable it is important to know the safe limits for a ‘clear solution’ to operate even if the kinetics are unfavorable. Relatively concentrated solutions are strongly non‐ideal and realistic calculations on solution equilibria necessitate the modeling of excess thermodynamic properties of these solutions. The excess model selected for aqueous species was Pitzer's ion‐interaction model and for the gas phase Tsonopoulos' model. Emphasis in data assessment was on the thermodynamic modeling of various binaries and ternaries to create a substantial database for the approximation of the quaternary system.
Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme
Authors: Kosinska Urszula , Carnrot Cecilia , Eriksson Staffan , Wang Liya , Eklund Hans
Published: December 2005

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell‐wall‐lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu‐TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu‐TK was determined in a feedback inhibitor complex, demonstrating that a lasso‐like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main‐chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso‐like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5′‐OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu‐TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2′‐Ara position, but larger substitutions at the 3′ position of the deoxyribose.
Computational processing and error reduction strategies for standardized quantitative data in biological networks
Authors: Schilling Marcel , Maiwald Thomas , Bohl Sebastian , Kollmann Markus , Kreutz Clemens , Timmer Jens , Klingmüller Ursula
Published: December 2005

High‐quality quantitative data generated under standardized conditions is critical for understanding dynamic cellular processes. We report strategies for error reduction, and algorithms for automated data processing and for establishing the widely used techniques of immunoprecipitation and immunoblotting as highly precise methods for the quantification of protein levels and modifications. To determine the stoichiometry of cellular components and to ensure comparability of experiments, relative signals are converted to absolute values. A major source for errors in blotting techniques are inhomogeneities of the gel and the transfer procedure leading to correlated errors. These correlations are prevented by randomized gel loading, which significantly reduces standard deviations. Further error reduction is achieved by using housekeeping proteins as normalizers or by adding purified proteins in immunoprecipitations as calibrators in combination with criteria‐based normalization. Additionally, we developed a computational tool for automated normalization, validation and integration of data derived from multiple immunoblots. In this way, large sets of quantitative data for dynamic pathway modeling can be generated, enabling the identification of systems properties and the prediction of targets for efficient intervention.
Topology, tinkering and evolution of the human transcription factor network
Authors: Rodriguez‐Caso Carlos , Medina Miguel A. , Solé Ricard V.
Published: December 2005

Patterns of protein interactions are organized around complex heterogeneous networks. Their architecture has been suggested to be of relevance in understanding the interactome and its functional organization, which pervades cellular robustness. Transcription factors are particularly relevant in this context, given their central role in gene regulation. Here we present the first topological study of the human protein–protein interacting transcription factor network built using the TRANSFAC database. We show that the network exhibits scale‐free and small‐world properties with a hierarchical and modular structure, which is built around a small number of key proteins. Most of these proteins are associated with proliferative diseases and are typically not linked to each other, thus reducing the propagation of failures through compartmentalization. Network modularity is consistent with common structural and functional features and the features are generated by two distinct evolutionary strategies: amplification and shuffling of interacting domains through tinkering and acquisition of specific interacting regions. The function of the regulatory complexes may have played an active role in choosing one of them.
Stream‐Stage Response Tests and Their Joint Interpretation with Pumping Tests
Authors: Rötting Tobias S. , Carrera Jesus , Bolzicco Jose , Salvany Josep Maria
Published: December 2005

Hydraulic head response to stream‐stage variations can be used to explore the hydraulic properties of stream‐aquifer systems at a relatively large scale. These stream‐stage response tests, also called flooding tests, are typically interpreted using one‐ or two‐dimensional models that assume flow perpendicular to the river. Therefore, they cannot be applied to systems that are both horizontally and vertically heterogeneous. In this work, we use the geostatistical inverse problem to jointly interpret data from stream‐stage response and pumping tests. The latter tests provide flow data (which are needed to resolve aquifer diffusivity into transmissivity and storage coefficient) and may supply supplementary small‐scale information. Here, we summarize the methodology for the design, execution, and joint numerical interpretation of these tests. Application to the Aznalcóllar case study allows us to demonstrate that the proposed methodology may help in responding to questions such as the continuity of aquitards, the role and continuity of highly permeable paleochannels, or the time evolution of stream‐aquifer interaction. These results expand the applicability and scope of stream‐stage response tests.
Where Do Periodic Variations in the Discharge of a Well Become Negligible?
Authors: Bakker Mark
Published: December 2005

