bluuebunny/crossref_metadata_2025 huggingface dataset:
The Different Electrophysiological Characteristics in Children with Wolff‐Parkinson‐White Syndrome Between Those with and Without Atrial Fibrillation
Authors: LEE PI‐CHANG , HWANG BETAU , TAI CHING‐TAI , CHIANG CHERN‐EN , YU WEN‐CHUNG , CHEN SHIH‐ANN
Published: February 2004

Atrioventricular reciprocating tachycardia (AVRT) is known to be the most common supraventricular tachycardias in childhood. Because AF with rapid ventricular response may degenerate to ventricular fibrillation through conduction of accessory pathways (APs), it can be potentially life‐threatening in some pediatric patients with WPW syndrome. However, information about WPW syndrome children associated with AF is limited. The purpose of this study was to investigate the specific electrophysiological characteristics in pediatric patients with WPW syndrome and AF. From July 1992 to February 2002, 51 pediatric patients with manifest WPW syndrome and documented AVRT underwent electrophysiological study and radiofrequency catheter ablation. In these patients, two (4%) were found to have several spontaneous episodes of AF recognized on 12‐lead standard ECG or 24‐hour Holter monitoring. Eleven (22%) patients had AF induced by rapid atrial pacing during the baseline procedure of electrophysiological study. The children with manifest WPW syndrome were divided into two groups: those with AF (group 1; n = 11) consisted of seven male and four female children (mean age 15 ± 3 years, range 10–18), and those without AF (group 2; n = 40) consisted of 22 boys and 18 girls (mean age 16 ± 3 years, range 7–18). The study excluded a patient who had Ebstein's anomaly associated with moderate tricuspid regurgitation and right atrial enlargement. The onset and duration of symptoms were not significantly different between the two groups. Comparing the electrophysiological characteristics, the atrial effective refractory period (ERP) was shorter in WPW syndrome children with AF (170 ± 36 vs 190 ± 38 ms, P = 0.041). This study demonstrated that the pediatric WPW syndrome patients with AF had different electrophysiological characteristics from those without AF. (PACE 2004; 27:235–239)
Cyclic Appropriate Mode Switching and Inappropriate Back Switching of a Biventricular Pacemaker During Atrial Tachyarrhythmia
Authors: ERVEN LIESELOT VAN , MOLHOEK SANDER G. , WALL ERNST E. VAN DER , SCHALIJ MARTIN J.
Published: February 2004

In patients with resynchronization devices and intact intrinsic AV conduction, atrial tachyarrhythmias may give rise to high ventricular rates, resulting in inhibition of (bi)ventricular pacing and concomitant lack of therapeutic effects of the device. This report presents a patient with atrial arrhythmias in whom mode switching and back switching of the biventricular pacemaker occurred, due to special timing of the atrial and ventricular deflections. This case report stresses the importance of strenuous treatment of atrial arrhythmias in patients with resynchronization devices. (PACE 2004; 27:249–251)
Atrial Standstill with Subnodal Conduction Defect and Diffuse Ventricular Fibrosis Managed with Low Right Atrial Pacing
Authors: APOSTOLOPOULOS THEODOROS , GATZOULIS KONSTANTINOS , GIALAFOS JOHN
Published: February 2004

In patients with congenital heart disease presenting with bradyarrhythmia, many electrophysiological abnormalities are found. This report presents a case of a man with surgically corrected single atrium and pulmonary valve stenosis, atrial standstill, delayed AV node conduction, first‐degree intra‐ and second‐degree infra‐His block, who was permanently paced from a restricted excitable area of the low right atrium, as the pacing threshold was unacceptable at any ventricular site. (PACE 2004; 27:256–258)
Electrical characterization of silicon-on-insulator structures with a nondamaging elastic–metal gate
Authors: Hillard Robert J. , Howland William H. , Tan Louison C. , Ye Win
Published: February 2004

