bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of coordinated molecular alterations in the development of androgen‐independent prostate cancer: an in vitro model that corroborates clinical observations
Authors: SHI YAN , CHATTERJEE SUNANDA J. , BRANDS FRANK H. , SHI SHAN‐RONG , POOTRAKUL LLANA , TAYLOR CLIVE R. , DATAR RAM , COTE RICHARD J.
Published: December 2005

OBJECTIVE

To investigate the role of potential downstream targets of HER‐2/neu, including the cell‐cycle regulator p27, proliferation‐associated protein Ki‐67, apoptosis inhibitor Bcl‐2, and signal‐transduction molecule Akt (which is associated with cell survival), as the development of androgen‐independent prostate cancer (AIPC) in patients who are initially responsive to androgen‐ablation therapy (AAT) is a significant clinical problem.

PATIENTS AND METHODS

Earlier studies showed that high levels of HER‐2/neu tyrosine kinase receptor expression as assessed by immunohistochemistry were significantly associated with the development of AIPC, and we hypothesised that HER‐2/neu overexpression provides an alternative proliferative stimulus upon androgen depletion. We established a unique clinical model system, comprising patients who received no AAT, or who had preoperative AAT, or those with advanced tumours resistant to AAT. To test our hypothesis in vitro, we stably transfected full‐length HER‐2/neu cDNA in androgen‐responsive LNCaP cells and examined the effects of HER‐2/neu overexpression on cell proliferation, apoptosis, androgen‐receptor activation, and Akt phosphorylation upon androgen deprivation by using immunohistochemistry and Western blot technique.

RESULTS

p27 expression was initially induced on exposure to AAT, and significantly decreased in AIPC (P < 0.001). There was also a significant increase in the Ki‐67 index in AIPC (P = 0.001). Elevated Bcl‐2 expression was closely associated with AAT (P = 0.002), suggesting that Bcl‐2 expression is induced on initial exposure to AAT. Further, Bcl‐2 expression was highest in hormone‐resistant cancers (P < 0.001). Using the HER‐2/neu transfected cell‐line model, we confirmed the mechanistic basis of the clinical observations which elucidate the pathway leading to HER‐2/neu‐mediated androgen independence. On androgen deprivation, the HER‐2/neu transfected cells had higher proliferation rates, lower G1 arrest, inhibited p27 up‐regulation, a lower apoptotic index, and higher Bcl‐2, prostate‐specific antigen and phosphorylated Akt expression than the mock‐transfected LNCaP cells.

CONCLUSION

This study suggests that prostate cancer cells undergo a series of coordinated changes after exposure to AAT, which eventually result in the development of androgen independence. Further, in support of previous results, it appears that a major factor in this process is the induction of HER‐2/neu overexpression, which occurs after initial exposure to AAT. HER‐2/neu may contribute to the development of androgen independence through: (i) maintaining cell proliferation; (ii) inhibiting apoptosis; and/or (iii) inducing AR activation in a ligand‐independent fashion. These effects may be mediated, at least in part, through activation of the PI3K/Akt pathway.
The role of Ni2+‐sensitive T‐type Ca2+ channels in the regulation of spontaneous excitation in detrusor smooth muscles of the guinea‐pig bladder
Authors: YANAI YOSHIMASA , HASHITANI HIKARU , KUBOTA YASUE , SASAKI SHOICHI , KOHRI KENJIRO , SUZUKI HIKARU
Published: December 2005

OBJECTIVE

To explore the role of Ni2+‐sensitive T‐type Ca2+ channels in the generation of spontaneous excitation of detrusor smooth muscles.

MATERIALS AND METHODS

In isolated detrusor smooth muscle bundles of the guinea‐pig bladder, changes in the membrane potential and muscle tension were measured using intracellular microelectrodes and isometric tension recording. Changes in the intracellular Ca2+ concentration were recorded from bundles loaded with the fluorescent dye fura‐PE3.

RESULTS

Detrusor smooth muscles had two types of spontaneous electrical activity, i.e. individual and bursting action potentials. Ni2+ (30 µm), a blocker for T‐type Ca2+ channels, reduced the frequency of individual action potentials without changing their amplitude. Higher concentrations of Ni2+ (100–300 µm) converted individual action potentials into the bursts, as did apamin (0.1 µm), a blocker of small‐conductance Ca2+‐activated K+ channels (SK). They also increased the amplitudes of spontaneous Ca2+ transients and corresponding contractions whilst reducing their frequencies. In preparations which generated bursting action potentials, nifedipine (1 µm) converted action potentials into spontaneous transient depolarizations (STDs), and subsequent applications of Ni2+ (100 µm) abolished STDs. Gadolinium (100 µm) and SKF96365 (10 µm), blockers for nonselective cation channels, and niflumic acid (100 µm), a blocker for Ca2+‐activated Cl– channels, had no effect on either the amplitude or frequency of spontaneous action potentials.