A rule of thumb is presented to determine where variations in the discharge of a pumping well have a significant influence on the flow in an aquifer. The rule of thumb relates the period of the variation of the discharge to the distance from the well beyond which the transient effect on the flow in the aquifer is insignificant. For example, when an irrigation well pumps intermittently during the growing season, the rule may be applied to determine the distance from the well beyond which flow in the aquifer can be simulated with an average discharge during the growing season; the distance from the well beyond which flow can be simulated with a steady, yearly averaged discharge can also be computed.
Grid Cell Distortion and MODFLOW’s Integrated Finite‐Difference Numerical Solution
Authors: Romero Dave M , Silver Steven E
Published: December 2005

The ground water flow model MODFLOW inherently implements a nongeneralized integrated finite‐difference (IFD) numerical scheme. The IFD numerical scheme allows for construction of finite‐difference model grids with curvilinear (piecewise linear) rows. The resulting grid comprises model cells in the shape of trapezoids and is distorted in comparison to a traditional MODFLOW finite‐difference grid. A version of MODFLOW‐88 (herein referred to as MODFLOW IFD) with the code adapted to make the one‐dimensional DELR and DELC arrays two dimensional, so that equivalent conductance between distorted grid cells can be calculated, is described. MODFLOW IFD is used to inspect the sensitivity of the numerical head and velocity solutions to the level of distortion in trapezoidal grid cells within a converging radial flow domain. A test problem designed for the analysis implements a grid oriented such that flow is parallel to columns with converging widths. The sensitivity analysis demonstrates MODFLOW IFD’s capacity to numerically derive a head solution and resulting intercell volumetric flow when the internal calculation of equivalent conductance accounts for the distortion of the grid cells. The sensitivity of the velocity solution to grid cell distortion indicates criteria for distorted grid design. In the radial flow test problem described, the numerical head solution is not sensitive to grid cell distortion. The accuracy of the velocity solution is sensitive to cell distortion with error <1% if the angle between the nonparallel sides of trapezoidal cells is <12.5°. The error of the velocity solution is related to the degree to which the spatial discretization of a curve is approximated with piecewise linear segments. Curvilinear finite‐difference grid construction adds versatility to spatial discretization of the flow domain. MODFLOW‐88's inherent IFD numerical scheme and the test problem results imply that more recent versions of MODFLOW 2000, with minor modifications, have the potential to make use of a curvilinear grid.
Important changes in medical x‐ray imaging facility shielding design methodology. A brief summary of recommendations in NCRP Report No. 147
Authors: Archer Benjamin R. , Gray Joel E.
Published: December 2005

The recently published Report No. 147 of The National Council on Radiation Protection and Measurements entitled “Structural shielding design for medical x‐ray imaging facilities” provides an update of shielding recommendations for x rays used for medical imaging. The goal of this report is to ensure that the shielding in these facilities limits radiation exposures to employees and members of the public to acceptable levels. Board certified medical and health physicists, as defined in this report, are the “qualified experts” who are competent to design radiation shielding for these facilities. As such, physicists must be aware of the new technical information and the changes from previous reports that Report No. 147 supersedes. In this article we summarize the new data, models and recommendations for the design of radiation barriers in medical imaging facilities that are presented in Report No. 147.
Optimization of radiotherapy dose‐time fractionation with consideration of tumor specific biology
Authors: Yang Yong , Xing Lei
Published: December 2005