This article explores electrical characterization methods for silicon-on-insulator (SOI) structures with a nondamaging elastic metal gate (EM gate). Important material electrical properties related to the top silicon layer, gate dielectric and interfaces, and buried oxide are addressed. The techniques utilized are currently under development for SOI and are based on EM-gate capacitance–voltage methods, current–voltage methods, and a back channel metal–oxide–semiconductor transistor that utilizes elastic probes to form a temporary source and drain.
Diffusion and segregation of shallow As and Sb junctions in silicon
Authors: Krüger D. , Rücker H. , Heinemann B. , Melnik V. , Kurps R. , Bolze D.
Published: February 2004

The diffusion and segregation of Sb and As is investigated after low-energy implantation and annealing, both rapid thermal processing and furnace annealing. We demonstrate that the absence of transient enhanced diffusion effects for Sb facilitates the fabrication of significantly shallower junctions with less dopant segregation to the surface. It is shown that Sb implantation can be used to fabricate low-resistivity ultrashallow junctions suitable for source/drain extensions in n-type metal–oxide–semiconductor field effect transistors.
Recent developments and applications of plasma immersion ion implantation
Authors: Chu Paul K.
Published: February 2004

Plasma immersion ion implantation (PIII) is an established technique in some niche microelectronics applications, such as synthesis of silicon on insulator. In other applications, such as shallow junction formation by plasma doping, trench doping, and others, PIII possesses unique advantages over conventional techniques. In the last few years, there have been significant breakthroughs in these areas. Recent developments in plasma doping, direct-current plasma immersion ion implantation that excels in planar sample processing, PIII of insulating materials, as well as plasma surface modification of biomaterials are reviewed in this article.
Tantalum film for x-ray lithography mask deposited by electron cyclotron resonance plasma source coupled with divided microwaves
Authors: Nishimura H. , Ono T. , Oda M. , Matsuo S.
Published: February 2004

An advanced electron cyclotron resonance (ECR) plasma source coupled with divided microwaves has been applied to Ta film formation. The feasibility of the Ta film as an x-ray mask absorber is investigated. The stress in an x-ray absorber material must be low, uniform, and stable. According to the results of an investigation on the stress-depth distribution in Ta film, the stress can be described as the sum of interfacial stress (that retained near the film–substrate interface) and bulk stress (that retained in the film bulk). It is found that these individual stresses can be independently controlled. The interfacial stress depends on the substrate temperature, working-gas pressure, and deposition rate. The bulk stress becomes compressive by adding Ar to the sputtering gas (Xe). A low stress film of 23 MPa (400 nm thick film) with no stress-depth distribution is obtained using optimized deposition parameters of 3.3×10−2 Pa of Xe, 1.75 sccm of Ar, and 280 °C substrate temperature. The ECR Ta film showed excellent stability against stress with a change in stress of less than 5 MPa after the x-ray mask fabrication process and synchrotron radiation irradiation. A highly accurate x-ray mask is fabricated by a wafer process using an ECR-deposited Ta film. The maximum image placement (IP) error of the x-ray mask is less than 60 nm, which includes the IP error induced by membrane shrinkage during back etching.
High-energy ion projection for deep ion implantation as a low cost high throughput alternative for subsequent epitaxy processes
Authors: Meijer Jan , Burchard Bernd , Ivanova Katja , Volland Burkhard E. , Rangelow Ivo W. , Rüb Michael , Deboy Gerald
Published: February 2004

Three-dimensional (3D) doping into depths up to 40 μm is of great interest for numerous device types. In particular, the production of high-power devices requires low cost vertically structured doping. State of the art epitaxial growth combined with diffusion and/or low-energy ion implantation is time consuming and cost intensive. We suggest 3D structured high-energy ion projection implantation as a simple cost effective and reliable alternative. This method allows controlled fast doping with high homogeneity and reproducibility. This article outlines some details of a feasibility study of the technique and discusses advantages and problems.
Leakage optimization of ultra-shallow junctions formed by solid phase epitaxial regrowth
Authors: Lindsay R. , Henson K. , Vandervorst W. , Maex K. , Pawlak B. J. , Duffy R. , Surdeanu R. , Stolk P. , Kittl J. A. , Giangrandi S. , Pages X. , van der Jeugd K.
Published: February 2004

