bluuebunny/crossref_metadata_2025 huggingface dataset:
Type‐Curve Interpretation of Multirate Single‐Hole Pneumatic Injection Tests in Unsaturated Fractured Rock
Authors: Illman Walter A. , Neuman Shlomo P.
Published: August 2005

More than 270 multirate single‐hole pneumatic injection tests were conducted by Guzman et al. (1996) within six shallow vertical and inclined boreholes in unsaturated fractured tuff at the Apache Leap Research Site (ALRS) near Superor, Arizona. The authors used steady‐state formulae to obtain air permeability values for the rock based solely on late pressure data from each test. We developed pressure and pressure‐derivative type‐curves for the interpretation of transient data from these tests, which account for storage in the test interval due to air compressibility and skin effect due to damage to the surrounding rock. We applied our type‐curves to pressure data from more than 40 of the tests to obtain information about air permeability, skin factor, phenomenology, and dimensionality of the flow regime on a nominal scale of 1 m in the immediate vicinity of each test interval. Our air permeabilities agreed well with those determined previously by means of steady‐ state formulae but correlated poorly with fracture density data. Nonlinear effects due to air compressibility were pronounced, but skin effect was generally small or nonexistent. Nonlinear two‐phase flow and inertia were evident but did not have a significant impact on type‐curve interpretation of the test data. Most of these data fit a continuum model of spherical airflow around the test interval, but a few fit a continuum model of radial flow or suggest the dominance of a single fracture. We take this to imply that fractures at the site are pneumatically well connected and can, for the most part, be treated as a porous continuum when analyzing airflow on scales of 1 m or more. This is supported by the finding that 1 m scale air permeability data from single‐hole tests at the ALRS are amenable to geostatistical analysis (Chen et al. 2000), which views them as a sample from a random field defined over a continuum. That the same holds true on larger scale is indicated by our ability to interpret cross‐hole pneumatic injection tests at the site by means of type‐curves and inverse methods (Illman et al. 1998), which treat the entire rock mass as a porous continuum.
Modification of Planck Blackbody Emissive Power and Intensity in Particulate Media Due to Multiple and Dependent Scattering
Authors: Prasher Ravi
Published: August 2005

The dispersion relation for an electromagnetic wave is obtained in particulate media using effective field approximation (EFA) and quasi-crystalline approximation (QCA). Due to multiple and dependent scattering the density of states, phase velocity and group velocity of photons are modified. Modification of these parameters modifies the Planck blackbody equilibrium radiation intensity and emissive power. Results show that EFA can accurately capture the dependence of density of states, phase velocity, and the group velocity on volume fraction of scatterers whereas QCA can capture the dependence of effective attenuation as well as density of states, phase velocity, and the group velocity. Comparisons of the temperature, heat flux, and effective attenuation are made between EFA, QCA, and work done by C. L. Tien and co-workers. Results show that heat flux and temperature predictions made by models in the literature for multiple and dependent scattering are not correct as these models do not take the modification of the equilibrium intensity into account. Finally we introduce a new model called dependent effective field approximation (DEFA) which accurately captures the effect of volume fraction on the equilibrium intensity and effective attenuation.
Local Pool Boiling Coefficients on the Outside Surface of a Horizontal Tube
Authors: Kang Myeong-Gie
Published: August 2005

Changes in local pool boiling heat transfer coefficients on the outside surface of a 51mmdia horizontal tube have been investigated experimentally in water at atmospheric pressure. The local values were determined at every 45deg from the very bottom to the uppermost of the tube periphery, and a representative value was suggested. The maximum and the minimum local coefficients were observed at the azimuthal angles of 45 and 180deg, respectively, from the tube bottom. The coefficient at 90deg can be suggested as the representative of the coefficients. The major mechanisms affecting heat transfer on the surface were liquid agitation and bubble coalescence. Moreover, it is identified that the data can be curve fitted as hb=C1+C2q″.
Self-Preserving Mixing Properties of Steady Round Nonbuoyant Turbulent Jets in Uniform Crossflows
Authors: Diez F. J. , Bernal L. P. , Faeth G. M.
Published: August 2005