CONCLUSIONS

The T‐type Ca2+ channel may have dual roles in generating spontaneous excitation in detrusor smooth muscles. First, activity of these channels may account for the preceding depolarizations that lead to action potentials. Second, Ca2+ influx through T‐type Ca2+ channels may couple functionally to SK channels, contributing to the stability of the resting membrane potential in detrusor smooth muscle. Thus, pharmacological manipulation of T‐type Ca2+ channels in detrusor smooth muscles could be of potential value for treating the overactive bladder.
FROM RATIONALITY TO BOUNDED RATIONALITY*
Authors: ZHENG ZIYU
Published: December 2005

We investigate the role of bounded rationality in asset pricing and information efficiency. We show that the market stays between the weak‐form efficiency and the semi‐strong‐form efficiency in a market with a single asymmetric information and without noise supply. We show the existence of an overall equilibrium in which the publicly available information can be priced and is ‘almost free’, and the majority of the agents choose to stay uninformed. We show the existence of a Nash competitive equilibrium in an information game.
OPTIMAL INVESTMENT MODELS WITH MINIMUM CONSUMPTION CRITERIA
Authors: FLEMING WENDELL H.
Published: December 2005

This paper considers a max‐min formulation of multistage optimal investment and consumption problems, with uncertainties in the form of variable productivities of capital and interest rates. The criterion of control performance is minimum consumption over time, weighted by a coefficient which indicates the likelihood of possible disturbance sequences. A dynamic programming method is used. Explicit results for a max‐min formulation of the Merton portfolio optimisation problem are obtained. A production‐consumption‐debt model arising in international finance is also considered.
Leading Indicators for Euro‐area Inflation and GDP Growth*
Authors: Banerjee Anindya , Marcellino Massimiliano , Masten Igor
Published: December 2005

In this paper, we evaluate the role of a set of variables as leading indicators for Euro‐area inflation and GDP growth. Our leading indicators are taken from the variables in the European Central Bank's (ECB) Euro‐area‐wide model database, plus a set of similar variables for the US. We compare the forecasting performance of each indicator ex post with that of purely autoregressive models. We also analyse three different approaches to combining the information from several indicators. First, ex post, we discuss the use as indicators of the estimated factors from a dynamic factor model for all the indicators. Secondly, within an ex ante framework, an automated model selection procedure is applied to models with a large set of indicators. No future information is used, future values of the regressors are forecast, and the choice of the indicators is based on their past forecasting records. Finally, we consider the forecasting performance of groups of indicators and factors and methods of pooling the ex ante single‐indicator or factor‐based forecasts. Some sensitivity analyses are also undertaken for different forecasting horizons and weighting schemes of forecasts to assess the robustness of the results.
Evaluating PcGets and RETINA as Automatic Model Selection Algorithms*
Authors: Castle Jennifer L.
Published: December 2005

The paper describes two automatic model selection algorithms, RETINA and PcGets, briefly discussing how the algorithms work and what their performance claims are. RETINA's Matlab implementation of the code is explained, then the program is compared with PcGets on the data in Perez‐Amaral, Gallo and White (2005, Econometric Theory, Vol. 21, pp. 262–277), ‘A Comparison of Complementary Automatic Modelling Methods: RETINA and PcGets’, and Hoover and Perez (1999, Econometrics Journal, Vol. 2, pp. 167–191), ‘Data Mining Reconsidered: Encompassing and the General‐to‐specific Approach to Specification Search’. Monte Carlo simulation results assess the null and non‐null rejection frequencies of the RETINA and PcGets model selection algorithms in the presence of nonlinear functions.
Impact of pre‐aging on the tensile and bending properties of AA 6061
Authors: Ou Bin‐Lung , Shen Chin‐Hui
Published: December 2005

