bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitriding stainless steels at moderate temperature: Time- and depth-resolved characterization of the near surface composition during the nitriding process
Authors: Parascandola S. , Kruse O. , Richter E. , Moeller W.
Published: July 2002

Time- and depth-resolved compositional analyses have been performed during the nitriding of stainless steel in order to improve understanding of the nitriding kinetics. It is directly observed that the surface oxide layer may act as a transport barrier. The experimental data are discussed on the basis of simple transport that considers the removal of the surface oxide layer due to sputtering, its regeneration from the residual gas, and the ballistic implantation depth of nitrogen.
Blueshift of In0.2Ga0.8N/GaN single quantum well band gap energy by rapid thermal annealing
Authors: Li G. , Chua S. J. , Teng J. H. , Wang W. , Feng Z. C. , Huang Y. H. , Osipowicz T.
Published: July 2002

Up to 15 nm blueshift in the low-temperature photoluminescence (LT-PL) peak wavelength of Si-doped In0.2Ga0.8N/GaN single quantum well (SQW) is reported after rapid thermal annealing in atmospheric N2 ambient at temperatures below 1000 °C. We found that the thermal annealing not only blueshifts the LT-PL peak wavelength but it also improves the optical properties in terms of an increase in LT-PL peak intensity and a reduction in the spectrum width. Under the same thermal annealing conditions, however, a dielectric encapsulant layer (a spin-on silica layer) suppresses the blueshift of the LT-PL peak wavelength of the In0.2Ga0.8N/GaN QW. The effect of the thermal annealing temperature and time on the blueshift of the LT-PL peak wavelength is also reported. The possible mechanism responsible for the thermal annealing effect on the band gap energy of the In0.2Ga0.8N/GaN SQW is discussed.
Mechanical response of thin films with helical microstructures
Authors: Seto M. W. , Robbie K. , Vick D. , Brett M. J. , Kuhn L.
Published: July 2002

A nanoindentation technique has been applied to demonstrate the mechanical actuation of thin films fabricated by glancing angle deposition. The films were comprised of a porous layer whose microstructures consisted of helical columns that resembled springs. Surmounting the porous layer was a dense capping layer which served to redistribute the force of the nanoindenter tip and prevent it from penetrating the microstructured film. Atomic force microscope images and force versus displacement curves confirmed that there was an elastic regime to the displacements in the films when low forces were applied. The loading behavior of the porous films was compared with that of dense, unstructured films and revealed a marked difference between the two. A study of the measurements and results enabled such properties as the spring constant and resonant frequencies to be estimated, establishing a basis for future developments and applications in wave-type devices or resonators constructed from these microstructured thin films.
Critical influence of plating bath temperature on Cu damascene electrodeposits
Authors: Jiang Qing-Tang , Mikkola Robert , Carpenter Brad
Published: July 2002

The plating bath temperature has a critical influence on the damascene gap fill quality. Scanning electron microscopy studies indicated that over a small temperature range from 20 to 30 °C defect-free electroplated Cu fill was obtained. The effect was attributable to the initial nucleation and the chemistry of the plating bath, particularly the functionality of the additives. Secondary ion mass spectroscopy analysis showed different impurity levels of H, C, O, S, and Cl incorporated within the films. X-ray diffraction analysis on blanket Cu films indicated that, although all the Cu films had roughly the same (111) texture strength after plating, after a high temperature anneal at 450 °C for 30 min, the films deposited at higher temperatures exhibited much stronger (111) texture than the others. However, x-ray pole figure analysis of Cu lines with 0.35 μm linewidth and 0.40 μm spacing indicated roughly the same (111) texture intensity after the anneal. The shortened transformation time and more tensile stress observed on blanket Cu films plated at low temperatures were due to smaller grains and less dense packing of deposits which were formed at low temperature plating. Severe anisotropy of the stress was measured in 0.35 μm Cu lines where the stress perpendicular to the trench and along the surface normal were only about half of the stress parallel to the trench. This indicates the existence of strong side wall interaction between Cu and the dielectrics.
Patterning of silicon nanopillars formed with a colloidal gold etch mask
Authors: Lewis P. A. , Ahmed H.
Published: July 2002