The self-preserving mixing properties of steady round nonbuoyant turbulent jets in uniform crossflows were investigated experimentally. The experiments involved steady round nonbuoyant fresh water jet sources injected into uniform and steady fresh water crossflows within the windowed test section of a water channel facility. Mean and fluctuating concentrations of source fluid were measured over cross sections of the flow using planar-laser-induced-fluorescence (PLIF). The self-preserving penetration properties of the flow were correlated successfully similar to Diez et al. [ASME J. Heat Transfer, 125, pp. 1046–1057 (2003)] whereas the self-preserving structure properties of the flow were correlated successfully based on scaling analysis due to Fischer et al. [Academic Press, New York, pp. 315–389 (1979)]; both approaches involve assumptions of no-slip convection in the cross stream direction (parallel to the crossflow) and a self-preserving nonbuoyant line puff having a conserved momentum force per unit length that moves in the streamwise direction (parallel to the initial source flow). The self-preserving flow structure consisted of two counter-rotating vortices, with their axes nearly aligned with the crossflow (horizontal) direction, that move away from the source in the streamwise direction due to the action of source momentum. Present measurements extended up to 260 and 440 source diameters from the source in the streamwise and cross stream directions, respectively, and yielded the following results: jet motion in the cross stream direction satisfied the no-slip convection approximation; geometrical features, such as the penetration of flow boundaries and the trajectories of the axes of the counter-rotating vortices, reached self-preserving behavior at streamwise distances greater than 40–50 source diameters from the source; and parameters associated with the structure of the flow, e.g., contours and profiles of mean and fluctuating concentrations of source fluid, reached self-preserving behavior at streamwise (vertical) distances from the source greater than 80 source diameters from the source. The counter-rotating vortex structure of the self-preserving flow was responsible for substantial increases in the rate of mixing of the source fluid with the ambient fluid compared to corresponding axisymmetric flows in still environments, e.g., transverse dimensions in the presence of the self-preserving counter-rotating vortex structure were 2–3 times larger than transverse dimensions in self-preserving axisymmetric flows at comparable conditions.
Theoretical and experimental vibration analysis for a piezoceramic disk partially covered with electrodes
Authors: Huang Chi-Hung
Published: August 2005

Using the linear two-dimensional electroelastic theory, the vibration characteristics of partially electrode-covered thin piezoceramic disks with traction-free boundary conditions are investigated by theoretical analysis, numerical calculation, and experimental measurement. Four types of piezoceramic disks are discussed owing to the different electrode-covered distribution and electrical boundary conditions. It is found that when an alternating electrical potential is applied to the piezoceramic disk with axisymmetric partial electrodes, not only extensional but also transverse vibrations occur in resonance. The electrode-covered distribution influences the extensional vibration characteristics of piezoceramic disks; however, this phenomenon is not present for the transverse vibration. In this paper the optical speckle interferometry and electrical impedance analysis are employed to validate the theoretical analysis. Numerical calculations based on the finite element method are performed to make comparison with the theoretical and experimental results. According to the theoretical calculation, the variations of extensional resonant frequencies and dynamic electromechanical coupling coefficients depending on the various electrode-covered ratios are also investigated in this work. For the type with circular partial electrodes being connected, it is shown that the coupling coefficients will reach a maximum at the fundamental mode and drop to zero at the overtone mode for certain electrode-covered ratios.
The effect of cross-channel synchrony on the perception of temporal regularity
Authors: Krumbholz Katrin , Bleeck Stefan , Patterson Roy D. , Senokozlieva Maria , Seither-Preisler Annemarie , Lütkenhöner Bernd
Published: August 2005

Temporal models of pitch are based on the assumption that the auditory system measures the time intervals between neural events, and that pitch corresponds to the most common time interval. The current experiments were designed to test whether time intervals are analyzed independently in each peripheral channel, or whether the time-interval analysis in one channel is affected by synchronous activity in other channels. Regular and irregular click trains were filtered into narrow frequency bands to produce target and flanker stimuli. The threshold for discriminating a regular target from an irregular distracter click train was measured in the presence of an irregular masker click train in the target band, as a function of the frequency separation between the target band and a flanker band. The flanker click train was either regular or irregular. The threshold for detecting the regular target was 5–7 dB lower when the flanker was regular. The data indicate that the detection of temporal regularity (and thus, pitch) involves cross-channel processes that can operate over widely separated channels. Model simulations suggest that these cross-channel processes occur after the time-interval extraction stage and that they depend on the similarity, or consistency, of the time-interval patterns in the relevant channels.
Experimental evidence of three-dimensional acoustic propagation caused by nonlinear internal waves
Authors: Frank Scott D. , Badiey Mohsen , Lynch James F. , Siegmann William L.
Published: August 2005