The impact of low temperature pre‐aging on the artificial age‐hardening of AA 6061 (Al–1.1Mg–0.63Si) has been investigated using differential scanning calorimetry (DSC) as well as by measuring the electrical resistivity, and confirmed by microstructure observation using scanning electron microscopy and transmission electron microscopy (TEM). Tensile tests were performed to determine the mechanical properties of the samples. During the pre‐aging treatment GP zones I formed at 0–30°C. These GP zones I retarded the precipitation of the intermediate β′ phase during artificial aging and led to a decrease in the strength of the alloy. During the pre‐aging treatment β′′ precipitates formed at 40–70°C. Increasing the pre‐aging temperature significantly increases the strength after artificial aging. On the other hand, increasing the pre‐aging temperature decreases the size of the precipitation‐free zones (PFZ) adjacent to the grain boundaries. It was found that the tensile strength was greater if the PFZs were smaller than if they were larger. Grain boundary precipitates (GBP) did not significantly contribute to the hardening or strengthening. The specimens subjected to a lower pre‐aging temperature followed by the same artificial aging step had better bendability than did the specimens subjected to a higher pre‐aging temperature.
Human airway trypsin‐like protease induces amphiregulin release through a mechanism involving protease‐activated receptor‐2‐mediated ERK activation and TNF α‐converting enzyme activity in airway epithelial cells
Authors: Chokki Manabu , Eguchi Hiroshi , Hamamura Ichiro , Mitsuhashi Hiroaki , Kamimura Takashi
Published: December 2005

Human airway trypsin‐like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease‐activated receptor‐2 (PAR‐2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR‐epidermal growth factor receptor pathway in the airway epithelial cell line NCI‐H292. In this study, the mechanisms by which HAT‐induced AR release can occur were investigated. HAT‐induced AR gene expression was mediated by extracellular signal‐regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR‐2 agonist peptide (PAR‐2 AP) induced ERK phosphorylation; further, desensitization of PAR‐2 with a brief exposure of cells to PAR‐2 AP resulted in inhibition of HAT‐induced ERK phosphorylation, suggesting that HAT activates ERK through PAR‐2. Moreover, PAR‐2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR‐2‐mediated activation of ERK is essential for HAT‐induced AR production. However, in contrast to HAT, PAR‐2 AP could not cause AR release into extracellular space; it appears that activation of PAR‐2 is not sufficient for HAT‐induced AR release. Finally, HAT‐induced AR release was eliminated by blockade of tumour necrosis factor α‐converting enzyme (TACE) by the TAPI‐1 and RNA interference, suggesting that TACE activity is necessary for HAT‐induced AR release. These observations show that HAT induces AR production through the PAR‐2 mediated ERK pathway, and then causes AR release by a TACE‐dependent mechanism.
Uniform Head in Horizontal and Vertical Wells
Authors: Steward David R. , Jin Wei
Published: December 2005

The steady‐state head within a fully penetrating well may be estimated by evaluating the Thiem equation at the radius of the well. A method is presented here to extend results from the Thiem equation to horizontal wells and to partially penetrating wells. The particular model used in this investigation is based upon the analytic element method; it accurately reproduces a boundary condition of uniform head along the cylindrical surface at the perforated face of the well. This model is exercised over a representative range of parameters including the well's length, radius, and pumping rate, and the aquifer's hydraulic conductivity and thickness. Results are presented in a set of figures and tables that compare the well's drawdown to the drawdown that would have been obtained using the Thiem solution with the same pumping rate and radius. A methodology is presented to estimate the head within a horizontal or partially penetrating well by adding a correction term to results that can be readily obtained from computer models of vertical fully penetrating wells. This approach may also be used to contrast the differences in head between horizontal and vertical wells of various lengths, radii, and placement elevations.
Optimized interstitial PDT prostate treatment planning with the Cimmino feasibility algorithm
Authors: Altschuler Martin D. , Zhu Timothy C. , Li Jun , Hahn Stephen M.
Published: December 2005

