bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of temporal and spatial expression patterns of amelogenin isoforms during mouse molar development
Authors: Iacob Stanca , Veis Arthur
Published: May 2006

Amelogenin synthesis is initiated in a restricted time frame during odontogenesis. Polypeptides translated from several alternatively spliced isoforms of amelogenin mRNA have been identified in ameloblasts and odontoblasts. Recent studies suggest that the isoforms deleting exons 6a, 6b, and 6c produce polypeptides that might exert regulatory functions governing the late stages of ameloblast and odontoblast differentiation. Herein, the spatial and temporal expression of mouse amelogenin mRNA isoforms M194, M180, M73, and M59 have been determined around the perinatal development period using splice form‐specific probes. Expression levels and distribution patterns varied with developmental stage and cell location. Amelogenin mRNA expression was most prominent within the enamel organ at boundaries between cell layers, beginning at the newborn stage (PN0.5). Odontoblasts supported the expression of M73 and M59 mRNA from developmental stages PN0.5 to PN1.5 (1 d of age). In contrast, ameloblasts expressed predominantly the M180 mRNA isoform with full exon 6 but devoid of exon 4. In the enamel organ, the stratum intermediun cells supported expression of the full‐length isoform, M194, including the full exon 6 and exon 4 sequences, and strikingly, expression of M180 message was inhibited. In conclusion, ameloblasts, odontoblasts, and stratum intermedium cells demonstrate selective alternative splicing patterns of the amelogenin pre‐mRNA transcript.
Expression and characterization of a Rana pipiens amelogenin protein
Authors: Diekwisch T. G. H. , Wang X. , Fan J. L. , Ito Y. , Luan X.
Published: May 2006

Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico‐chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens. In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli. Our newly generated R. pipiens amelogenin‐specific antisera resolved a major 19‐kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue‐specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence‐based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41‐nm radius subunits compared with 14‐nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self‐assembly in contrast with a homogeneous ‘stippled’ matrix in mouse amelogenin matrix self‐assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico‐chemical properties which may be related to specific modes of crystal formation in frog enamel.
Anti‐inflammatory properties of enamel matrix derivative in human blood
Authors: Myhre A. E. , Lyngstadaas S. P. , Dahle M. K. , Stuestøl J. F. , Foster S. J. , Thiemermann C. , Lilleaasen P. , Wang J. E. , Aasen A. O.
Published: May 2006

Background and Objective:  Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro‐inflammatory cytokine [tumor necrosis factor (TNF)‐α], an anti‐inflammatory cytokine (interleukin‐10) and a chemokine (interleukin‐ 8) in whole human blood challenged by bacterial cell wall components.

Material and Methods:  Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8‐(4‐chlorophenyl)thio‐cAMP (8‐CPT‐cAMP). TNF‐α, interleukin‐8 and interleukin‐10 were analysed from plasma by enzyme‐linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA).

Results:  We found that EMD attenuated the release of TNF‐α and interleukin‐8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin‐10 was unchanged. Enamel matrix derivative also produced a four‐fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8‐CPT‐cAMP attenuated the formation of TNF‐α, but not of interleukin‐10, in blood challenged by lipopolysaccharide.

Conclusion:  Enamel matrix derivative limits the release of pro‐inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti‐inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells.
One-Equation Subgrid Scale Model Using Dynamic Procedure for the Energy Production
Authors: Kajishima Takeo , Nomachi Takayuki
Published: May 2006

The transport equation of subgrid scale (SGS) kinetic energy, KSGS, is used for the large-eddy simulation (LES), considering its consistency with dynamic procedure. The dynamically determined parameter is suitable for describing the energy transfer from resolved turbulence to SGS portion. Thus the procedure is applied to the production term in the transport equation of KSGS, while the eddy viscosity in the filtered equation of motion is determined indirectly through KSGS. The statistically derived model for KSGS equation is adopted for the basis of our improvement. Computational examination has been conducted for fully developed turbulent flow in a plane channel. Agreement with DNS database was satisfactory. Moreover, in a channel on solid body rotation, our model reasonably reproduced the decay of SGS turbulence in the vicinity of the suction side.
Stresses in Proton Exchange Membranes Due to Hygro-Thermal Loading
Authors: Tang Yaliang , Santare Michael H. , Karlsson Anette M. , Cleghorn Simon , Johnson William B.
Published: May 2006