A natural lithography technique utilizing colloidal gold particles as etch masks has been integrated with standard microfabrication techniques for the fabrication of silicon nanopillars in selected regions. We have demonstrated four patterning techniques, which enable the pillars to be formed either recessed into the substrate or protruding above the plane. A high density array of patterned pillar regions was obtained using a combination of the colloidal gold etch mask and e-beam lithography. The ultimate objective of the patterning techniques is to incorporate the colloids and the resulting nanopillars into microelectronic devices. We have achieved this goal by fabricating a prototype field emission device utilizing the silicon nanopillars as nanocathodes.
Fabrication of high performance microlenses for an integrated capillary channel electrochromatograph with fluorescence detection
Authors: Wendt J. R. , Warren M. E. , Sweatt W. C. , Bailey C. G. , Matzke C. M. , Arnold D. W. , Allerman A. A. , Carter T. R. , Asbill R. E. , Samora S.
Published: July 2002

We describe the microfabrication of an extremely compact optical system as a key element in an integrated capillary channel electrochromatograph with fluorescence detection. The optical system consists of a vertical cavity surface-emitting laser (VCSEL), two high performance microlenses, and a commercial photodetector. The microlenses are multilevel diffractive optics patterned by electron beam lithography and etched by reactive ion etching in fused silica. The design uses substrate-mode propagation within the fused silica substrate. Two generations of optical subsystems are described. The first generation design has a 6 mm optical length and is integrated directly onto the capillary channel-containing substrate. The second generation design separates the optical system onto its own substrate module and the optical path length is further compressed to 3.5 mm. The first generation design has been tested using direct fluorescence detection with a 750 nm VCSEL pumping a 10−4 M solution of CY-7 dye. The observed signal-to-noise ratio of better than 100:1 demonstrates that the background signal from scattered pump light is low despite the compact size of the optical system and is adequate for system sensitivity requirements.
Rare earth focused ion beam implantation utilizing Er and Pr liquid alloy ion sources
Authors: Chao L. C. , Lee B. K. , Chi C. J. , Cheng J. , Chyr I. , Steckl A. J.
Published: July 2002

We have developed procedures for the fabrication of Er–Ni and Pr–Pt liquid alloy ion sources (LAIS). Er2+beam with target current of ∼100 pA and Pr2+ beams with target current of ∼200 pA were obtained, which correspond to 50% and 80% of the total target current, respectively. Both Er–Ni and Pr–Pt alloys oxidize quickly once exposed to air. Er–Ni source lifetimes were generally larger than 200 μA h. The source lifetimes of Pr–Pt LAISs were approximately 30 μA h, limited by oxide contamination and wettability problems. Visible photoluminescence has been observed from Er- or Pr-doped GaN, Al2O3, and ZBLAN glass using focused ion beam direct write implantation.
Projection reduction exposure with variable axis immersion lenses: Next generation lithography
Authors: Pfeiffer H. C. , Dhaliwal R. S. , Golladay S. D. , Doran S. K. , Gordon M. S. , Groves T. R. , Kendall R. A. , Lieberman J. E. , Petric P. F. , Pinckney D. J. , Quickle R. J. , Robinson C. F. , Rockrohr J. D. , Senesi J. J. , Stickel W. , Tressler E. V. , Tanimoto A. , Yamaguchi T. , Okamoto K. , Suzuki K. , Okino T. , Kawata S. , Morita K. , Suziki S. C. , Shimizu H. , Kojima S. , Varnell G. , Novak W. T. , Stumbo D. P. , Sogard M.
Published: July 2002