The 1995 SWARM experiment collected high quality environmental and acoustic data. One goal was to investigate nonlinear internal wave effects on acoustic signals. This study continues an investigation of broadband airgun data from the two southwest propagation tracks. One notable feature of the experiment is that a packet of nonlinear internal waves crossed these tracks at two different incidence angles. Observed variations for the lower angle track were modeled using two-dimensional parabolic equation calculations in a previous study. The higher incidence angle is close to critical for total internal reflection, suggesting that acoustic horizontal refraction occurs as nonlinear internal waves traverse this track. Three-dimensional adiabatic mode parabolic equation calculations reproduce principal features of observed acoustic intensity variations. The correspondence between data and simulation results provides strong evidence of the actual occurrence of horizontal refraction due to nonlinear internal waves.
Touch and temporal behavior of grand piano actions
Authors: Goebl Werner , Bresin Roberto , Galembo Alexander
Published: August 2005

This study investigated the temporal behavior of grand piano actions from different manufacturers under different touch conditions and dynamic levels. An experimental setup consisting of accelerometers and a calibrated microphone was used to capture key and hammer movements, as well as the sound signal. Five selected keys were played by pianists with two types of touch (“pressed touch” versus “struck touch”) over the entire dynamic range. Discrete measurements were extracted from the accelerometer data for each of the over 2300 recorded tones (e.g., finger-key, hammer-string, and key bottom contact times, maximum hammer velocity). Travel times of the hammer (from finger-key to hammer-string) as a function of maximum hammer velocity varied clearly between the two types of touch, but only slightly between pianos. A travel time approximation used in earlier work [Goebl W., (2001). J. Acoust. Soc. Am. 110, 563–572] derived from a computer-controlled piano was verified. Constant temporal behavior over type of touch and low compression properties of the parts of the action (reflected in key bottom contact times) were hypothesized to be indicators for instrumental quality.
Time-reversal focusing of elastic surface waves
Authors: Norville Pelham D. , Scott Waymond R.
Published: August 2005

Time-reversal focusing is experimentally applied in an elastic medium in the presence of multiple scattering objects on the order of a wavelength in size. The effectiveness of time-reversal is compared to time-delayed focusing and uniform excitation of the transducer array for focusing energy to a desired location within the medium. A filter is also designed to improve the bandwidth of the excitation signal. Time-reversal focusing is investigated in the context of an elastic-wave landmine detection system. Results are presented demonstrating the advantages and limitations of time-reversal in excitation of a resonance in a TS-50 landmine buried in the medium. A special case is presented for a landmine buried in shadow regions where uniform excitation fails to illuminate the target while time-reversal focusing yields improved target illumination.
A passive acoustic monitoring method applied to observation and group size estimation of finless porpoises
Authors: Wang Kexiong , Wang Ding , Akamatsu Tomonari , Li Songhai , Xiao Jianqiang
Published: August 2005

The present study aimed at determining the detection capabilities of an acoustic observation system to recognize porpoises under local riverine conditions and compare the results with sighting observations. Arrays of three to five acoustic data loggers were stationed across the main channel of the Tian-e-zhou Oxbow of China’s Yangtze River at intervals of 100–150m to record sonar signals of free-ranging finless porpoises (Neophocaena phocaenoides). Acoustic observations, concurrent with visual observations, were conducted at two occasions on 20–22 October 2003 and 17–19 October 2004. During a total of 42h of observation, 316 finless porpoises were sighted and 7041 sonar signals were recorded by loggers. The acoustic data loggers recorded ultrasonic signals of porpoises clearly, and detected the presence of porpoises with a correct detection level of 77.6% and a false alarm level of 5.8% within an effective distance of 150m. Results indicated that the stationed passive acoustic observation method was effective in detecting the presence of porpoises and showed potential in estimating the group size. A positive linear correlation between the number of recorded signals and the group size of sighted porpoises was indicated, although it is faced with some uncertainty and requires further investigation.
A model for estimating ultrasound attenuation along the propagation path to the fetus from backscattered waveforms
Authors: Bigelow Timothy A. , O’Brien William D.
Published: August 2005