Durability of the proton exchange membrane (PEM) is a major technical barrier to the commercial viability of polymer electrolyte membrane fuel cells (PEMFC) for stationary and transportation applications. In order to reach Department of Energy objectives for automotive PEMFCs, an operating design lifetime of at least 5000h over a broad temperature range is required. Reaching these lifetimes is an extremely difficult technical challenge. Though good progress has been made in recent years, there are still issues that need to be addressed to assure successful, economically viable, long-term operation of PEM fuel cells. Fuel cell lifetime is currently limited by gradual degradation of both the chemical and hygro-thermomechanical properties of the membranes. Eventually the system fails due to a critical reduction of the voltage or mechanical damage. However, the hygro-thermomechanical loading of the membranes and how this effects the lifetime of the fuel cell is not understood. The long-term objective of the research is to establish a fundamental understanding of the mechanical processes in degradation and how they influence the lifetime of PEMFCs based on perfluorosulfuric acid membrane. In this paper, we discuss the finite element models developed to investigate the in situ stresses in polymer membranes.
Forced Oscillations in a Mixed-Compression Inlet at Mach 3.5 for Pulse Detonation Engine Systems
Authors: Nori Venkata , Lerma Nelson , Gustavsson Jonas , Segal Corin , Fernandez Rene
Published: May 2006

The effects of oscillatory backpressure on the air induction system for pulse detonation engines were examined for a two-dimensional, mixed-compression configuration at a freestream Mach number of 3.5. The pressure perturbations at the diffuser exit were produced by injecting air through four ports located at the corners of the exit cross section. The frequency, coupling of the ports and airflow rates through the ports were varied, simulating the operation of detonation tubes. A terminal normal shock in the diffuser oscillated in the excited inlet, causing large pressure fluctuation amplitudes at some locations. Large injection mass flows resulted in inlet flow oscillations throughout the inlet, increased the spillage, yet did not cause inlet unstart.
Power Law Turbulent Velocity Profile in Transitional Rough Pipes
Authors: Afzal Noor , Seena Abu , Bushra Afzal
Published: May 2006

Alternate power law velocity profile u+=Aζα in transitional rough pipe fully turbulent flow, has been proposed, in terms of new appropriate inner rough wall variables (ζ=Z+∕ϕ, uϕ=u∕ϕ), and new parameters Rϕ=Rτ∕ϕ termed as the roughness friction Reynolds number, Reϕ=Re∕ϕ termed as the roughness Reynolds number and ϕ termed as roughness scale (along with normal wall coordinate Z=y+ϵr where ϵr is the shift of the origin of boundary layer due to the rough wall, Z+=Zuτ∕ν and u+=u∕uτ). The envelope of the power law shows that the power law constants α and A depend on the parameter Rϕ (i.e., α=α(Rϕ) and A=A(Rϕ)) but explicitly independent of the wall roughness parameter h∕δ (roughness height h in pipe of radius δ). The roughness scale ϕ has been related to the roughness function ΔU+ of Clauser representing the velocity shift caused by wall roughness. The present results of the velocity profile, just slightly above the wall roughness level h, remain valid for all types of wall roughness. The data of Nikuradse for sand-grain roughness, in transitional and fully rough pipes, has been considered, which provides good support to the predictions of an alternate power law velocity profile, based on single parameter Rϕ, the roughness friction Reynolds number.
Miniature Single-Disk Viscous Pump (Single-DVP), Performance Characterization
Authors: Blanchard Danny , Ligrani Phil , Gale Bruce
Published: May 2006

The development and testing of a rotating single-disk viscous pump are described. This pump consists of a 10.16mm diameter spinning disk, and a pump chamber, which are separated by a small gap that forms the fluid passage. The walls of the pump chamber form a C-shaped channel with an inner radius of 1.19mm, an outer radius of 2.38mm, and a depth of 40, 73, 117, or 246μm. Fluid inlet and outlet ports are located at the ends of the C-shaped channel. Experimental flow rate and pressure rise data are obtained for rotational speeds from 100to5000rpm, fluid chamber heights from 40to246μm, flow rates from 0to4.75ml∕min, pressure rises from 0to31.1kPa, and fluid viscosities from 1to62mPas. An analytical expression for the net flow rate and pressure rise, as dependent on the fluid chamber geometry, disk rotational speed, and fluid viscosity, is derived and found to agree with the experimental data. The flow rate and pressure rise of the pump vary nearly linearly with rotational speed. The volumetric flow rate does not change significantly with changes in fluid viscosity for the same rotational speed and pumping circuit. Advantages of the disk pumps include simplicity, ease of manufacture, ability to produce continuous flow with a flow rate that does not vary significantly in time, and ability to pump biological samples without significant alteration or destruction of cells, protein suspension, or other delicate matter.
Multisectored interstitial ultrasound applicators for dynamic angular control of thermal therapy
Authors: Kinsey Adam M. , Diederich Chris J. , Tyreus P. Daniel , Nau William H. , Rieke Viola , Pauly Kim Butts
Published: May 2006