Projection reduction exposure with variable axis immersion lenses (PREVAIL) represents the high throughput e-beam projection approach to NGL, which IBM is pursuing in cooperation with Nikon as alliance partner; another e-beam projection approach is SCALPEL pursued by Lucent Technologies. This article discusses the challenges and accomplishments of the PREVAIL project. It will focus on the results obtained with the proof of concept (POC) system. This system was developed to demonstrate key technical building blocks required for high throughput, high resolution e-beam step, and scan projection lithography. The supreme challenge facing all e-beam lithography approaches has been and still is throughput. Since the throughput of e-beam projection systems is severely limited by the available optical field size, the key to success is the ability to overcome this limitation. The PREVAIL technique overcomes field-limiting off-axis aberrations through the use of variable axis lenses, which electronically shift the optical axis simultaneously with the deflected beam, so that the beam effectively remains on axis. This technique developed by IBM has been successfully applied to probe-forming shaped beam systems (EL-4). It had to be modified and extended to provide the larger beam deflections and the wider beam images at the wafer plane used in projection reduction systems. The POC system projects sequentially 1×1 mm2 subfields, selected at the reticle, in 4:1 reduction mode onto the wafer, exposing and resolving patterns of 80 nm lines and spaces in resist; each subfield contains 107 pixels. The deflection capability demonstrated permits electronic selection of 20 1 mm subfields at the reticle and projection of these 20 subfields onto the wafer exposing a field with 5 mm scan length. The resist images provide proof that PREVAIL effectively eliminates off-axis aberrations affecting resolution, since the deflected and undeflected images are indistinguishable. PREVAIL also controls off-axis aberrations affecting placement accuracy of pixels, since distortions of the deflected subfield are corrected to within 12 nm. A high emittance gun has been developed to provide uniform illumination of the patterned subfield, and to fill the large numerical aperture of the projection optics required to significantly reduce beam blur caused by Coulomb interaction.
Scratching on polystyrene thin film without bumps using atomic force microscopy
Authors: Iwata Futoshi , Matsumoto Tarou , Ogawa Ryuhei , Sasaki Akira
Published: July 2002

Polystyrene (PS) thin film modified with a novel ultrasonic scratching method has been studied using an atomic force microscope (AFM). Ultrasonic scratching can be realized by a combination of an AFM and a quartz crystal resonator (QCR). The PS thin film was deposited on the surface of an AT-cut QCR which oscillates with mechanical shear deformation at its resonant frequency (6.5 MHz) with amplitude of a few nm. The oscillating surface is scratched with the AFM’s diamond tip to generate an effective cutting force. Scratching without a QCR oscillation forms bumps on the PS surface, which is known well to be a unique phenomenon of scratched polymer surfaces. However, ultrasonic scratching can result in the carving of PS thin films without the formation of bumps. The depressed bottom carved by ultrasonic scratching is flat in comparison with that scratched without surface oscillations. Furthermore, on the previously modified area of ultrasonic scratching, bumps are not formed, even after overlapping of the scratching without surface oscillations.
Laser operation at room temperature of self-organized In0.1Ga0.9As/(GaAs)6(AlAs)1 quantum wires grown on (775)B-oriented GaAs substrates by molecular beam epitaxy
Authors: Ohno Yasuhide , Higashiwaki Masataka , Shimomura Satoshi , Hiyamizu Satoshi , Ikawa Seiji
Published: July 2002

In 0.1 Ga 0.9 As/(GaAs) 6 (AlAs) 1 quantum wires (QWRs) were naturally formed in a 3.0-nm- or 4.8-nm-thick In0.1Ga0.9As/(GaAs)6(AlAs)1 quantum well grown on a (775)B-oriented GaAs substrate by molecular beam epitaxy which has a corrugated AlAs-on-InGaAs upper interface (a period of about 40 nm and a vertical amplitude of about 2 nm) and a flat InGaAs-on-AlAs lower interface. Strong polarization dependence [P≡(I∥−I⊥)/(I∥+I⊥)=0.15] of photoluminescence spectrum from the (775)B InGaAs QWR structures (Lw=3.0 nm) was observed at 11 K, indicating their good one dimensionality. Graded index separate confinement heterostructure-type self-organized (775)B In0.1Ga0.9As/(GaAs)6(AlAs)1QWR lasers were fabricated, and they showed laser oscillation with threshold current densities of 1.7–3.1 kA/cm2 and lasing wavelengths of 833–868 nm at room temperature (27 °C) under pulsed current condition.
Urals Yield Secret of a Lasting Bond
Authors: Kerr Richard A.
Published: July 2002

By probing the rock beneath the Ural Mountains with artificial seismic waves, an international team of researchers has drawn a portrait of the mountains' deep underpinnings. As they report in this issue (beginning on (
220
)), their deep cross section reveals why the Urals, which mark the 250-million-year-old suture between Europe and Asia, never rifted apart again. Unlike most continental bonds, the Urals are built on strong, thick crust
Element-Specific Magnetic Anisotropy Determined by Transverse Magnetic Circular X-ray Dichroism
Authors: Dürr H. A. , Guo G. Y. , van der Laan G. , Lee J. , Lauhoff G. , Bland J. A. C.
Published: July 2002