Ultra-shallow p+ junctions formed by solid phase epitaxial regrowth (SPER) have promise for sub-65 nm CMOS technologies. Due to above-equilibrium solid solubilities and minimal diffusion, such junctions can far outperform spike-annealed junctions in terms of resistance, abruptness, and depth. However, the low-temperature annealing does not dissolve the end of range defects creating concerns for junction leakage in the device. In this work, we show how SPER junctions can be optimized to meet the ITRS junction profile and low-power leakage requirements of the 45 nm CMOS node [International Technology Roadmap for Semiconductors (Semiconductor Industry Association, San Jose, CA, 2001)]. Diode leakage is shown to decrease with Ge amorphization depth and B dose and energy. Leakage is shown to increase dramatically with the background doping level. Increasing the regrowth, or post-annealing, thermal budget improves leakage and can be optimized to avoid deactivation. The inclusion of a preanneal does not affect the junction leakage, however co-implanting F increases leakage. The influence of each is explained using various physical and electrical characterization techniques. Optimizing these parameters gives a junction of 9 nm with an Rs of 698 Ω/sq and area leakage of 1E-6 A/cm2 with HALO doping.
Study on poly depletion in sub-0.1 μm metal–oxide–semiconductor field effect transistors by scanning capacitance microscopy
Authors: Wang Y. G. , Edwards H. , Ukraintsev V. , Wu J. , Chen J. , Waller J. , Woodall D. , Scott D. B. , Machala C. , Ekbote S. , Tsao A.
Published: February 2004

We study the relative changes in active concentrations in the n- and p-type poly gates of 90 nm node metal–oxide–semiconductor field effect transistors (MOSFETs) by scanning capacitance microscopy on beveled and backside etched samples. We polished and etched from the backside of MOSFETs to expose the bottom of poly gates. Measurements at different Lg show that the boron and phosphorus concentrations in short-channel devices are lower than in long-channel ones. By using staircase calibration standards and NIST FACTC2D, we estimate that boron concentration is reduced by about 5× while phosphorus concentration is reduced by about 2× when Lg shrinks from 10 μm to 50 nm. The effects of high-frequency voltage on quantitatively determining the dopant concentration are discussed. By beveling the poly, we are able to get the lateral dc/dv profiles in Lg=50 nm MOSFETs. The active boron concentration is lower at the p-poly edge while the phosphorus profile is flatter in n-poly.
Model for photoresist trim etch in inductively coupled CF4/O2 plasma
Authors: Rauf Shahid
Published: February 2004

A two-dimensional model for photoresist (PR) trim etching in an inductively coupled CF4/O2 plasma is described. The model couples a Hybrid Plasma Equipment Model based simulation of the plasma reactor with a string-based model for PR feature surface evolution. The etch mechanism in the feature profile simulation considers chemical etching and physical sputtering of PR, fluorocarbon polymer deposition on PR, polymer etching, and etching and redeposition of bottom antireflecting coating (ARC). O atoms are the primary PR trim etch precursor in the mechanism and F atoms play a major supporting role. Modeling results show that polymer deposition impacts feature profile and trim etch rate under conditions where large amount of CFx radicals are generated in the plasma. Since trim etching is primarily driven by neutral atoms who have a broad angular distribution, closely spaced feature sidewalls can become tapered during trimming and dense features trim at a slower rate than isolated structures. PR trim etch rate exhibits nonmonotonic dependence on CF4 fraction in the CF4/O2 gas mixture. F extraction of H from PR enhances O atom reactivity with PR at low CF4 fractions, enhancing PR trim etch rate with increasing CF4 fraction. When CF4 fraction increases beyond 10%, trim etch rate decreases due to O depletion and polymer deposition. Trim etch rate increases monotonically with coil power and saturates at large coil powers. This dependence has been linked to O2 dissociation characteristics and polymer deposition. Increase in rf bias voltage enhances ion energy, which results in ARC layer etching and PR sputtering. Consequent ARC redeposition on PR sidewalls slows down trim etching of PR.
Advanced processing techniques for through-wafer interconnects
Authors: Burkett S. L. , Qiao X. , Temple D. , Stoner B. , McGuire G.
Published: February 2004