Dynamic angular control of thermal ablation and hyperthermia therapy with current interstitial heating technology is limited in capability, and often relies upon nonadjustable angular power deposition patterns and/or mechanical manipulation of the heating device. The objective of this study was to investigate the potential of multisectored tubular interstitial ultrasound devices to provide control of the angular heating distribution without device manipulation. Multisectored tubular transducers with independent sector power control were incorporated into modified versions of internally cooled ( OD) and catheter‐cooled ( OD) interstitial ultrasound applicators in this work. The heating capabilities of these multisectored devices were evaluated by measurements of acoustic output properties, measurements of thermal lesions produced in ex vivo tissue samples, biothermal simulations of thermal ablation and hyperthermia treatments, and MR temperature imaging of ex vivo and in vivo experiments. Acoustic beam measurements of each applicator type displayed a 35°–40° acoustic dead zone between each independent sector, with negligible mechanical or electrical coupling. Thermal lesions produced in ex vivo liver tissue with one, two, or three sectors activated ranged from in radius with contiguous zones of coagulation between active sectors. The simulations demonstrated the degree of angular control possible by using variable power levels applied to each sector, variable duration of applied constant power to individual sectors, respectively, or a multipoint temperature controller to vary the power applied to each sector. Despite the acoustic dead zone between sectors, the simulations also showed that the variance from the maximum lesion radius with three elements activated is within 4%–13% for tissue perfusions from . Simulations of hyperthermia with maximum tissue temperatures of and displayed radial penetration up to of the steady‐state contour. Thermal characterizations of trisectored applicators in ex vivo and in vivo muscle, using real‐time MR thermal imaging, reinforced angular controllability and negligible radial variance of the heating pattern from the applicators, demonstrated effective heating penetration, and displayed MR compatibility. The multisectored interstitial ultrasound applicators developed in this study demonstrated a significant degree of dynamic angular control of a heating pattern without device manipulation, while maintaining heat penetration consistent with previously reported results from other interstitial ultrasound applicators.
Monte Carlo calculation of the ionization chamber response to beam using PENELOPE
Authors: Yi Chul‐Young , Hah Suck‐Ho , Yeom Min Sun
Published: May 2006

The calculation of the ionization chamber response is one of key factors to develop a primary standard of air kerma. Using Monte Carlo code PENELOPE, we simulated the cavity response of the plane parallel ionization chamber to the monoenergetic beam incident normally on a flat surface of the chamber. Two simulation techniques, namely, the uniform interaction technique and the reentrance technique, were introduced. The effect of the input parameters such as C1 (average angular deflection in a single step between hard elastic events), C2 (maximum average fractional energy loss in a step), (maximum step length) and (the lower energy of secondary electrons created as a result of a hard collision) on the simulated cavity dose was evaluated. We found that the simulated cavity response of the graphite and solid air chambers obtained by PENELOPE to the monoenergetic beam could be consistent with the value expected from the cross‐sections of PENELOPE to within 0.2% (one standard deviation) when and were selected carefully.
An analytic model for the response of a CZT detector in diagnostic energy dispersive x‐ray spectroscopy
Authors: LeClair Robert J. , Wang Yinkun , Zhao Peiying , Boileau Michel , Wang Lilie , Fleurot Fabrice
Published: May 2006