Accurate estimates of the ultrasound pressure and/or intensity incident on the developing fetus on a patient-specific basis could improve the diagnostic potential of medical ultrasound by allowing the clinician to increase the transmit power while still avoiding the potential for harmful bioeffects. Neglecting nonlinear effects, the pressure/intensity can be estimated if an accurate estimate of the attenuation along the propagation path (i.e., total attenuation) can be obtained. Herein, a method for determining the total attenuation from the backscattered power spectrum from the developing fetus is proposed. The boundaries between amnion and either the fetus’ skull or soft tissue are each modeled as planar impedance boundaries at an unknown orientation with respect to the sound beam. A mathematical analysis demonstrates that the normalized returned voltage spectrum from this model is independent of the planes orientation. Hence, the total attenuation can be estimated by comparing the location of the spectral peak in the reflection from the fetus to the location of the spectral peak in a reflection obtained from a rigid plane in a water bath. The independence of the attenuation estimate and plane orientation is then demonstrated experimentally using a Plexiglas plate, a rat’s skull, and a tissue-mimicking phantom.
A time-domain model of transient acoustic wave propagation in double-layered porous media
Authors: Fellah Z. E. A. , Wirgin A. , Fellah M. , Sebaa N. , Depollier C. , Lauriks W.
Published: August 2005

This paper concerns a time-domain model of transient wave propagation in double-layered porous materials. An analytical derivation of reflection and transmission scattering operators is given in the time domain. These scattering kernels are the medium’s responses to an incident acoustic pulse. The expressions obtained take into account the multiple reflections occurring at the interfaces of the double-layered material. The double-layered porous media consist of two slabs of homogeneous isotropic porous materials with a rigid frame. Each porous slab is described by a temporal equivalent fluid model, in which the acoustic wave propagates only in the fluid saturating the material. In this model, the inertial effects are described by the tortuosity; the viscous and thermal losses of the medium are described by two susceptibility kernels which depend on the viscous and thermal characteristic lengths. Experimental and numerical results are given for waves transmitted and reflected by double-layered porous media formed by air-saturated plastic foam samples.
Acoustic axes in triclinic anisotropy
Authors: Vavryčuk Václav
Published: August 2005

Calculation of acoustic axes in triclinic elastic anisotropy is considerably more complicated than for anisotropy of higher symmetry. While one polynomial equation of the 6th order is solved in monoclinic anisotropy, we have to solve two coupled polynomial equations of the 6th order in two variables in triclinic anisotropy. Furthermore, some solutions of the equations are spurious and must be discarded. In this way we obtain 16 isolated acoustic axes, which can run in real or complex directions. The real/complex acoustic axes describe the propagation of homogeneous/inhomogeneous plane waves and are associated with a linear/elliptical polarization of waves in their vicinity. The most frequent number of real acoustic axes is 8 for strong triclinic anisotropy and 4 to 6 for weak triclinic anisotropy. Examples of anisotropy with no or 16 real acoustic axes are presented.
Modeling vibration isolator performance with hybrid finite element/statistical energy (FE-SEA) analysis
Authors: Gardner Bryce , Shorter Phil , Cotoni Vincent
Published: August 2005

Vibration isolators are often modeled as simple single degree of freedom systems. Such an approach is often adequate for characterizing the low frequency performance of a vibration isolator (assuming that the effective spring stiffness and damping loss factor of the isolator can be obtained). However, at mid to high frequencies, two problems are often encountered. The first is that the impedance of the structures connected to the isolator become important (assumptions of rigid body behavior of the components of interest are generally not valid). The second is that the internal dynamic behavior of the isolator becomes important (internal resonances of the isolator degrade the isolation performance). This paper describes the development of a hybrid FE-SEA model of an in situ vibration isolator that addresses these problems. The key factors affecting the mid and high frequency performance of an isolator are investigated.
Regulation of Blood Glucose by Hypothalamic Pyruvate Metabolism
Authors: Lam Tony K. T. , Gutierrez-Juarez Roger , Pocai Alessandro , Rossetti Luciano
Published: August 2005