The primary aim of this study was to determine whether optimized photodynamic therapy (PDT) treatment planning (seeking optimized positions, lengths, and strengths of the light sources to satisfy a given dose prescription) can improve dose coverage to the prostate and the sparing of critical organs relative to what can be achieved by the standard PDT plan. The Cimmino algorithm and search procedures based on that algorithm were tested for this purpose. A phase I motexafin lutetium (MLu)‐mediated photodynamic therapy protocol is ongoing at the University of Pennsylvania. PDT for the prostate is performed with cylindrical diffusing fibers of various lengths inserted perpendicular to a base plate to obtain longitudinal coverage by a matrix of parallel catheters. The standard plan for the protocol uses sources of equal strength with equal spaced loading. Uniform optical properties were assumed. Our algorithms produce plans that cover the prostate and spare the urethra and rectum with less discrepancy from the dose prescription than the standard plan. The Cimmino feasibility algorithm is fast enough that changes to the treatment plan may be made in the operating room before and during PDT to optimize light delivery.
The UF series of tomographic computational phantoms of pediatric patients
Authors: Lee Choonik , Williams Jonathan L. , Lee Choonsik , Bolch Wesley E.
Published: December 2005

Two classes of anthropomorphic computational phantoms exist for use in Monte Carlo radiation transport simulations: tomographic voxel phantoms based upon three‐dimensional (3D) medical images, and stylized mathematical phantoms based upon 3D surface equations for internal organ definition. Tomographic phantoms have shown distinct advantages over the stylized phantoms regarding their similarity to real human anatomy. However, while a number of adult tomographic phantoms have been developed since the early 1990s, very few pediatric tomographic phantoms are presently available to support dosimetry in pediatric diagnostic and therapy examinations. As part of a larger effort to construct a series of tomographic phantoms of pediatric patients, five phantoms of different ages ( male, female, female, male, and male) have been constructed from computed tomography (CT) image data of live patients using an IDL‐based image segmentation tool. Lungs, bones, and adipose tissue were automatically segmented through use of window leveling of the original CT numbers. Additional organs were segmented either semiautomatically or manually with the aid of both anatomical knowledge and available image‐processing techniques. Layers of skin were created by adding voxels along the exterior contour of the bodies. The phantoms were created from fused images taken from head and chest‐abdomen‐pelvis CT exams of the same individuals ( and phantoms) or of two different individuals of the same sex and similar age (, , and phantoms). For each model, the resolution and slice positions of the image sets were adjusted based upon their anatomical coverage and then fused to a single head‐torso image set. The resolutions of the phantoms for the , , , , and are , , , , and , respectively. While organ masses can be matched to reference values in both stylized and tomographic phantoms, side‐by‐side comparisons of organ doses in both phantom classes indicate that organ shape and positioning are equally important parameters to consider in accurate determinations of organ absorbed dose from external photon irradiation. Preliminary studies of external photon irradiation of the phantom indicate significant departures of organ dose coefficients from that predicted by the existing stylized phantom series. Notable differences between pediatric stylized and tomographic phantoms include anterior‐posterior (AP) and right lateral (RLAT) irradiation of the stomach wall, left lateral (LLAT) and right lateral (RLAT) irradiation of the thyroid, and AP and posterior‐anterior (PA) irradiation of the urinary bladder.
Reduction of false positives by internal features for polyp detection in CT‐based virtual colonoscopy
Authors: Wang Zigang , Liang Zhengrong , Li Lihong , Li Xiang , Li Bin , Anderson Joseph , Harrington Donald
Published: December 2005

In this paper, we present a computer‐aided detection (CAD) method to extract and use internal features to reduce false positive (FP) rate generated by surface‐based measures on the inner colon wall in computed tomographic (CT) colonography. Firstly, a new shape descriptionglobal curvature, which can provide an overall shape description of the colon wall, is introduced to improve the detection of suspicious patches on the colon wall whose geometrical features are similar to that of the colonic polyps. By a ray‐driven edge finder, the volume of each detected patch is extracted as a fitted ellipsoid model. Within the ellipsoid model, CT image density distribution is analyzed. Three types of (geometrical, morphological, and textural) internal features are extracted and applied to eliminate the FPs from the detected patches. The presented CAD method was tested by a total of 153 patient datasets in which 45 patients were found with 61 polyps of sizes 4–30 mm by optical colonoscopy. For a 100% detection sensitivity (on polyps), the presented CAD method had an average FPs of 2.68 per patient dataset and eliminated 93.1% of FPs generated by the surface‐based measures. The presented CAD method was also evaluated by different polyp sizes. For polyp sizes of 10–30 mm, the method achieved mean number of FPs per dataset of 2.0 with 100% sensitivity. For polyp sizes of 4–10 mm, the method achieved 3.44 FP per dataset with 100% sensitivity.
Radiation dose reduction in four‐dimensional computed tomography
Authors: Li T. , Schreibmann E. , Thorndyke B. , Tillman G. , Boyer A. , Koong A. , Goodman K. , Xing L.
Published: December 2005