A detector (CZTD) can be very useful for measuring diagnostic x‐ray spectra. The semiconductor detector does, however, exhibit poor hole transport properties and fluorescence generation upon atomic de‐excitations. This article describes an analytic model to characterize these two phenomena that occur when a CZTD is exposed to diagnostic x rays. The analytical detector response functions compare well with those obtained via Monte Carlo calculations. The response functions were applied to 50, 80, and x‐ray spectra. Two spectra were measured; one with no filtration and the other with Al filtration. The unfiltered spectrum was numerically filtered with of Al in order to see whether the recovered spectrum resembled the filtered spectrum actually measured. A deviation curve was obtained by subtracting one curve from the other on an energy bin by bin basis. The deviation pattern fluctuated around the zero line when corrections were applied to both spectra. Significant deviations from zero towards the lower energies were observed when the uncorrected spectra were used. Beside visual observations, the exposure obtained using the numerically attenuated unfiltered beam was compared to the exposure calculated with the actual filtered beam. The percent differences were 0.8% when corrections were applied and 25% for no corrections. The model can be used to correct diagnostic x‐ray spectra measured with a detector.
A multipinhole small animal SPECT system with submillimeter spatial resolution
Authors: Funk Tobias , Després Philippe , Barber William C. , Shah Kanai S. , Hasegawa Bruce H.
Published: May 2006

Single photon emission computed tomography (SPECT) is an important technology for molecular imaging studies of small animals. In this arena, there is an increasing demand for high performance imaging systems that offer improved spatial resolution and detection efficiency. We have designed a multipinhole small animal imaging system based on position sensitive avalanche photodiode (PSAPD) detectors with the goal of submillimeter spatial resolution and high detection efficiency, which will allow us to minimize the radiation dose to the animal and to shorten the time needed for the imaging study. Our design will usePSAPD detector modules coupled to thallium‐doped cesium iodidescintillators, which can achieve an intrinsic spatial resolution ofat. These detectors will be arranged in rings of 24 modules each; the animal is positioned in the center of the 9 stationary detector rings which capture projection data from the animal with a cylindrical tungsten multipinhole collimator. The animal is supported on a bed which can be rocked about the central axis to increase angular sampling of the object. In contrast to conventional SPECT pinhole systems, in our design each pinhole views only a portion of the object. However, the ensemble of projection data from all of the multipinhole detectors provide angular sampling that is sufficient to reconstruct tomographic data from the object. The performance of this multipinhole PSAPD imaging system was simulated using a ray tracing program that models the appropriate point spread functions and then was compared against the performance of a dual‐headed pinhole SPECT system. The detection efficiency of both systems was simulated and projection data of a hot rod phantom were generated and reconstructed to assess spatial resolution. Appropriate Poisson noise was added to the data to simulate an acquisition time ofand an activity ofdistributed in the phantom. Both sets of data were reconstructed with an ML‐EM reconstruction algorithm. In addition, the imaging performance of both systems was evaluated with a uniformity phantom and a realistic digital mouse phantom. Simulations show that our proposed system produces a spatial resolution ofand an average detection efficiency of. In contrast, simulations of the dual‐headed pinhole SPECT system produce a spatial resolution ofand an average detection efficiency of. These results suggest that our novel design will achieve high spatial resolution and will improve the detection efficiency by more than an order of magnitude compared to a dual‐headed pinhole SPECT system. We expect that this system can perform SPECT with submillimeter spatial resolution, high throughput, and low radiation dose suitable forin vivoimaging of small animals.
Inverse plan optimization accounting for random geometric uncertainties with a multiple instance geometry approximation (MIGA)
Authors: McShan D. L. , Kessler M. L. , Vineberg K. , Fraass B. A.
Published: May 2006

Radiotherapy treatment plans that are optimized to be highly conformal based on a static patient geometry can be degraded by setup errors and/or intratreatment motion, particularly for IMRT plans. To achieve improved plans in the face of geometrical uncertainties, direct simulation of multiple instances of the patient anatomy (to account for setup and/or motion uncertainties) is used within the inverse planning process. This multiple instance geometry approximation (MIGA) method uses two or more instances of the patient anatomy and optimizes a single beam arrangement for all instances concurrently. Each anatomical instance can represent expected extremes or a weighted distribution of geometries. The current implementation supports mapping between instances that include distortions, but this report is limited to the use of rigid body translations/rotations. For inverse planning, the method uses beamlet dose calculations for each instance, with the resulting doses combined using a weighted sum of the results for the multiple instances. Beamlet intensities are then optimized using the inverse planning system based on the cost for the composite dose distribution. MIGA can simulate various types of geometrical uncertainties, including random setup error and intratreatment motion. A limited number of instances are necessary to simulate Gaussian‐distributed errors. IMRT plans optimized using MIGA show significantly less degradation in the face of geometrical errors, and are robust to the expected (simulated) motions. Results for a complex head/neck plan involving multiple target volumes and numerous normal structures are significantly improved when the MIGA method of inverse planning is used. Inverse planning using MIGA can lead to significant improvements over the use of simple PTV volume expansions for inclusion of geometrical uncertainties into inverse planning, since it can account for the correlated motions of the entire anatomical representation. The optimized plan results reflect the differing patient geometry situations which can be important near the surface or heterogeneities. For certain clinical situations, the MIGA optimization approach can correct for a significant part of the degradation of the plan caused by the setup uncertainties.
Efficient on‐line setup correction strategies using plan‐intent functions
Authors: Keller Harry , Jaffray David A. , Rosewall Tara , White Elizabeth
Published: May 2006