The brain keenly depends on glucose for energy, and mammalians have redundant systems to control glucose production. An increase in circulating glucose inhibits glucose production in the liver, but this negative feedback is impaired in type 2 diabetes. Here we report that a primary increase in hypothalamic glucose levels lowers blood glucose through inhibition of glucose production in rats. The effect of glucose requires its conversion to lactate followed by stimulation of pyruvate metabolism, which leads to activation of adenosine triphosphate (ATP)–sensitive potassium channels. Thus, interventions designed to enhance the hypothalamic sensing of glucose may improve glucose homeostasis in diabetes.
The Pyrite-Type High-Pressure Form of Silica
Authors: Kuwayama Yasuhiro , Hirose Kei , Sata Nagayoshi , Ohishi Yasuo
Published: August 2005

Silica (SiO
2
) exhibits extensive polymorphism at elevated pressures. X-ray diffraction measurements showed that a high-pressure form with a pyrite-type structure, denser than other known silica phases, is stable above 268 giga-pascals and 1800 kelvin. The silicon coordination number increases from 6 in the α-PbO
2
-type phase to 6+2 in the pyrite-type phase, leading to a large increase in density by about 5% at the phase transition.
Ultrafast Dynamics of Solute-Solvent Complexation Observed at Thermal Equilibrium in Real Time
Authors: Zheng Junrong , Kwak Kyungwon , Asbury John , Chen Xin , Piletic Ivan R. , Fayer M. D.
Published: August 2005

In general, the formation and dissociation of solute-solvent complexes have been too rapid to measure without disturbing the thermal equilibrium. We were able to do so with the use of two-dimensional infrared vibrational echo spectroscopy, an ultrafast vibrational analog of two-dimensional nuclear magnetic resonance spectroscopy. The equilibrium dynamics of phenol complexation to benzene in a benzene–carbon tetrachloride solvent mixture were measured in real time by the appearance of off-diagonal peaks in the two-dimensional vibrational echo spectrum of the phenol hydroxyl stretch. The dissociation time constant τ
d
for the phenol-benzene complex was 8 picoseconds. Adding two electron-donating methyl groups to the benzene nearly tripled the value of τ
d
and stabilized the complex, whereas bromobenzene, with an electron-withdrawing bromo group, formed a slightly weaker complex with a slightly lower τ
d
. The spectroscopic method holds promise for studying a wide variety of other fast chemical exchange processes.
Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by
Methylobacterium
sp. Strain JS178
Authors: Fournier Diane , Trott Sandra , Hawari Jalal , Spain Jim
Published: August 2005

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C
2
H
5
N
3
O
3
) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various
Rhodococcus
spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus
Methylobacterium
. The strain, named
Methylobacterium
sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N
2
O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO
2
−
), or ammonium (NH
4
+
) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [
14
C]NDAB produced in situ from [
14
C]RDX by
Rhodococcus
sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.
Impact of Plant Species and Site on Rhizosphere-Associated Fungi Antagonistic to
Verticillium dahliae
Kleb
Authors: Berg Gabriele , Zachow Christin , Lottmann Jana , Götz Monika , Costa Rodrigo , Smalla Kornelia
Published: August 2005