Four‐dimensional (4D) CT is useful in many clinical situations, where detailed abdominal and thoracic imaging is needed over the course of the respiratory cycle. However, it usually delivers a larger radiation dose than the standard three‐dimensional (3D) CT, since multiple scans at each couch position are required in order to provide the temporal information. Our purpose in this work is to develop a method to perform 4D CT scans at relatively low current, hence reducing the radiation exposure of the patients. To deal with the increased statistical noise caused by the low current, we proposed a novel 4D penalized weighted least square (4D‐PWLS) smoothing method, which can incorporate both spatial and phase information. The 4D images at different phases were registered to the same phase via a deformable model, thereby, a regularization term combining temporal and spatial neighbors can be designed for the 4D‐PWLS objective function. The proposed method was tested with phantom experiments and a patient study, and superior noise suppression and resolution preservation were observed. A quantitative evaluation of the benefit of the proposed method to 4D radiotherapy and 4D PET/CT imaging are under investigation.
Feasibility of four‐dimensional conformal planning for robotic radiosurgery
Authors: Schlaefer A. , Fisseler J. , Dieterich S. , Shiomi H. , Cleary K. , Schweikard A.
Published: December 2005

Organ motion can have a severe impact on the dose delivered by radiation therapy, and different procedures have been developed to address its effects. Conventional techniques include breath hold methods and gating. A different approach is the compensation for target motion by moving the treatment beams synchronously. Practical results have been reported for robot based radiosurgery, where a linear accelerator mounted on a robotic arm delivers the dose. However, not all organs move in the same way, which results in a relative motion of the beams with respect to the body and the tissues in the proximity of the tumor. This relative motion can severely effect the dose delivered to critical structures. We propose a method to incorporate motion in the treatment planning for robotic radiosurgery to avoid potential overdosing of organs surrounding the target. The method takes into account the motion of all considered volumes, which is discretized for dose calculations. Similarly, the beam motion is taken into account and the aggregated dose coefficient over all discrete steps is used for planning. We simulated the treatment of a moving target with three different planning methods. First, we computed beam weights based on a 3D planning situation and simulated treatment with organ motion and the beams moving synchronously to the target. Second, beam weights were computed by the 4D planning method incorporating the organ and beam motion and treatment was simulated for beams moving synchronously to the target. Third, the beam weights were determined by the 4D planning method with the beams fixed during planning and simulation. For comparison we also give results for the 3D treatment plan if there was no organ motion and when the plan is delivered by fixed beams in the presence of organ motion. The results indicate that the new 4D method is preferable and can further improve the overall conformality of motion compensated robotic radiosurgery.
2D‐3D registration of coronary angiograms for cardiac procedure planning and guidance
Authors: Turgeon Guy‐Anne , Lehmann Glen , Guiraudon Gerard , Drangova Maria , Holdsworth David , Peters Terry
Published: December 2005

We present a completely automated 2D‐3D registration technique that accurately maps a patient‐specific heart model, created from preoperative images, to the patient's orientation in the operating room. This mapping is based on the registration of preoperatively acquired 3D vascular data with intraoperatively acquired angiograms. Registration using both single and dual‐plane angiograms is explored using simulated but realistic datasets that were created from clinical images. Heart deformations and cardiac phase mismatches are taken into account in our validation using a digital 4D human heart model. In an ideal situation where the pre‐ and intraoperative images were acquired at identical time points within the cardiac cycle, the single‐plane and the dual‐plane registrations resulted in 3D root‐mean‐square (rms) errors of and , respectively. When a 10% timing offset was added between the pre‐ and the intraoperative acquisitions, the single‐plane registration approach resulted in inaccurate registrations in the out‐of‐plane axis, whereas the dual‐plane registration exhibited a 98% success rate with a 3D rms error of . When all potential sources of error were included, namely, the anatomical background, timing offset, and typical errors in the vascular tree reconstruction, the dual‐plane registration performed at 94% with an accuracy of .
Determination of penumbral widths from ion chamber measurements
Authors: Herrup D. , Chu J. , Cheung H. , Pankuch M.
Published: December 2005