With the introduction of image‐guided radiation therapy (IGRT) delivery systems on‐line set‐up correction strategies have gained in popularity. Usually, the correction workload of these strategies is high compared to off‐line strategies as daily setup corrections have to be performed based on a predefined action level. In this work, it is proposed that on‐line strategies must not only be judged in terms of workload but also in terms of efficacy. While workload can be easily predicted for such strategies, the efficacy must ultimately reflect the efficiency with which the original treatment plan intent is met. The purpose of this work is to investigate the tradeoff between workload and efficacy of three different on‐line set‐up correction strategies: The common fixed action level strategy and two novel on‐line setup correction strategies, i.e., a dose‐volume histogram (DVH) constraint and an equivalent uniform dose (EUD) score strategy that aim directly for better compliance with original treatment plan intent. All strategies were reformulated in terms of a score function that reflected treatment plan intent. A retrospective study was conducted on 5 prostate patients (7‐field conformal, , 42 fractions). PTV margins were except in the posterior direction . The original treatment plan intent for these patients was defined using a set of DVH constraints. The results show that the on‐line setup correction strategy based on a fixed action level of resulted in a considerable correction workload. For larger action levels, a dose benefit (in terms of EUD) in the rectum and bladder was observed for all patients which is clinically “fortuitous” but difficult to take advantage of. In contrast, the application of the two novel strategies generally resulted in a controlled decrease of the dose to the rectum and the bladder with a smaller workload. It is concluded that using information about target anatomy and the planned dose distribution allows the design of specific correction strategies that are better tailored to the individual patient and that comply effectively with initial treatment plan intent.
Model‐based image reconstruction for four‐dimensional PET
Authors: Li Tianfang , Thorndyke Brian , Schreibmann Eduard , Yang Yong , Xing Lei
Published: May 2006

Positron emission tonography (PET) is useful in diagnosis and radiation treatment planning for a variety of cancers. For patients with cancers in thoracic or upper abdominal region, the respiratory motion produces large distortions in the tumor shape and size, affecting the accuracy in both diagnosis and treatment. Four‐dimensional (4D) (gated) PET aims to reduce the motion artifacts and to provide accurate measurement of the tumor volume and the tracer concentration. A major issue in 4D PET is the lack of statistics. Since the collected photons are divided into several frames in the 4D PET scan, the quality of each reconstructed frame degrades as the number of frames increases. The increased noise in each frame heavily degrades the quantitative accuracy of the PET imaging. In this work, we propose a method to enhance the performance of 4D PET by developing a new technique of 4D PET reconstruction with incorporation of an organ motion model derived from 4D‐CT images. The method is based on the well‐known maximum‐likelihood expectation‐maximization (ML‐EM) algorithm. During the processes of forward‐ and backward‐projection in the ML‐EM iterations, all projection data acquired at different phases are combined together to update the emission map with the aid of deformable model, the statistics is therefore greatly improved. The proposed algorithm was first evaluated with computer simulations using a mathematical dynamic phantom. Experiment with a moving physical phantom was then carried out to demonstrate the accuracy of the proposed method and the increase of signal‐to‐noise ratio over three‐dimensional PET. Finally, the 4D PET reconstruction was applied to a patient case.
3D interfractional patient position verification using 2D‐3D registration of orthogonal images
Authors: Jans H.‐S. , Syme A. M. , Rathee S. , Fallone B. G.
Published: May 2006