Fungi with antagonistic activity toward plant pathogens play an essential role in plant growth and health. To analyze the effects of the plant species and the site on the abundance and composition of fungi with antagonistic activity toward
Verticillium dahliae
, fungi were isolated from oilseed rape and strawberry rhizosphere and bulk soil from three different locations in Germany over two growing seasons. A total of 4,320 microfungi screened for in vitro antagonism toward
Verticillium
resulted in 911 active isolates. This high proportion of fungi antagonistic toward the pathogen
V. dahliae
was found for bulk and rhizosphere soil at all sites. A plant- and site-dependent specificity of the composition of antagonistic morphotypes and their genotypic diversity was found. The strawberry rhizosphere was characterized by preferential occurrence of
Penicillium
and
Paecilomyces
isolates and low numbers of morphotypes (
n
= 31) and species (
n
= 13), while
Monographella
isolates were most frequently obtained from the rhizosphere of oilseed rape, for which higher numbers of morphotypes (n = 41) and species (n = 17) were found.
Trichoderma
strains displayed high diversity in all soils, but a high degree of plant specificity was shown by BOX-PCR fingerprints. The diversity of rhizosphere-associated antagonists was lower than that of antagonists in bulk soil, suggesting that some fungi were specifically enriched in each rhizosphere. A broad spectrum of new
Verticillium
antagonists was identified, and the implications of the data for biocontrol applications are discussed.
Functional Importance of Deletion Mutant Genotypes in an Insect Nucleopolyhedrovirus Population
Authors: Simón Oihane , Williams Trevor , López-Ferber Miguel , Caballero Primitivo
Published: August 2005

A Nicaraguan isolate of a nucleopolyhedrovirus (SfNIC) that attacks the fall armyworm,
Spodoptera frugiperda
, survives as a mixture of nine genotypes (SfNIC A to I) that all present genomic deletions, except variant B (complete genotype). Sequencing of cloned restriction fragments revealed that genotypic variants lack between 5 and 16 of the open reading frames present in a contiguous sequence of 18 kb of the SfNIC genome. The absence of oral infectivity of SfNIC-C and -D variants is related to the deletion of the
pif
and/or
pif-2
gene, while that of SfNIC-G remains unexplained. The presence of open reading frame 10, homolog of Se030, also appeared to influence pathogenicity in certain variants. Previous studies demonstrated a significant positive interaction between genotypes B and C. We compared the median lethal concentration of single genotypes (A, B, C, D, and F) and co-occluded genotype mixtures (B+A, B+D, B+F, A+C, and F+C in a 3:1 ratio). Mixtures B+A and B+D showed increased pathogenicity, although only B+D restored the activity of the mixture to that of the natural population. Mixtures of two deletion variants (A+C and F+C) did not show interactions in pathogenicity. We conclude that minority genotypes have an important influence on the overall pathogenicity of the population. These results clearly demonstrate the value of retaining genotypic diversity in virus-based bioinsecticides.
Simultaneous Display of Multiple Foreign Peptides in the FliD Capping and FliC Filament Proteins of the
Escherichia coli
Flagellum
Authors: Majander Katariina , Korhonen Timo K. , Westerlund-Wikström Benita
Published: August 2005

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from
Staphylococcus aureus
at the tip and fragments of YadA from
Yersinia enterocolitica
as well as SlpA from
Lactobacillus crispatus
along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.
Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus
Paecilomyces variotii
CBS115145
Authors: García-Peña Inés , Hernández Sergio , Auria Richard , Revah Sergio
Published: August 2005

A biofiltration system inoculated with the mold
Paecilomyces variotii
CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m
3
of biofilter/h, which was higher than the values usually reported for bacteria.
P. variotii
assimilated
m
- and
p
-cresols but not the
o
isomer. Initial toluene hydroxylation occurred both on the methyl group and through the
p
-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and
p
-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O
2
/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O
2
/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of
P. variotii
were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.
In Vitro Study of Lipid Biosynthesis in an Anaerobically Methane-Oxidizing Microbial Mat
Authors: Blumenberg Martin , Seifert Richard , Nauhaus Katja , Pape Thomas , Michaelis Walter
Published: August 2005

The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro
13
CH
4
labeling study (δ
13
CH
4
, ∼5,400‰) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the
13
C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1Δ11]; difference between the δ
13
C at the start and the end of the experiment [Δδ
13
C
start-end
], ∼160‰). In contrast, bacterial glycerol diethers exhibited only slight changes in δ
13
C (Δδ
13
C
start-end
, ∼10‰). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol (Δδ
13
C
start-end
, ∼25‰), a monounsaturated archaeol, and biphytanes, whereas for
sn
-2-hydroxyarchaeol there was considerably less change in the δ
13
C (Δδ
13
C
start-end
, ∼2‰). Moreover, an increase in the uptake of
13
C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in
13
C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater
13
C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.
Secretion of Human Serum Albumin by
Kluyveromyces lactis
Overexpressing
KlPDI1
and
KlERO1
Authors: Lodi Tiziana , Neglia Barbara , Donnini Claudia
Published: August 2005