The fabrication of interconnects used in future microelectronic devices and for three-dimensional (3D) integration of these components will require advanced integrated circuit (IC) processing techniques. One attractive approach to providing increased connectivity is to use through-wafer interconnects. The primary challenge to implementing 3D stacking is the formation of these high aspect ratio interconnects with a sufficiently small diameter and, consequently, with sufficiently high density. Processing techniques to fabricate through-wafer interconnects in silicon for applications that require multichip stacking or contacts on both sides of the wafer will be described. The techniques used for fabrication of the vertical interconnects are compatible with complementary metal–oxide–semiconductor technology and executed within the thermal budget of a completed IC. The processing techniques to be described include: deep silicon etching (DRIE) to form small diameter vias, insulator lining, adhesion/barrier layer deposition, seed layer deposition, copper electroplating, and chemical mechanical planarization of copper. Through-wafer copper interconnects have been formed with via diameters of 50–100 μm on 4 and 6 in. silicon wafers containing no active devices and at temperatures no greater than 200 °C.
Carrier profiling via scanning tunneling spectroscopy: Comparison with scanning capacitance microscopy
Authors: Liu F. Y. , Griffin P. B. , Plummer J. D. , Lyding J. W. , Moran J. M. , Richards J. F. , Kulig L.
Published: February 2004

Tunneling measurements were taken on a hydrogen terminated, 2×1 reconstructed, Si(100) surface, formed by an in situ passivation technique. I–V characteristics on this surface are shown to be sensitive to the electronic structure at atomic length scales. Tunneling measurements across a pn junction clearly delineate a transition of width in close agreement with that predicted by process and device simulators. In contrast, a scanning capacitance microscopy profile on the same sample exhibits a significantly larger transition width.
Growth of Si wires on a Si(111) substrate under ultrahigh vacuum condition
Authors: Park H. D. , Hogan T. P.
Published: February 2004

The Si wires were grown on a Si(111) substrate using the pulsed laser deposition system. Gold was used as a metal catalyst to form the silicon wires utilizing the vapor-liquid-solid growth mechanism. Samples were grown under ultrahigh vacuum conditions at a growth temperature of 800 °C. Typical dimensions of the grown Si wires were 200–500 nm in diameter and 1.6 μm in height.
Integrins Regulate Rac Targeting by Internalization of Membrane Domains
Authors: del Pozo Miguel A. , Alderson Nazilla B. , Kiosses William B. , Chiang Hui-Hsien , Anderson Richard G. W. , Schwartz Martin A.
Published: February 2004

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.
Extracellular Replication ofListeria monocytogenesin the Murine Gall Bladder
Authors: Hardy Jonathan , Francis Kevin P. , DeBoer Monica , Chu Pauline , Gibbs Karine , Contag Christopher H.
Published: February 2004

The bacteriumListeria monocytogenescan cause a life-threatening systemic illness in humans. Despite decades of progress in animal models of listeriosis, much remains unknown about the processes of infection and colonization. Here, we report thatL. monocytogenescan replicate in the murine gall bladder and provide evidence that its replication there is extracellular and intraluminal. In vivo bioluminescence imaging was employed to determine the location of the infection over time in live animals, revealing strong signals from the gall bladder over a period of several days, in diseased as well as asymptomatic animals. The data suggest thatL. monocytogenesmay be carried in the human gall bladder.
Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil
Authors: Okano Yutaka , Hristova Krassimira R. , Leutenegger Christian M. , Jackson Louise E. , Denison R. Ford , Gebreyesus Binyam , Lebauer David , Scow Kate M.
Published: February 2004