The “four Rs” of radiobiology play an important role in the design of radiation therapy treatment protocol. The purpose of this work is to explore their influence on external beam radiotherapy for fast and slowly proliferating tumors and develop an optimization framework for tumor‐biology specific dose‐time‐fractionation scheme. The linear quadratic model is used to describe radiation response of tumor, in which the time dependence of sublethal damage repair and the redistribution and reoxygenation effects are included. The optimum radiotherapeutic strategy is defined as the treatment scheme that maximizes tumor biologically effective dose (BED) while keeping normal tissue BED constant. The influence of different model parameters on total dose, overall treatment time, fraction size, and intervals is also studied. The results showed that, for fast proliferating tumors, the optimum overall time is similar to the assumed kickoff time and almost independent of interval patterns. Significant increase in tumor control can be achieved using accelerated schemes for the tumors with doubling time smaller than 3 days, but little is gained for those with doubling time greater than 5 days. The incomplete repair of normal tissues between two consecutive fractions in standard fractionation has almost no influence on the fractional doses, even for the hyperfractionation with an interval time of 8 h. However, when the resensitization effect is included, the fractional doses at the beginning and end of each irradiated week become obviously higher than others in the optimum scheme and the hyperfractionation scheme has little advantage over the standard or hypofractionation one. For slowly proliferating tumors, provided that the ratio of the tumor is comparable to that of the normal tissues, a hypofractionation is more favorable. The overall treatment time should be larger than a minimum, which is predominantly determined by the resensitization time. The proposed technique provides a useful tool to systematically optimize radiotherapy for fast and slow proliferating tumors and sheds important insight into the complex problem of dose‐time fractionation.
Contrast cancellation technique applied to digital x‐ray imaging using silicon strip detectors
Authors: Avila C. , Lopez J. , Sanabria J. C. , Baldazzi G. , Bollini D. , Gombia M. , Cabal A. E. , Ceballos C. , Diaz Garcia A. , Gambaccini M. , Taibi A. , Sarnelli A. , Tuffanelli A. , Giubellino P. , Marzari‐Chiesa A. , Prino F. , Tomassi E. , Grybos P. , Idzik M. , Swientek K. , Wiacek P. , Montaño L. M. , Ramello L. , Sitta M.
Published: December 2005

Dual‐energy mammographic imaging experimental tests have been performed using a compact dichromatic imaging system based on a conventional x‐ray tube, a mosaic crystal, and a 384‐strip silicon detector equipped with full‐custom electronics with single photon counting capability. For simulating mammal tissue, a three‐component phantom, made of Plexiglass, polyethylene, and water, has been used. Images have been collected with three different pairs of x‐ray energies: , , and . A Monte Carlo simulation of the experiment has also been carried out using the MCNP‐4C transport code. The Alvarez‐Macovski algorithm has been applied both to experimental and simulated data to remove the contrast between two of the phantom materials so as to enhance the visibility of the third one.
Calculation and validation of the use of effective attenuation coefficient for attenuation correction in In‐111 SPECT
Authors: Seo Youngho , Wong Kenneth H. , Hasegawa Bruce H.
Published: December 2005

Nuclear medicine tracers usingas a radiolabel are increasing in their use, especially in the domain of oncologic imaging. In these applications, it often is critical to have the capability of quantifying radionuclide uptake and being able to relate it to the biological properties of the tumor. However, images from single photon emission computed tomography (SPECT) can be degraded by photon attenuation, photon scattering, and collimator blurring; without compensation for these effects, image quality can be degraded, and accurate and precise quantification is impossible. Although attenuation correction for SPECT is becoming more common, most implementations can only model single energy radionuclides such asand. Thus, attenuation correction foris challenging because it emits two photons (171 and) at nearly equal rates (90.2% and 94% emission probabilities). In this paper, we present a method of calculating a single “effective” attenuation coefficient for the dual‐energy emissions of, and that can be used to correct for photon attenuation in radionuclide images acquired with this radionuclide. Using this methodology, we can derive an effective linear attenuation coefficientand an effective photon energybased on the emission probabilities and linear attenuation coefficients of thephotons. This approach allows us to treat the emissions fromas a single photon with an effective energy of. We obtained emission projection data from a tank filled with a uniform solution of. The projection data were reconstructed using an iterative maximum‐likelihood algorithm with no attenuation correction, and with attenuation correction assuming photon energies of 171, 245, and(the derived). The reconstructed tomographic images demonstrate that the use of no attenuation correction, or correction assuming photon energies of 171 orintroduces inaccuracies into the reconstructed radioactivity distribution when compared against the effective energy method. In summary, this work provides both a theoretical framework and experimental methodology of attenuation correction for the dual‐energy emissions from. Although these results are specific to, the foundation could easily be extended to other multiple‐energy isotopes.
Technique factors and their relationship to radiation dose in pendant geometry breast CT
Authors: Boone John M. , Kwan Alexander L. C. , Seibert J. Anthony , Shah Nikula , Lindfors Karen K. , Nelson Thomas R.
Published: December 2005