We have investigated methods of reconstructing beam profiles in the penumbral region using a set of axially symmetric chambers, differing only in the detector radius. In principal, the transfer functions, or kernels, of such chambers should be functions of radius only. Three chambers of radii 0.297, 0.556, and 0.714 cm have been used. The transfer functions of the chambers can be determined by deconvolving the profiles measured with each detector with the PPMC profile. The results indicate that the transfer functions can be parametrized accurately as a Gaussian cutoff at , with the average radius of the chamber. Deconvolution of the measured profiles with the transfer functions yields a profile that agrees with the PPMC profile to over the 20–80% penumbra.
Regulation and Characterization of Two Nitroreductase Genes,
nprA
and
nprB
, of
Rhodobacter capsulatus
Authors: Pérez-Reinado Eva , Blasco Rafael , Castillo Francisco , Moreno-Vivián Conrado , Roldán M. Dolores
Published: December 2005

Among photosynthetic bacteria, strains B10 and E1F1 of
Rhodobacter capsulatus
photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of
R. capsulatus
, two putative genes coding for possible nitroreductases were isolated from
R. capsulatus
B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the
nprB
gene seems to be constitutive, whereas
nprA
gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative
mar
/
sox
boxes in the possible promoter region of the
nprA
gene and the induction of
nprA
gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the
nprA
gene for the two-component MarRA regulatory system, which in
Escherichia coli
controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the
nprA
gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.
Infectious Swine Hepatitis E Virus Is Present in Pig Manure Storage Facilities on United States Farms, but Evidence of Water Contamination Is Lacking
Authors: Kasorndorkbua C. , Opriessnig T. , Huang F. F. , Guenette D. K. , Thomas P. J. , Meng X.-J. , Halbur P. G.
Published: December 2005

Fresh feces, manure slurry (from earthen lagoons and/or concrete pits), and drinking and surface water samples were collected from 28 pig farms in the Midwestern United States. All samples were tested for hepatitis E virus (HEV) RNA by reverse transcription-PCR. Seven of 28 farms had fecal samples that contained HEV. Of 22 farms where pit samples were accessible, 15 contained HEV, and of 8 farms that had lagoons, 3 contained HEV. The highest virus titers were 10 and 10
3
genome equivalents per 60 ml of manure slurry in lagoon and pit samples, respectively. None of the water samples tested HEV positive. To determine the infectivity of the HEV found in the positive farm 19 lagoon (designated L19) or farm 12 pit (designated P12) samples, pigs were inoculated either intravenously (
n
= 3) or orally (
n
= 3) with the L19 or P12 manure slurry. Four pigs inoculated intravenously with prototype swine HEV served as positive controls. All positive-control pigs shed HEV in feces and 3 of 4 developed anti-HEV antibodies. Two pigs in the intravenously inoculated P12 group shed HEV in feces, and one of the pigs seroconverted to anti-HEV antibodies. None of the pigs in the negative-control, L19 oral, L19 intravenous, or P12 oral group shed HEV in feces. The findings indicate that HEV found in pig manure slurry was infectious when inoculated intravenously. Pit manure slurry is a potential source of HEV infection and for contamination of the environment. Contamination of drinking or surface water with HEV was not found on or near the pig farms.
Coaggregation Facilitates Interspecies Hydrogen Transfer betweenPelotomaculum thermopropionicumandMethanothermobacter thermautotrophicus
Authors: Ishii Shun'ichi , Kosaka Tomoyuki , Hori Katsutoshi , Hotta Yasuaki , Watanabe Kazuya
Published: December 2005

A thermophilic syntrophic bacterium,Pelotomaculum thermopropionicumstrain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen,Methanothermobacter thermautotrophicusstrain ΔH. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with ΔH cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and ΔH cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Fick's diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 μm, while the allowable distances for ethanol and propanol oxidation were 16 μm and 32 μm, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 μm (at a concentration in the mid-exponential growth phase of the coculture of 5 × 107cells ml−1), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.
Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences
Authors: Slavica Anita , Dib Iskandar , Nidetzky Bernd
Published: December 2005

One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing stress-induced oxidation inTrigonopsis variabilisd-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.
Distribution of Genes Encoding Putative Virulence Factors and Fragment Length Polymorphisms in the
vrrA
Gene among Brazilian Isolates of
Bacillus cereus
and
Bacillus thuringiensis
Authors: Zahner Viviane , Cabral Diana Aparecida , Régua-Mangia Adriana Hamond , Rabinovitch Leon , Moreau Gaétan , McIntosh Douglas
Published: December 2005