The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast
Kluyveromyces lactis
and evaluated the secretion of heterologous proteins.
KlERO1
, an orthologue of
Saccharomyces cerevisiae ERO1
, was cloned by functional complementation of the
ts
phenotype of an
Scero1
mutant. The expression of
KlERO1
was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either
PDI1
or
ERO1
led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of
KlERO1
did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the
ERO1
genes of
S. cerevisiae
and
K. lactis
are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of
KlERO1
copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of
KlERO1
and
KlPDI1
is important for the production of high levels of heterologous proteins of this type.
Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1
Authors: Murli Sumati , MacMillan Karen S. , Hu Zhihao , Ashley Gary W. , Dong Steven D. , Kealey James T. , Reeves Christopher D. , Kennedy Jonathan
Published: August 2005

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both
Escherichia coli
and
Streptomyces coelicolor
and fed a variety of acyl-thioesters, several novel 15-
R
-6-dEB analogues were produced. The simpler “monoketide” acyl-thioester substrates required for this route of 15-
R
-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1
o
for the production of 15-
R
-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to
N
-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both
E. coli
and
S. coelicolor
.
Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR
Authors: Fuhrman Jed A. , Liang Xiaolin , Noble Rachel T.
Published: August 2005

Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (
r
2
= 0.99) in fresh water and 23% (
r
2
= 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <10
4
enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%,
r
2
= 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%;
r
2
= 0.93; HA 12%,
r
2
= 0.87). The optimized method was used with 1-liter field samples from two very different freshwater “creeks” that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.
Analysis of the
hupSL
Operon of the Nonheterocystous Cyanobacterium
Lyngbya majuscula
CCAP 1446/4: Regulation of Transcription and Expression under a Light-Dark Regimen
Authors: Leitão Elsa , Oxelfelt Fredrik , Oliveira Paulo , Moradas-Ferreira Pedro , Tamagnini Paula
Published: August 2005

This work presents the characterization of an uptake hydrogenase from a marine filamentous nonheterocystous cyanobacterium,
Lyngbya majuscula
CCAP 1446/4. The structural genes encoding the uptake hydrogenase (
hupSL
) were isolated and characterized, and regulatory sequences were identified upstream of
hupS
. In silico analysis highlighted various sets of long repetitive sequences within the
hupSL
intergenic region and downstream of
hupL
. The transcriptional regulator that operates global nitrogen control in cyanobacteria (NtcA) was shown to bind to the promoter region, indicating its involvement in the transcriptional regulation of
hupSL
. Under N
2
-fixing conditions and a 12-h light/12-h dark regime, H
2
uptake activity was shown to follow a daily pattern with a clear maximum towards the end of the dark period, preceded by an increase in the transcript levels initiated in the end of the light phase. Novel antibodies directed against HupL of
Lyngbya majuscula
CCAP 1446/4 were used to monitor the protein levels throughout the 24-h period. The results suggest that protein turnover occurs, with degradation taking place during the light phase and de novo synthesis occurring during the dark phase, coinciding with the pattern of H
2
uptake. Taking into account our results and the established correlation between the uptake hydrogenase activity and N
2
fixation in cyanobacteria, it seems probable that both processes are confined to the dark period in aerobically grown cells of
Lyngbya majuscula
CCAP 1446/4.
Culturability and Coexistence of Colony-Forming and Single-Cell Marine Bacterioplankton
Authors: Simu Karin , Holmfeldt Karin , Zweifel Ulla Li , Hagström Åke
Published: August 2005