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (
amoA
) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10
5
cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH
4
)
2
SO
4
application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10
6
cells/g of dry soil to peak values (day 7) of 35 × 10
6
and 66 × 10
6
cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10
9
and 2.2 × 10
9
cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10
6
to 38.0 × 10
6
cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH
4
+
h
−1
cell
−1
and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10
6
, 17.5 × 10
6
, and 1.7 × 10
6
cells/mol of NH
4
+
in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.
Cyanophage Diversity, Inferred from
g20
Gene Analyses, in the Largest Natural Lake in France, Lake Bourget
Authors: Dorigo Ursula , Jacquet Stéphan , Humbert Jean-François
Published: February 2004

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the
g20
gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus
g20
genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the
g20
gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.
Induction of Methyl
Tertiary
Butyl Ether (MTBE)-Oxidizing Activity in
Mycobacterium vaccae
JOB5 by MTBE
Authors: Johnson Erika L. , Smith Christy A. , O'Reilly Kirk T. , Hyman Michael R.
Published: February 2004

Alkane-grown cells of
Mycobacterium vaccae
JOB5 cometabolically degrade the gasoline oxygenate methyl
tertiary
butyl ether (MTBE) through the activities of an alkane-inducible monooxygenase and other enzymes in the alkane oxidation pathway. In this study we examined the effects of MTBE on the MTBE-oxidizing activity of
M. vaccae
JOB5 grown on diverse nonalkane substrates. Carbon-limited cultures were grown on glycerol, lactate, several sugars, and tricarboxylic acid cycle intermediates, both in the presence and absence of MTBE. In all MTBE-containing cultures, MTBE consumption occurred and
tertiary
butyl alcohol (TBA) and
tertiary
butyl formate accumulated in the culture medium. Acetylene, a specific inactivator of alkane- and MTBE-oxidizing activities, fully inhibited MTBE consumption and product accumulation but had no other apparent effects on culture growth. The MTBE-dependent stimulation of MTBE-oxidizing activity in fructose- and glycerol-grown cells was saturable with respect to MTBE concentration (50% saturation level = 2.4 to 2.75 mM), and the onset of MTBE oxidation in glycerol-grown cells was inhibited by both rifampin and chloramphenicol. Other oxygenates (TBA and
tertiary
amyl methyl ether) also induced the enzyme activity required for their own degradation in glycerol-grown cells. Presence of MTBE also promoted MTBE oxidation in cells grown on organic acids, compounds that are often found in anaerobic, gasoline-contaminated environments. Experiments with acid-grown cells suggested induction of MTBE-oxidizing activity by MTBE is subject to catabolite repression. The results of this study are discussed in terms of their potential implications towards our understanding of the role of cometabolism in MTBE and TBA biodegradation in gasoline-contaminated environments.
Inactivation of the
lys7
Gene, Encoding Saccharopine Reductase in
Penicillium chrysogenum
, Leads to Accumulation of the Secondary Metabolite Precursors Piperideine-6-Carboxylic Acid and Pipecolic Acid from α-Aminoadipic Acid
Authors: Naranjo Leopoldo , Martín de Valmaseda Eva , Casqueiro Javier , Ullán Ricardo V. , Lamas-Maceiras Mónica , Bañuelos Oscar , Martín Juan F.
Published: February 2004

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid.
Penicillium chrysogenum
has a very active α-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The
lys7
gene, encoding saccharopine reductase in
P. chrysogenum
, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named
P. chrysogenum
SR1
−
) showed the presence of a mutant
lys7
gene lacking about 1,000 bp in the 3′-end region.
P. chrysogenum
SR1
−
lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact
lys7
gene in an autonomously replicating plasmid.
P. chrysogenum
SR1
−
was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant
P. chrysogenum
SR1
−
was grown with
l-
lysine as the sole nitrogen source and supplemented with
dl
-α-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1
−
with a
lys2
-defective mutant provided evidence showing that
P. chrysogenum
synthesizes pipecolic acid from α-aminoadipic acid and not from
l-
lysine catabolism.
Development and Validation of Experimental Protocols for Use of Cardinal Models for Prediction of Microorganism Growth in Food Products
Authors: Pinon Anthony , Zwietering Marcel , Perrier Louise , Membré Jeanne-Marie , Leporq Benoît , Mettler Eric , Thuault Dominique , Coroller Louis , Stahl Valérie , Vialette Michèle
Published: February 2004