Reproducible positioning of the patient during fractionated external beam radiation therapy is imperative to ensure that the delivered dose distribution matches the planned one. In this paper, we expand on a 2D‐3D image registration method to verify a patient's setup in three dimensions (rotations and translations) using orthogonal portal images and megavoltage digitally reconstructed radiographs (MDRRs) derived from CT data. The accuracy of 2D‐3D registration was improved by employing additional image preprocessing steps and a parabolic fit to interpolate the parameter space of the cost function utilized for registration. Using a humanoid phantom, precision for registration of three‐dimensional translations was found to be better than (1 s.d.) for any axis when no rotations were present. Three‐dimensional rotations about any axis were registered with a precision of better than (1 s.d.) when no translations were present. Combined rotations and translations of up to and were registered with and accuracy for each axis. The influence of setup translations on registration of rotations and vice versa was also investigated and mostly agrees with a simple geometric model. Additionally, the dependence of registration accuracy on three cost functions, angular spacing between MDRRs, pixel size, and field‐of‐view, was examined. Best results were achieved by mutual information using angular spacing and a field‐of‐view with . Approximating patient motion as rigid transformation, the registration method is applied to two treatment plans and the patients’ setup errors are determined. Their magnitude was found to be and for any axis in all of the six fractions measured for each treatment plan.
Environmental Monitoring by Wireless Communication Networks
Authors: Messer Hagit , Zinevich Artem , Alpert Pinhas
Published: May 2006

The global spread of wireless networks brings a great opportunity for their use in environmental studies. Weather, atmospheric conditions, and constituents cause propagation impairments on radio links. As such, while providing communication facilities, existing wireless communication systems can be used as a widely distributed, high-resolution atmospheric observation network, operating in real time with minimum supervision and without additional cost. Here we demonstrate how measurements of the received signal level, which are made in a cellular network, provide reliable measurements for surface rainfall. We compare the estimated rainfall intensity with radar and rain gauge measurements.
Identification of Poly(cis-1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a FunctionallcpHomologue fromNocardia farcinicaStrain E1
Authors: Ibrahim Ebaid M. A. , Arenskötter Matthias , Luftmann Heinrich , Steinbüchel Alexander
Published: May 2006

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships toActinomadura nitritigenes,Nocardia farcinica, andThermomonospora curvata. While strains related toN. farcinicaexhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genusGordonia, strains related toA. nitritigenesandT. curvataformed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50°C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearingprotein) fromStreptomycessp. strain K30 was identified inNocardia farcinicaE1.Streptomyces lividansTK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.
Genomic and Proteomic Analyses of the Agarolytic System Expressed bySaccharophagus degradans2-40
Authors: Ekborg Nathan A. , Taylor Larry E. , Longmire Atkinson G. , Henrissat Bernard , Weiner Ronald M. , Hutcheson Steven W.
Published: May 2006

Saccharophagus degradans2-40 (formerlyMicrobulbifer degradans2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of theS. degradans2-40 agarolytic system were identified. Five agarases were identified in theS. degradans2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed thataga50Aandaga86Ewere essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a β-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system ofS. degradans2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an α-neoagarobiose hydrolase to release galactose from agarose.
High-Throughput Screen for Poly-3-Hydroxybutyrate in
Escherichia coli
and
Synechocystis
sp. Strain PCC6803
Authors: Tyo Keith E. , Zhou Hang , Stephanopoulos Gregory N.
Published: May 2006

A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium
Synechocystis
sp. strain PCC6803 and the eubacterium
Escherichia coli
to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In
Synechocystis
, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In
E. coli
, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high-throughput evaluation of combinatorial libraries of
Synechocystis
and
E. coli
using fluorescence-activated cell sorting to identify mutants with increased PHB-accumulating properties.
Occurrence of Natural
Bacillus thuringiensis
Contaminants and Residues of
Bacillus thuringiensis
-Based Insecticides on Fresh Fruits and Vegetables
Authors: Frederiksen Kristine , Rosenquist Hanne , Jørgensen Kirsten , Wilcks Andrea
Published: May 2006

A total of 128
Bacillus cereus
-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as
Bacillus thuringiensis
on the basis of their content of
cry
genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50
B. thuringiensis
strains were of the same subtype as
B. thuringiensis
strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from
B. thuringiensis
subsp.
kurstaki
HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with
B. thuringiensis
subsp.
aizawai
present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other
B. thuringiensis
and
B. cereus
-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of
B. thuringiensis
-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.
Assessment of Toluene/Biphenyl Dioxygenase Gene Diversity in Benzene-Polluted Soils: Links between Benzene Biodegradation and Genes Similar to Those Encoding Isopropylbenzene Dioxygenases
Authors: Witzig Robert , Junca Howard , Hecht Hans-Jürgen , Pieper Dietmar H.
Published: May 2006