One hundred twenty-one strains of the
Bacillus cereus
complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (
bceT
,
hblA
,
nheBC
,
plc
,
sph
, and
vip3A
) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the
vrrA
open reading frame as the target. Amplicons were generated from isolates of
B. cereus
and
Bacillus thuringiensis
for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in
Bacillus thuringiensis
than in
B. cereus
. The
vip3A
sequence, which encodes an insecticidal toxin, was detected exclusively in
B. thuringiensis
. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that
B. cereus
and
B. thuringiensis
should be considered to represent a single species.
Impact of the Microscale Distribution of a
Pseudomonas
Strain Introduced into Soil on Potential Contacts with Indigenous Bacteria
Authors: Dechesne Arnaud , Pallud Céline , Bertolla Franck , Grundmann Geneviève L.
Published: December 2005

Soil bioaugmentation is a promising approach in soil bioremediation and agriculture. Nevertheless, our knowledge of the fate and activity of introduced bacteria in soil and thus of their impact on the soil environment is still limited. The microscale spatial distribution of introduced bacteria has rarely been studied, although it determines the encounter probability between introduced cells and any components of the soil ecosystem and thus plays a role in the ecology of introduced bacteria. For example, conjugal gene transfer from introduced bacteria to indigenous bacteria requires cell-to-cell contact, the probability of which depends on their spatial distribution. To quantitatively characterize the microscale distribution of an introduced bacterial population and its dynamics, a
gfp
-tagged derivative of
Pseudomonas putida
KT2440 was introduced by percolation in repacked soil columns. Initially, the introduced population was less widely spread at the microscale level than two model indigenous functional communities: the 2,4-dichlorophenoxyacetic acid degraders and the nitrifiers (each at 10
6
CFU g
−1
soil). When the soil was percolated with a substrate metabolizable by
P. putida
or incubated for 1 month, the microscale distribution of introduced bacteria was modified towards a more widely dispersed distribution. The quantitative data indicate that the microscale spatial distribution of an introduced strain may strongly limit its contacts with the members of an indigenous bacterial community. This could constitute an explanation to the low number of indigenous transconjugants found most of time when a plasmid-donor strain is introduced into soil.
Stable Isotopic Studies of
n
-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture
Authors: Davidova Irene A. , Gieg Lisa M. , Nanny Mark , Kropp Kevin G. , Suflita Joseph M.
Published: December 2005

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated
n
-alkanes (C
6
to C
12
) and 1-
13
C-labeled
n
-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of
d
14
-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated
n
-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-
13
C-labeled
n
-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that
13
C nuclei from 1-
13
C-labeled
n
-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that
13
C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol.
177:
235-243, 2002) for metabolism of deuterated
n
-hexane by a denitrifying bacterium.
UniFrac: a New Phylogenetic Method for Comparing Microbial Communities
Authors: Lozupone Catherine , Knight Rob
Published: December 2005

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.
Contingency Locus in
ctsR
of
Listeria monocytogenes
Scott A: a Strategy for Occurrence of Abundant Piezotolerant Isolates within Clonal Populations
Authors: Karatzas Kimon A. G. , Valdramidis Vasileios P. , Wells-Bennik Marjon H. J.
Published: December 2005

In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of
Listeria monocytogenes
lacks a codon in the class 3 heat shock regulator gene
ctsR
. This mutation in the region that encodes four consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and reduced virulence. The aim of the present study was to determine whether mutations in
ctsR
are frequently associated with piezotolerance in
L. monocytogenes
. Wild-type cultures of
L. monocytogenes
were therefore exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant mutants were estimated to be present in the initial wild-type population at frequencies of >10
−5
. Subsequent sequencing of the
ctsR
gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e.,
n
= 21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion in the glycine-encoding region of
ctsR
, identical to what was found in our previous study. Interestingly, 2 of 21 mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area (both leading to frameshifts and a truncated
ctsR
), and an in-frame 114-bp deletion encoding a drastically shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A piezotolerant phenotype could not be linked to mutations in
ctsR
in 8 of 33 isolates, indicating that other thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance of
ctsR
in piezotolerance and stress tolerance of
L. monocytogenes
, and it demonstrates that short-sequence repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation within
L. monocytogenes
cultures, thus playing an important role in survival.
Adaptive Responses to Oxygen Stress in Obligatory Anaerobes
Clostridium acetobutylicum
and
Clostridium aminovalericum
Authors: Kawasaki Shinji , Watamura Yusuke , Ono Masaki , Watanabe Toshihiro , Takeda Kouji , Niimura Youichi
Published: December 2005