An experimental protocol to validate secondary-model application to foods was suggested.
Escherichia coli
,
Listeria monocytogenes
,
Bacillus cereus
,
Clostridium perfringens
, and
Salmonella
were observed in various food categories, such as meat, dairy, egg, or seafood products. The secondary model validated in this study was based on the gamma concept, in which the environmental factors temperature, pH, and water activity (aw) were introduced as individual terms with microbe-dependent parameters, and the effect of foodstuffs on the growth rates of these species was described with a food- and microbe-dependent parameter. This food-oriented approach was carried out by challenge testing, generally at 15 and 10°C for
L. monocytogenes
,
E. coli
,
B. cereus
, and
Salmonella
and at 25 and 20°C for
C. perfringens
. About 222 kinetics in foods were generated. The results were compared to simulations generated by existing software, such as PMP. The bias factor was also calculated. The methodology to obtain a food-dependent parameter (fitting step) and therefore to compare results given by models with new independent data (validation step) is discussed in regard to its food safety application. The proposed methods were used within the French national program of predictive microbiology, Sym′Previus, to include challenge test results in the database and to obtain predictive models designed for microbial growth in food products.
Biodegradation of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Ring Cleavage Product 4-Nitro-2,4-Diazabutanal by
Phanerochaete chrysosporium
Authors: Fournier Diane , Halasz Annamaria , Spain Jim , Spanggord Ronald J. , Bottaro Jeffrey C. , Hawari Jalal
Published: February 2004

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by
Rhodococcus
sp. strain DN22 produces CO
2
and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH
2
NHNO
2
, in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing
Phanerochaete chrysosporium
. A soil sample taken from an ammunition plant contained RDX (342 μmol kg
−1
), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 μmol kg
−1
), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 μmol kg
−1
), and traces of NDAB (3.8 μmol kg
−1
). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 ± 22 μmol kg
−1
) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N
2
O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N
2
O. To determine C stoichiometry, we first generated [
14
C]NDAB in situ by incubating [
14
C]RDX with strain DN22, followed by incubation with the fungus. The production of
14
CO
2
increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus
P. chrysosporium
may constitute the basis for the development of bioremediation technologies.
Detection and Characterization of a Dehalogenating Microorganism by Terminal Restriction Fragment Length Polymorphism Fingerprinting of 16S rRNA in a Sulfidogenic, 2-Bromophenol-Utilizing Enrichment
Authors: Fennell Donna E. , Rhee Sung-Keun , Ahn Young-Beom , Häggblom Max M. , Kerkhof Lee J.
Published: February 2004

Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (aDesulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a secondDesulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.
Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme
Authors: Fujita Yasuya , Ito Junji , Ueda Mitsuyoshi , Fukuda Hideki , Kondo Akihiko
Published: February 2004

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast
Saccharomyces cerevisiae
. When a cell surface display system based on α-agglutinin was used,
Trichoderma reesei
endoglucanase II and cellobiohydrolase II and
Aspergillus aculeatus
β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.
Electron Transfer by
Desulfobulbus propionicus
to Fe(III) and Graphite Electrodes
Authors: Holmes Dawn E. , Bond Daniel R. , Lovley Derek R.
Published: February 2004

Desulfobulbus propionicus
was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of
Desulfobulbaceae
species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.
Evaluation of Colilert-18 for Detection of Coliforms and
Eschericha coli
in Subtropical Freshwater
Authors: Chao Kuo-Kuang , Chao Chen-Ching , Chao Wei-Liang
Published: February 2004

The accuracy of Colilert-18 as a test for coliforms and
Escherichia coli
in subtropical freshwater was evaluated by using API 20E strips and fatty acid methyl ester analysis. The false-positive and -negative rates of detection were 7.4 and 3.5%, respectively, for
E. coli
and 9.6 and 6.3%, respectively, for coliforms.
Stabilization of Penicillin G Acylase from
Escherichia coli
: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment
Authors: Abian Olga , Grazú Valeria , Hermoso Juan , González Ramón , García José Luis , Fernández-Lafuente Roberto , Guisán José Manuel
Published: February 2004

Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4°C, pH 5 at 60°C, pH 7 at 55°C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.
Growth Characteristics ofBartonella henselaein a Novel Liquid Medium: Primary Isolation, Growth-Phase-Dependent Phage Induction, and Metabolic Studies
Authors: Chenoweth M. R. , Somerville G. A. , Krause D. C. , O'Reilly K. L. , Gherardini F. C.
Published: February 2004

Bartonella henselaeis a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolateB. henselaedirectly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 × 108CFU/ml) and permits the isolation ofB. henselaefrom the blood of infected cats. During the development of this medium, we observed thatB. henselaedid not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest,B. henselaedepleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phaseB. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed thatB. henselaehas a complete TCA cycle. Taken together, these data suggestB. henselaemay catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation ofB. henselaeand basic research into the pathogenesis of the bacterium.
Observing Growth and Division of Large Numbers of Individual Bacteria by Image Analysis
Authors: Elfwing A. , LeMarc Y. , Baranyi J. , Ballagi A.
Published: February 2004

We describe a method that enabled us to observe large numbers of individual bacterial cells during a long period of cell growth and proliferation. We designed a flow chamber in which the cells attached to a transparent solid surface. The flow chamber was mounted on a microscope equipped with a digital camera. The shear force of the flow removed the daughter cells, making it possible to monitor the consecutive divisions of a single cell. In this way, kinetic parameters and their distributions, as well as some physiological characteristics of the bacteria, could be analyzed based on more than 1,000 single-cell observations. The method which we developed enabled us to study the history effect on the distribution of the lag times of single cells.
Genes Coding for Hepatotoxic Heptapeptides (Microcystins) in the Cyanobacterium
Anabaena
Strain 90
Authors: Rouhiainen Leo , Vakkilainen Tanja , Siemer Berit Lumbye , Buikema William , Haselkorn Robert , Sivonen Kaarina
Published: February 2004

The cluster of microcystin synthetase genes from
Anabaena
strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (
mcyA
-
mcyB
-
mcyC
) is transcribed in the opposite direction from the second operon (
mcyG
-
mcyD
-
mcyJ
-
mcyE
-
mcyF
-
mcyI
) and the third operon (
mcyH
). The genes
mcyA
,
mcyB
, and
mcyC
encode nonribosomal peptide synthetases (NRPS), while
mcyD
codes for a polyketide synthase (PKS), and
mcyG
and
mcyE
are mixed NRPS-PKS genes. The genes
mcyJ
,
mcyF
, and
mcyI
are similar to genes coding for a methyltransferase, an aspartate racemase, and a
d
-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of
mcyB
coding for the adenylation domain was found to be 96% identical with the corresponding part of
mcyC
, suggesting a recent duplication of this fragment and a replacement in
mcyB
. In
Anabaena
strain 90, the order of the domains encoded by the genes in the two sets (from
mcyG
to
mcyI
and from
mcyA
to
mcyC
) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in
Anabaena
strain 90 differs from the arrangement found in two other cyanobacterial species,
Microcystis aeruginosa
and
Planktothrix agardhii
. The average sequence match between the microcystin synthetase genes of
Anabaena
strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.
Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom
Rhizosolenia setigera
Authors: Nagasaki Keizo , Tomaru Yuji , Katanozaka Noriaki , Shirai Yoko , Nishida Kensho , Itakura Shigeru , Yamaguchi Mineo
Published: February 2004

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom
Rhizosolenia setigera
(
R. setigera
RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 μmol of photons m
−2
s
−1
with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.
Variation in Sulfide Tolerance of Photosystem II in Phylogenetically Diverse Cyanobacteria from Sulfidic Habitats
Authors: Miller Scott R. , Bebout Brad M.
Published: February 2004