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic α subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant α subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase α subunit CumA1 of
Pseudomonas fluorescens
IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from
Pseudomonas fluorescens
IP01, to the inner surface of the substrate-binding pocket. Isolates containing an α subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.
Improvement of an Unusual Twin-Arginine Transporter Leader Peptide by a Codon-Based Randomization Approach
Authors: Monroy-Lagos Olga , Soberon Xavier , Gaytan Paul , Osuna Joel
Published: May 2006

Secretion of
Escherichia coli
penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with respect to the wild-type value).
Longitudinal Farm Study of Extended-Spectrum β-Lactamase-Mediated Resistance
Authors: Liebana E. , Batchelor M. , Hopkins K. L. , Clifton-Hadley F. A. , Teale C. J. , Foster A. , Barker L. , Threlfall E. J. , Davies R. H.
Published: May 2006

Extended-spectrum β-lactamase (ESBL)-mediated resistance is of considerable importance in human medicine. Recently, such enzymes have been reported in bacteria from animals. We describe a longitudinal study of a dairy farm suffering calf scour with high mortality rates. In November 2004, two
Escherichia coli
isolates with resistance to a wide range of β-lactams (including amoxicillin-clavulanate and cefotaxime) were isolated from scouring calves. Testing by PCR and sequence analysis confirmed the isolates as being both
bla
CTX-M14/17
and
bla
TEM-35
(IRT-4)
positive. They had indistinguishable plasmid and pulsed-field gel electrophoresis (PFGE) profiles. Transferability studies demonstrated that
bla
CTX-M
was located on a conjugative 65-MDa IncK plasmid. Following a farm visit in December 2004, 31/48 calves and 2/60 cows were positive for
E. coli
with
bla
CTX-M
. Also, 5/48 calf and 28/60 cow samples yielded
bla
CTX
- and
bla
TEM
-negative
E. coli
isolates that were resistant to cefotaxime, and sequence analysis confirmed that these presented mutations in the promoter region of the chromosomal
ampC
gene. Fingerprinting showed 11 different PFGE types (seven in
bla
CTX-M
-positive isolates). Six different PFGE clones conjugated the same
bla
CTX-M
-positive IncK plasmid. One clone carried a different-sized,
bla
CTX-M
-positive, transformable plasmid. This is the first report of
bla
CTX-M
from livestock in the United Kingdom, and this report demonstrates the complexity of ESBL epidemiology. Results indicate that horizontal plasmid transfer between strains as well as horizontal gene transfer between plasmids have contributed to the spread of resistance. We have also shown that some clones can persist for months, suggesting that clonal spread also contributes to the perpetuation of resistance.
Emergence of
Cryptococcus gattii
in a Novel Environment Provides Clues to Its Incubation Period
Authors: MacDougall L. , Fyfe M.
Published: May 2006

Cryptococcus gattii
emerged in 1999 in a distinct ecologic environment on Vancouver Island, Canada. Estimates of incubation period were derived from an analysis of travelers with discreet exposures to this region. Individual case incubation periods ranged from 2 to 11 months, with a median of 6 to 7 months.
Use of an Acute Seroconversion Panel To Evaluate a Third-Generation Enzyme-Linked Immunoassay for Detection of Human Immunodeficiency Virus-Specific Antibodies Relative to Multiple Other Assays
Authors: Louie Brian , Pandori Mark W. , Wong Ernest , Klausner Jeffrey D. , Liska Sally
Published: May 2006

A human immunodeficiency virus type 1 (HIV-1)/HIV-2 antibody screening assay, the Genetic Systems HIV-1/HIV-2 PLUS O EIA, was compared to several established screening or confirmatory tests using an acute HIV seroconversion panel. The HIV-1/HIV-2 PLUS O EIA showed an improved sensitivity over all tested antibody screening methods, and detected antibody in 7 of 19 specimens found to be negative by a first-generation EIA but positive for the presence of HIV RNA.
Isolation, Characterization, and Epidemiological Assessment of Shiga Toxin-Producing
Escherichia coli
O84 Isolates from New Zealand
Authors: Cookson Adrian L. , Croucher Dawn , Pope Chris , Bennett Jenny , Thomson-Carter Fiona , Attwood Graeme T.
Published: May 2006