The use of breast computed tomography (CT) as an alternative to mammography in some patients is being studied at several institutions. However, the radiation dosimetry issues associated with breast CT are markedly different than in the case of mammography. In this study, the spectral properties of an operational breast CT scanner were characterized both by physical measurement and computer modeling of the kVp‐dependent spectra, from 40 to 110 kVp (Be window W anode with 0.30 mm added Cu filtration). Previously reported conversion factors, normalized glandular dose for CT‐DgN(ct), derived from Monte Carlo methods, were used in concert with the output spectra of the breast scanner to compute the mean glandular dose to the breast based upon different combinations of x‐ray technique factors (kVp and mAs). The mean glandular dose (MGD) was measured as a function of the compressed breast thickness (2–8 cm) and three different breast compositions (0%, 50%, and 100% glandular fractions) in four clinical mammography systems in our institution. The average MGD from these four systems was used to compute the technique factors for breast CT systems that would match the two‐view mammographic dose levels. For a 14 cm diameter breast (equivalent to a 5 cm thick compressed breast in mammography), air kerma levels at the breast CT scanner's isocenter (468 mm from the source) of 4.4, 6.4, and 9.0 mGy were found to deliver equivalent mammography doses for 0%, 50%, and 100% glandular breasts (respectively) at 80 kVp. At 80 kVp (where air kerma was at the isocenter), 57 mAs (integrated over the entire scan) was required to match the mammography dose for a 14 cm 50% glandular breast. At 50 kVp, 360 mAs is required to match mammographic dose levels. Tables are provided for both air kerma at the isocenter and mAs for 0%, 50%, and 100% glandular breasts. Other issues that impact breast CT technique factors are also discussed.
Correction of pixel sensitivity variation and off‐axis response for amorphous silicon EPID dosimetry
Authors: Greer Peter B.
Published: December 2005

The aim of this work is to determine the pixel sensitivity variation and off‐axis dose response of an amorphous silicon electronic portal imaging device (EPID), and develop a correction method to improve EPID dosimetry. The uncorrected or raw pixel response of the aS500 amorphous silicon EPID shows differences in response (sensitivity) of individual pixels as well as a large off‐axis differential response with respect to an ion chamber in water. Both can be corrected by division of raw images by the flood‐field (FF) image. However, this leads to two problems for dosimetry: (1) the beam profile is present in both the raw image and FF image, and hence is “washed out” of the corrected image, and (2) any mismatch of EPID position between dosimetry and FF calibration means that the beam profile and off‐axis response in the raw image and FF are misaligned. This causes artifacts in FF division and dosimetric errors. A method was developed to measure the off‐axis response and pixel sensitivity variation separately to allow correction of images at any EPID position while retaining beam profile information. The pixel sensitivity variation is applied to the imager plane and is independent of imager position. The off‐axis response depends on the imager plane position relative to the beam central axis. The pixel sensitivities were derived from multiple images of the same symmetric field acquired with the detector displaced laterally between each image. The off‐axis response was measured by acquiring off‐axis raw images (FF correction removed) and dividing out the off‐axis beam fluence and previously determined pixel sensitivity differences. The dosimetric errors due to lateral and vertical detector displacement with the conventional FF calibration method were measured and compared to the new method. Corrected EPID profiles were then compared to beam profiles measured with ion chamber in water for open fields. The EPID was found to have a large off‐axis differential response with respect to an ion chamber in water, particularly for . This increased to 13% at off‐axis for , and 3.5% for at the isocenter plane. The dosimetric errors introduced by detector displacement with conventional FF calibration were found to be approximately of lateral detector displacement and of vertical displacement. These were reduced to less than 1% for any position with the new correction method. Corrected EPID images agreed with ion‐chamber measurements to within 2% (excluding penumbra and low‐dose areas outside the field) for various field sizes. The new correction method gives consistent dosimetry for any EPID position and retains beam profile information in the image.
Liver segmentation for CT images using GVF snake
Authors: Liu Fan , Zhao Binsheng , Kijewski Peter K. , Wang Liang , Schwartz Lawrence H.
Published: December 2005