Understanding of the magnetocrystalline anisotropy in magnetic materials (the influence of different elemental components on the direction of easy magnetization) can be greatly enhanced by measuring the orbital moment anisotropy of the elemental constituents. A circular x-ray dichroism technique is presented that allows the determination of the microscopic origin of the spin reorientation transition in ultrathin single-crystalline cobalt/nickel films. The stronger anisotropy contribution of a much thinner cobalt layer redirects the easy magnetization direction of the entire film.
Localization-Delocalization Transition in Quantum Dots
Authors: Zhitenev N. B. , Brodsky M. , Ashoori R. C. , Pfeiffer L. N. , West K. W.
Published: July 2002

Single-electron capacitance spectroscopy precisely measures the energies required to add individual electrons to a quantum dot. The spatial extent of electronic wave functions is probed by investigating the dependence of these energies on changes in the dot confining potential. For low electron densities, electrons occupy distinct spatial sites localized within the dot. At higher densities, the electrons become delocalized, and all wave functions are spread over the full dot area. Near the delocalization transition, the last remaining localized states exist at the perimeter of the dot. Unexpectedly, these electrons appear to bind with electrons in the dot center.
Something to Be Done: Treating HIV/AIDS
Authors: Bloom David E. , Associates River Path
Published: July 2002

Largely because of disparities in access to drug treatment and care, AIDS morbidity and mortality have fallen in the developed world but continue to rise among developing countries. Achieving more equitable access to AIDS drugs is hindered by high drug prices, technical complexities related to the provision of health care, and conflict among stakeholders. Recognition that health is vital to the prospects of the emerging global society must be combined with new mechanisms to help all stakeholders work together cooperatively. Tiered drugs pricing should be coupled with investment in health services. An independent “Global Task Force,” able to act as an “active think tank,” could build consensus about the way forward.
Monotherapy with Intravenous Followed by Oral High-Dose Ciprofloxacin versus Combination Therapy with Ceftazidime plus Amikacin as Initial Empiric Therapy for Granulocytopenic Patients with Fever
Authors: Giamarellou Helen , Bassaris Harry P. , Petrikkos George , Busch Wilhelm , Voulgarelis Michel , Antoniadou Anastasia , Grouzi Elisabeth , Zoumbos Nikolaos
Published: July 2002

The aim of the present study was to obtain clinical experience with the use of high-dose ciprofloxacin as monotherapy for the treatment of febrile neutropenia episodes (granulocyte count, <500/mm
3
) compared to a standard regimen and to clarify whether ciprofloxacin administration may be switched to the oral route. In a prospective randomized study ciprofloxacin was given at 400 mg three times a day (t.i.d.) for at least 72 h followed by oral administration at 750 mg twice a day (b.i.d). That regimen was compared with ceftazidime given intravenously at 2 g t.i.d. plus amikacin given intravenously at 500 mg b.i.d. The frequency of successful clinical response without modification at the end of therapy was almost identical for ciprofloxacin (50% [62 of 124 patients]) compared with that for ceftazidime plus amikacin (50.8% [62 of 122 patients]) in an intent-to-treat analysis; the frequencies were 48.3% (57 of 118 patients) versus 49.6% (56 of 113 patients), respectively, in a per-protocol analysis (
P
values for one-sided equivalence, 0.0485 and 0.0516, respectively; δ = 10%), with no significant differences among patients with bacteremia and other microbiologically or clinically documented infections and fever of unknown origin. For 82 (66.1%) patients, it was possible to switch from parenteral ciprofloxacin to the oral ciprofloxacin, and the response was successful for 61 (74.4%) patients. The efficacies of the regimens against streptococcal bacteremias were 16.6% (one of six patients) for the ciprofloxacin group and 33.3% (one of three patients) for the combination group (it was not statistically significant), with one breakthrough streptococcal bacteremia observed among the ciprofloxacin-treated patients. Adverse events were mostly self-limited and were observed in 27 (20.6%) ciprofloxacin-treated patients and 26 (19.7%) patients who were receiving the combination. This study demonstrates that high-dose ciprofloxacin given intravenously for at least 3 days and then by the oral route is therapeutically equivalent to the routine regimen of intraveneous ceftazidime plus amikacin even in febrile patients with severe neutropenia (polymorphonuclear leukocyte count, <100 mm
3
). However, it is very important that before an empirical therapy is chosen each hospital determine bacteriologic predominance and perform resistance surveillance.
Susceptibilities of Human Cytomegalovirus Clinical Isolates to BAY38-4766, BAY43-9695, and Ganciclovir
Authors: McSharry James J. , McDonough Ann , Olson Betty , Hallenberger Sabine , Reefschlaeger Juergen , Bender Wolfgang , Drusano George L.
Published: July 2002