Shiga toxin-producing
Escherichia coli
O84 isolates (
n
= 22) were examined using culture- and molecularly based methods in order to compare their phenotypic and genotypic characteristics. These analyses directly linked Shiga toxin-producing
Escherichia coli
O84 isolates from cattle and sheep with human isolates indicating that New Zealand livestock may be a reservoir of infection.
Peak doubling in the nuclear magnetic resonance spectra of certain phosphorus esters
Authors: Jardine R. V. , Gray A. H. , Reesor J. B.
Published: May 2006

Doubling of nuclear magnetic resonances of certain mixed phosphorus esters has been observed and has been attributed to the presence of rotational conformers occupying "up" and "down" orientations of the ester moieties. Treatment of these esters with uranyl nitrate results in the expected resonances, although chemically shifted.This is believed to be due to the "freezing" of rotational conformers by complex formation. Dependence of the observed resonances upon temperature supports this hypothesis. There appears to be some steric control over the relative population of the contributing conformers.
Preparative conversion of oximes to parent carbonyl compounds by cerium(IV) oxidation
Authors: Bird J. W. , Diaper D. G. M.
Published: May 2006

Oximes and semicarbazones are oxidized by ceric ammonium nitrate to the parent carbonyl compound in good yield. Suitable solvent systems include aqueous alcohols, acetonitrile, and acetic acid. The reaction is rapid at 0° or below. This procedure for regeneration of aldehydes and ketones from these derivatives may be preferable to the well-known hydrolytic procedures in some cases. The stoichiometry of the reaction was studied by isothermal calorimetry; two equivalents of oxidant are consumed by one mole of oxime evolving approximately 40 kcal/mole. A smaller consumption of oxidant by hexane-2,5-dione dioxime was attributed to stabilization of the monoxime as a nitroso chelate. Gaseous products were studied by infrared spectroscopy and nitrous oxide was identified as the sole product absorbing between 3 and 15 μ. A reaction mechanism is proposed involving one-electron oxidation to the iminoxy radical, oxidized by a second one-electron step to the α-hydroxy nitroso compound. Dimerization and elimination of hyponitrous acid gives the carbonyl compound. Nitrocyclohexane was absent from the oxidation product from cyclohexanone oxime. Carvone oxime was oxidized to carvone without racemization or significant attack of the olefin groups.
Infrared spectra of diphenylethine and diphenylethine-d10
Authors: Pecile Cesare , Lunelli Bruno
Published: May 2006

The infrared spectra of diphenylethine and diphenylethine-d10 have been measured in the 4000–33 cm−1 region both for solution and solid samples. Polarized spectra of oriented crystals, (001) crystal plane, allow segregation of most bands into their respective symmetry classes. The slightness of correlation crystal field effects suggested by the comparison between crystal and solution spectra is confirmed by measurements on dilute isotopic solutions. The oriented gas model predictions are a useful guide despite some well-identified deviations.The assignment of the infrared active fundamentals is proposed on the basis of the dichroic indications and the isotopic shifts. Most of the relevant absorption frequencies of diphenylethine and diphenylethine-d10 are recognized as characteristic of a monosubstituted benzene and the spectra are interpretable on the basis of two phenyl groups weakly interacting through the acetylenic bridge.
Infrared spectra of photodimers of anthracene, benzo[a]anthracene, and tetracene
Authors: Singh Surjit , Sandorfy C.
Published: May 2006

Infrared spectra of the photodimers of anthracene, anthracene-d10, tetracene, and benzo[a]anthracene are reported in the region 4000–200 cm−1. An attempt is made to correlate the observed bands with the proposed structure of the anthracene dimer. The infrared spectra indicate that the dimers of benzo[a]anthracene and tetracene have similar structures.
Nuclear charge dispersion in the fission of 232Th by protons of energy 20–85 MeV
Authors: Benjamin P. P. , Marsden D. A. , Porile N. T. , Yaffe L.
Published: May 2006

232Th was irradiated with protons of energy 20–85 MeV and the independent yields of 130Cs, 132Cs, 134Csm, 134Csg, 136Cs, 138Cs, 86Rb, and the cumulative yields of 137Cs and 138Xe were measured radio-chemically. Charge dispersion curves were constructed at various energies and, as in previous studies, the broadening with increasing energy and shift of the most probable charge towards stability were observed. The most probable charge of a fission fragment of a given mass number lies closer to the line of β-stability in the case of thorium fission than in uranium. Detailed calculations in an attempt to correlate the experimental data with equal charge displacement (e.c.d.) and unchanged charge displacement (u.c.d.) charge distribution postulates were attempted. The results are not conclusive, lying in between the two, but favoring u.c.d. at the higher energies.