Accurate liver segmentation on computed tomography (CT) images is a challenging task especially at sites where surrounding tissues (e.g., stomach, kidney) have densities similar to that of the liver and lesions reside at the liver edges. We have developed a method for semiautomatic delineation of the liver contours on contrast‐enhanced CT images. The method utilizes a snake algorithm with a gradient vector flow (GVF) field as its external force. To improve the performance of the GVF snake in the segmentation of the liver contour, an edge map was obtained with a Canny edge detector, followed by modifications using a liver template and a concavity removal algorithm. With the modified edge map, for which unwanted edges inside the liver were eliminated, the GVF field was computed and an initial liver contour was formed. The snake algorithm was then applied to obtain the actual liver contour. This algorithm was extended to segment the liver volume in a slice‐by‐slice fashion, where the result of the preceding slice constrained the segmentation of the adjacent slice. 551 two‐dimensional liver images from 20 volumetric images with colorectal metastases spreading throughout the livers were delineated using this method, and also manually by a radiologist for evaluation. The difference ratio, which is defined as the percentage ratio of mismatching volume between the computer and the radiologist's results, ranged from 2.9% to 7.6% with a median value of 5.3%.
High‐speed photography during ultrasound illustrates potential therapeutic applications of microbubbles
Authors: Postema Michiel , van Wamel Annemieke , ten Cate Folkert J. , de Jong Nico
Published: December 2005

Ultrasound contrast agents consist of microscopically small encapsulated bubbles that oscillate upon insonification. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical microbubbles have been studied with the aid of high‐speed photography. We filmed medical microbubbles at higher frame rates than the ultrasonic frequency transmitted. Microbubbles with thin lipid shells have been observed to act as microsyringes during one single ultrasonic cycle. This jetting phenomenon presumably causes sonoporation. Furthermore, we observed that the gas content can be forced out of albumin‐encapsulated microbubbles. These free bubbles have been observed to jet, too. It is concluded that microbubbles might act as a vehicle to carry a drug in gas phase to a region of interest, where it has to be released by diagnostic ultrasound. This opens up a whole new area of potential applications of diagnostic ultrasound related to targeted imaging and therapeutic delivery of drugs such as nitric oxide.
Microdosimetric evaluation of the carbon beam at HIMAC
Authors: Endo Satoru , Tanaka Kenichi , Ishikawa Masayori , Hoshi Masaharu , Onizuka Yoshihiko , Takada Masashi , Yamaguchi Hiroshi , Hayabuchi Naofumi , Maeda Naoko , Shizuma Kiyoshi
Published: December 2005

Microdosimetric single event spectra were determined as a function of depth in an acrylic phantom for the carbon beam at HIMAC using a tissue equivalent proportional counter (TEPC) coupled to a scintillation counter system. The fragments produced by the carbon beam were identified by the ‐time of flight distribution obtained from two scintillation counters which were positioned at the up‐ and down‐stream of the TEPC. Lineal energy distribution for the carbon beam and its five fragments, namely, proton, helium, lithium, beryllium, and boron ions, were measured in the lineal‐energy range of at five phantom depths between 0 and . The dose distribution for the carbon beam and its fragments were obtained separately. The relative biological effectiveness (RBE) of the carbon beam in the phantom was calculated using a response function. The maximum RBE for the carbon beam was found to be about 5 near the Bragg peak. It was observed to rapidly decrease for Bragg peaks occurring at deeper positions in the phantom. The dose from the beam fragments accounted for about 30% to the total dose, however, its contribution to the RBE was less than 17%.
2‐Step IMAT and 2‐Step IMRT in three dimensions
Authors: Bratengeier Klaus
Published: December 2005