Culturability and coexistence of bacterioplankton exhibiting different life strategies were investigated in the Baltic Sea and Skagerrak Sea. Bacterial numbers were estimated using a dilution-to-extinction culturing assay (DCA) and calculated as the most probable number, based on six different methods to detect bacterial growth in the DCA. Irrespective of the method used to detect growth, the fraction of multiplying cells never exceeded 10%, using the total count of 4′,6′-diamidino-2-phenylindole (DAPI)-stainable cells as a reference. Furthermore, the data also showed that non-colony-forming bacteria made up the majority of the viable cells, confirming molecular results showing dominance of non-colony-forming bacteria in clone libraries. The results obtained are in agreement with previous observations, indicating that bacterial assemblages in seawater are dominated by small, active subpopulations coexisting with a large group of inactive cells. The ratio of colony-forming to non-colony-forming bacteria was approximately 10 to 20 times higher in the brackish Baltic Sea than in the Skagerrak Sea. These two sea areas differ in (for example) their levels of bacterial production, dissolved organic carbon, and salinity. We suggest that the relative importance of colony-forming versus non-colony-forming bacterioplankton may be linked to environmental characteristics.
Insights into the Biosynthesis of the Benzoquinone Ansamycins Geldanamycin and Herbimycin, Obtained by Gene Sequencing and Disruption
Authors: Rascher Andreas , Hu Zhihao , Buchanan Greg O. , Reid Ralph , Hutchinson C. Richard
Published: August 2005

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different
Streptomyces hygroscopicus
strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from
S. hygroscopicus
AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (
gdmM
and
gdmL
) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.
Presence and Characterization of a Mosaic Genomic Island Which Distinguishes Sorbitol-Fermenting Enterohemorrhagic
Escherichia coli
O157:H
−
from
E. coli
O157:H7
Authors: Janka Andreas , Becker Georg , Sonntag Anne-Katharina , Bielaszewska Martina , Dobrindt Ulrich , Karch Helge
Published: August 2005

A mosaic genomic island comprising
Shigella
resistance locus (SRL) sequences flanked by segments of
Escherichia coli
O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic
Escherichia coli
(EHEC) O157:H
−
strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H
−
from EHEC O157:H7.
Production of a Recombinant Major Inner Capsid Protein for Serological Detection of Epizootic Hemorrhagic Disease Virus
Authors: Luo Lizhong , Sabara Marta I.
Published: August 2005

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.
Improvement of Lyme Borreliosis Serodiagnosis by a Newly Developed Recombinant Immunoglobulin G (IgG) and IgM Line Immunoblot Assay and Addition of VlsE and DbpA Homologues
Authors: Goettner Gereon , Schulte-Spechtel Ulrike , Hillermann Ruth , Liegl Gabi , Wilske Bettina , Fingerle Volker
Published: August 2005

We developed and evaluated a recombinant
Borrelia
line immunoblot assay based on 18 homologues of seven different antigens, i.e., p100, p58, p41i, BmpA, VlsE, OspC, and DbpA. Each recombinant antigen can be detected separately and is distinct even from homologues with identical molecular weights. This blot was compared to the recently described recombinant
Borrelia
Western immunoblot assay (U. Schulte-Spechtel, G. Lehnert, G. Liegl, V. Fingerle, C. Heimerl, B. J. Johnson, and B. Wilske, J. Clin. Microbiol. 41:1299-1303, 2003). To verify sensitivity and specificity, both blots were evaluated for reactivity with
Borrelia
-specific immunoglobulin G (IgG) and IgM antibodies with 85 sera from patients with different manifestations of Lyme borreliosis and 110 controls. According to European interpretation criteria for
Borrelia
Western blots, which define a serum as positive when it recognizes at least two bands, sensitivity increased significantly from 70.6% (Western blot) to 84.7% (line blot) for IgG (
P
= 0.042) and from 40.0% (Western blot) to 73.8% (line blot) for IgM (
P
< 0.005). The increased sensitivity for IgG detection is due to the new line blot technique, whereas the improvement in detection of IgM is mainly achieved through incorporation of the additional antigens. Notably, the recombinant VlsE of
Borrelia garinii
strain PBi displayed the highest sensitivity of all antigens tested for IgG detection and is also one of the most useful antigens for IgM. Due to its excellent sensitivity and specificity combined with ease of evaluation, this line immunoblot assay offers a useful improvement in serodiagnosis of Lyme borreliosis.