Clostridium acetobutylicum
and
Clostridium aminovalericum
, both obligatory anaerobes, grow normally after growth conditions are changed from anoxic to microoxic, where the cells consume oxygen proficiently. In
C. aminovalericum
, a gene encoding a previously characterized H
2
O-forming NADH oxidase, designated
noxA
, was cloned and sequenced. The expression of
noxA
was strongly upregulated within 10 min after the growth conditions were altered to a microoxic state, indicating that
C. aminovalericum
NoxA is involved in oxygen metabolism. In
C. acetobutylicum
, genes suggested to be involved in oxygen metabolism and genes for reactive oxygen species (ROS) scavenging were chosen from the genome database. Although no clear orthologue of
C. aminovalericum
NoxA was found, Northern blot analysis identified many O
2
-responsive genes (e.g., a gene cluster [
CAC2448
to
CAC2452
] encoding an NADH rubredoxin oxidoreductase-A-type flavoprotein-desulfoferrodoxin homologue-MerR family-like protein-flavodoxin, an operon [
CAC1547
to
CAC1549
] encoding a thioredoxin-thioredoxin reductase-glutathione peroxidase-like protein, an operon [
CAC1570
and
CAC1571
] encoding two glutathione peroxidase-like proteins, and genes encoding thiol peroxidase, bacterioferritin comigratory proteins, and superoxide dismutase) whose expression was quickly and synchronously upregulated within 10 min after flushing with 5% O
2.
The corresponding enzyme activities, such as NAD(P)H-dependent peroxide (H
2
O
2
and alkyl hydroperoxides) reductase, were highly induced, indicating that microoxic growth of
C. acetobutylicum
is associated with the expression of a number of genes for oxygen metabolism and ROS scavenging.
Inhibition ofHelicobacter pyloriand Associated Urease by Oregano and Cranberry Phytochemical Synergies
Authors: Lin Y. T. , Kwon Y. I. , Labbe R. G. , Shetty K.
Published: December 2005

Ulcer-associated dyspepsia is caused by infection withHelicobacter pylori. H. pyloriis linked to a majority of peptic ulcers. Antibiotic treatment does not always inhibit or killH. pyloriwith potential for antibiotic resistance. The objective of this study was to determine the potential for using phenolic phytochemical extracts to inhibitH. pyloriin a laboratory medium. Our approach involved the development of a specific phenolic profile with optimization of different ratios of extract mixtures from oregano and cranberry. Subsequently, antimicrobial activity and antimicrobial-linked urease inhibition ability were evaluated. The results indicated that the antimicrobial activity was greater in extract mixtures than in individual extracts of each species. The results also indicate that the synergistic contribution of oregano and cranberry phenolics may be more important for inhibition than any species-specific phenolic concentration. Further, based on plate assay, the likely mode of action may be through urease inhibition and disruption of energy production by inhibition of proline dehydrogenase at the plasma membrane.
Heterologous Leaky Production of Transglutaminase in
Lactococcus lactis
Significantly Enhances the Growth Performance of the Host
Authors: Fu Rui-Yan , Chen Jian , Li Yin
Published: December 2005

This study describes a novel strategy to improve the growth performance of
Lactococcus lactis
by heterologous production of food-grade transglutaminase. The
mtg
gene from
Streptoverticillium mobaraense
that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into
L. lactis
subsp.
cremoris
NZ9000. The leaky expression of the
mtg
gene from the
nisA
promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of
L. lactis
, which is an important step for the purposes of producing proteins of commercial interest using
L. lactis
as a host.
Identification of “CandidatusThioturbo danicus,” a Microaerophilic Bacterium That Builds Conspicuous Veils on Sulfidic Sediments
Authors: Muyzer Gerard , Yildirim Esengül , van Dongen Udo , Kühl Michael , Thar Roland
Published: December 2005

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the ε-subdivision of theProteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “CandidatusThioturbo danicus.”
Analysis of
fae
and
fhcD
Genes in Mono Lake, California
Authors: Nercessian Olivier , Kalyuzhnaya Marina G. , Joye Samantha B. , Lidstrom Mary E. , Chistoserdova Ludmila
Published: December 2005

Genes for two enzymes of the tetrahydromethanopterin-linked C
1
transfer pathway (
fae
and
fhcD
) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for
fae
and
fhcD
was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).