In two dimensions, 2‐Step Intensity Modulated Arc Therapy (2‐Step IMAT) and 2‐Step Intensity Modulated Radiation Therapy (IMRT) were shown to be powerful methods for the optimization of plans with organs at risk (OAR) (partially) surrounded by a target volume (PTV). In three dimensions, some additional boundary conditions have to be considered to establish 2‐Step IMAT as an optimization method. A further aim was to create rules for ad hoc adaptations of an IMRT plan to a daily changing PTV–OAR constellation. As a test model, a cylindrically symmetric PTV–OAR combination was used. The centrally placed OAR can adapt arbitrary diameters with different gap widths toward the PTV. Along the rotation axis the OAR diameter can vary, the OAR can even vanish at some axis positions, leaving a circular PTV. The width and weight of the second segment were the free parameters to optimize. The objective function to minimize was the root of the integral of the squared difference of the dose in the target volume and a reference dose. For the problem, two local minima exist. Therefore, as a secondary criteria, the magnitude of hot and cold spots were taken into account. As a result, the solution with a larger segment width was recommended. From plane to plane for varying radii of PTV and OAR and for different gaps between them, different sets of weights and widths were optimal. Because only one weight for one segment shall be used for all planes (respectively leaf pairs), a strategy for complex three‐dimensional (3‐D) cases was established to choose a global weight. In a second step, a suitable segment width was chosen, minimizing for this global weight. The concept was demonstrated in a planning study for a cylindrically symmetric example with a large range of different radii of an OAR along the patient axis. The method is discussed for some classes of tumor/organ at risk combinations. Noncylindrically symmetric cases were treated exemplarily. The product of width and weight of the additional segment as well as the integral across the segment profile was demonstrated to be an important value. This product was up to a factor of 3 larger than in the 2‐D case. Even in three dimensions, the optimized 2‐Step IMAT increased the homogeneity of the dose distribution in the PTV profoundly. Rules for adaptation to varying target‐OAR combinations were deduced. It can be concluded that 2‐Step IMAT and 2‐Step IMRT are also applicable in three dimensions. In the majority of cases, weights between 0.5 and 2 will occur for the additional segment. The width–weight product of the second segment is always smaller than the normalized radius of the OAR. The width–weight product of the additional segment is strictly connected to the relevant diameter of the organ at risk and the target volume. The derived formulas can be helpful to adapt an IMRT plan to altering target shapes.
Dimethylsulfoniopropionate Turnover Is Linked to the Composition and Dynamics of the Bacterioplankton Assemblage during a Microcosm Phytoplankton Bloom
Authors: Pinhassi Jarone , Simó Rafel , González José M. , Vila Maria , Alonso-Sáez Laura , Kiene Ronald P. , Moran Mary Ann , Pedrós-Alió Carlos
Published: December 2005

Processing of the phytoplankton-derived organic sulfur compound dimethylsulfoniopropionate (DMSP) by bacteria was studied in seawater microcosms in the coastal Gulf of Mexico (Alabama). Modest phytoplankton blooms (peak chlorophyll
a
[Chl
a
] concentrations of ∼2.5 μg liter
−1
) were induced in nutrient-enriched microcosms, while phytoplankton biomass remained low in unamended controls (Chl
a
concentrations of ∼0.34 μg liter
−1
). Particulate DMSP concentrations reached 96 nM in the enriched microcosms but remained approximately 14 nM in the controls. Bacterial biomass production increased in parallel with the increase in particulate DMSP, and nutrient limitation bioassays in the initial water showed that enrichment with DMSP or glucose caused a similar stimulation of bacterial growth. Concomitantly, increased bacterial consumption rate constants of dissolved DMSP (up to 20 day
−1
) and dimethylsulfide (DMS) (up to 6.5 day
−1
) were observed. Nevertheless, higher DMSP S assimilation efficiencies and higher contribution of DMSP to bacterial S demand were found in the controls compared to the enriched microcosms. This indicated that marine bacterioplankton may rely more on DMSP as a source of S under oligotrophic conditions than under the senescence phase of phytoplankton blooms. Phylogenetic analysis of the bacterial assemblages in all microcosms showed that the DMSP-rich algal bloom favored the occurrence of various
Roseobacter
members, flavobacteria (
Bacteroidetes
phylum), and oligotrophic marine
Gammaproteobacteria
. Our observations suggest that the composition of the bacterial assemblage and the relative contribution of DMSP to the overall dissolved organic sulfur/organic matter pool control how efficiently bacteria assimilate DMSP S and thereby potentially divert it from DMS production.
Soil Microbial Community Responses to Additions of Organic Carbon Substrates and Heavy Metals (Pb and Cr)
Authors: Nakatsu Cindy H. , Carmosini Nadia , Baldwin Brett , Beasley Federico , Kourtev Peter , Konopka Allan
Published: December 2005

Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the
Actinomycetales
were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.
Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community
Authors: DeRito Christopher M. , Pumphrey Graham M. , Madsen Eugene L.
Published: December 2005

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured
13
CO
2
respired from [
13
C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that
13
C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [
13
C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [
13
C]phenol, and (iii) 12 daily doses of [
13
C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted
13
CO
2
evolution by a factor of 10. Furthermore, imaging of
13
C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated
13
C into their biomass. PCR amplification and 16S rRNA gene sequencing of
13
C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera
Kocuria
and
Staphylococcus
, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus
Pseudomonas
. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.
Modeling Surface Growth of
Escherichia coli
on Agar Plates
Authors: Fujikawa Hiroshi , Morozumi Satoshi
Published: December 2005

Surface growth of
Escherichia coli
cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H. Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34°C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves.
Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification
Authors: Maruyama Fumito , Kenzaka Takehiko , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao
Published: December 2005

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count singleEscherichia colicells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes insideE. colicells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA.E. colicells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.
Triphenylmethane Reductase from
Citrobacter
sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in
Escherichia coli
Authors: Jang Moon-Sun , Lee Young-Mi , Kim Cheorl-Ho , Lee Jai-Heon , Kang Dong-Woo , Kim Seok-Jo , Lee Young-Choon
Published: December 2005

We purified to homogeneity an enzyme from
Citrobacter
sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from
Listeria monocytogenes
strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in
Escherichia coli
, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.
l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321
Authors: Sonke Theo , Ernste Sandra , Tandler Renate F. , Kaptein Bernard , Peeters Wilco P. H. , van Assema Friso B. J. , Wubbolts Marcel G. , Schoemaker Hans E.
Published: December 2005

An industrially attractivel-specific amidase was purified to homogeneity fromOchrobactrum anthropiNCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+(to 80%), Mn2+(to 400%), and Mg2+(to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of α-hydrogen- and (bulky) α,α-disubstituted α-amino acid amides, α-hydroxy acid amides, and α-N-hydroxyamino acid amides. In all cases, only thel-enantiomer was hydrolyzed, resulting inEvalues of more than 150. Simple aliphatic amides, β-amino and β-hydroxy acid amides, and dipeptides were not converted. The gene encoding thisl-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression inEscherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.
Molecular Analysis of the O-Antigen Gene Cluster ofEscherichia coliO86:B7 and Characterization of the Chain Length Determinant Gene (wzz)
Authors: Guo Hongjie , Yi Wen , Shao Jun , Lu Yuquan , Zhang Wenpeng , Song Jing , Wang Peng George
Published: December 2005

Escherichia coliO86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster betweengalFandhisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions betweengalFandgndare identical, includingwzygenes. However, deletion of the twowzygenes revealed thatwzyin O86:B7 is responsible for the polymerization of the O antigen, while the deletion ofwzyin O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functionalwzygene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that thewzzgenes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.
Exogenous Isoleucine and Fatty Acid Shortening Ensure the High Content of Anteiso-C
15:0
Fatty Acid Required for Low-Temperature Growth of
Listeria monocytogenes
Authors: Zhu Kun , Ding Xiang , Julotok Mudcharee , Wilkinson Brian J.
Published: December 2005

Previous studies have demonstrated that the branched-chain fatty acid anteiso-C
15:0
plays a critical role in the growth of
Listeria monocytogenes
at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C
15:0
solely at the expense of anteiso-C
17:0
. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10°C. Under this condition, the increase of anteiso-C
15:0
at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain α-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of β-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C
15:0
at the expense of anteiso-C
17:0
.