Physiological and molecular phylogenetic approaches were used to investigate variation among 12 cyanobacterial strains in their tolerance of sulfide, an inhibitor of oxygenic photosynthesis. Cyanobacteria from sulfidic habitats were found to be phylogenetically diverse and exhibited an approximately 50-fold variation in photosystem II performance in the presence of sulfide. Whereas the degree of tolerance was positively correlated with sulfide levels in the environment, a strain's phenotype could not be predicted from the tolerance of its closest relatives. These observations suggest that sulfide tolerance is a dynamic trait primarily shaped by environmental variation. Despite differences in absolute tolerance, similarities among strains in the effects of sulfide on chlorophyll fluorescence induction indicated a common mode of toxicity. Based on similarities with treatments known to disrupt the oxygen-evolving complex, it was concluded that sulfide toxicity resulted from inhibition of the donor side of photosystem II.
Degradation Pathway and Generation of Monohydroxamic Acids from the Trihydroxamate Siderophore Deferrioxamine B
Authors: Pierwola Agnes , Krupinski Tomasz , Zalupski Peter , Chiarelli Michael , Castignetti Domenic
Published: February 2004

Siderophores are avid ferric ion-chelating molecules that sequester the metal for microbes. Microbes elicit siderophores in numerous and different environments, but the means by which these molecules reenter the carbon and nitrogen cycles is poorly understood. The metabolism of the trihydroxamic acid siderophore deferrioxamine B by a
Mesorhizobium loti
isolated from soil was investigated. Specifically, the pathway by which the compound is cleaved into its constituent monohydroxamates was examined. High-performance liquid chromatography and mass-spectroscopy analyses demonstrated that
M. loti
enzyme preparations degraded deferrioxamine B, yielding a mass-to-charge (
m/z
) 361 dihydroxamic acid intermediate and an
m/z
219 monohydroxamate. The dihydroxamic acid was further degraded to yield a second molecule of the
m/z
219 monohydroxamate as well as an
m/z
161 monohydroxamate. These studies indicate that the dissimilation of deferrioxamine B by
M. loti
proceeds by a specific, achiral degradation and likely represents the reversal by which hydroxamate siderophores are thought to be synthesized.
A New Alkali-Thermostable Azoreductase from
Bacillus
sp. Strain SF
Authors: Maier Jürgen , Kandelbauer Andreas , Erlacher Angelika , Cavaco-Paulo Artur , Gübitz Georg M.
Published: February 2004

A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a
Bacillus
sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain,
Bacillus
sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80°C. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a
Bacillus
sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(
R
)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO
2
substituents, especially in the
ortho
position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.
Lead Precipitation by
Vibrio harveyi
: Evidence for Novel Quorum-Sensing Interactions
Authors: Mire Chad E. , Tourjee Jeanette A. , O'Brien William F. , Ramanujachary Kandalam V. , Hecht Gregory B.
Published: February 2004

Three pleiotropic, quorum sensing-defective
Vibrio harveyi
mutants were observed to precipitate soluble Pb
2+
as an insoluble compound. The compound was purified and subjected to X-ray diffraction and elemental analyses. These assays identified the precipitated compound as Pb
9
(PO
4
)
6
, an unusual and complex lead phosphate salt that is produced synthetically at temperatures of ca. 200°C. Regulation of the precipitation phenotype was also examined. Introduction of a
luxO
::
kan
allele into one of the mutants abolished lead precipitation, indicating that the well-characterized autoinducer 1 (AI1)-AI2 quorum-sensing system can block lead precipitation in dense cell populations. Interestingly, the
V. harveyi
D1 mutant, a strain defective for secretion of both AI1 and AI2, was shown to be an effective
trans
inhibitor of lead precipitation. This suggests that a previously undescribed
V. harveyi
autoinducer, referred to as AI3, can also negatively regulate lead precipitation. Experiments with heterologous bacterial populations demonstrated that many different species are capable of
trans
regulating the
V. harveyi
lead precipitation phenotype. Moreover, one of the
V. harveyi
mutants in this study exhibited little or no response to intercellular signals from other
V. harveyi
inocula but was quite responsive to some of the heterologous bacteria. Based on these observations, we propose that
V. harveyi
carries at least one quorum sensor that is specifically dedicated to receiving cross-species communication.