BAY38-4766 and BAY43-9695 are nonnucleosidic compounds with activities against human cytomegalovirus (HCMV). Two phenotypic assays were used to determine the drug susceptibilities of 36 HCMV clinical isolates to the BAY compounds and ganciclovir. Using either assay, both BAY compounds at a concentration of approximately 1 μM inhibited the replication of all 36 HCMV clinical isolates, including 11 ganciclovir-resistant clinical isolates, by 50%.
High Prevalence of Inducible Erythromycin Resistance among
Streptococcus bovis
Isolates in Taiwan
Authors: Teng Lee-Jene , Hsueh Po-Ren , Ho Shen-Wu , Luh Kwen-Tay
Published: July 2002

Susceptibilities to 13 antimicrobial agents were determined by measurement of MICs for 60 isolates of
Streptococcus bovis
from blood cultures. Thirty-eight isolates (63.3%) had high-level resistance to erythromycin (MICs, ≥128 μg/ml). Among the 38 erythromycin-resistant strains, 21 isolates (55%) had inducible resistance to macrolides-lincosamides-streptogramin B (iMLS isolates) and 17 (45%) had constitutive resistance to macrolides-lincosamides-streptogramin B (cMLS isolates). Tetracycline resistance was also found among all of the erythromycin-resistant strains. None of the strains displayed resistance to penicillin, chloramphenicol, or vancomycin. Detection of erythromycin resistance genes by PCR and sequencing indicated that all 17 cMLS isolates were positive for the
ermB
gene and that 7 of 21 iMLS isolates carried the
ermB
gene and the remaining 14 iMLS isolates carried the
ermT
gene. Sequence analysis of amplified partial
ermB
fragments (594 bp) from
S. bovis
isolates revealed a 99.8% nucleotide identity and a 100% amino acid homology compared with the sequences from gene banks. The sequences of amplified fragments with primers targeted for
ermC
were shown to be very similar to that of
ermGT
(
ermT
) from
Lactobacillus reuteri
(98.5% nucleotide identity). This is the first report to describe the detection of the
ermT
class of erythromycin resistance determinants in
S. bovis
. The high rate of inducible erythromycin resistance among
S. bovis
isolates in Taiwan was not reported before. The iMLS
S. bovis
isolates were shown to be heterogeneous by randomly amplified polymorphic DNA analysis. These results indicate that the prevalence of inducible erythromycin resistance in
S. bovis
in Taiwan is very high and that most of the resistant strains carry the
ermT
or the
ermB
gene.
In Vitro Activities of Approved and Investigational Antifungal Agents against 44 Clinical Isolates of Basidiomycetous Fungi
Authors: González Gloria M. , Sutton Deanna A. , Thompson Elizabeth , Tijerina Rolando , Rinaldi Michael G.
Published: July 2002

The in vitro activity of amphotericin B, fluconazole, flucytosine, itraconazole, voriconazole, and posaconazole was evaluated against 44 clinical isolates of filamentous basidiomycetous fungi. No statistically significant differences were noted between
Schizophyllum commune
(
n
= 5),
Coprinus
species (
n
= 8),
Bjerkandera adusta
(
n
= 14), and sterile, uncharacterized basidiomycetes (
n
= 17).
Interspecies Recombination Contributes Minimally to Fluoroquinolone Resistance in
Streptococcus pneumoniae
Authors: Bast Darrin J. , de Azavedo Joyce C. S. , Tam Tiffany Y. , Kilburn Laurie , Duncan Carla , Mandell Lionel A. , Davidson Ross J. , Low Donald E.
Published: July 2002

Analysis of 71 ciprofloxacin-resistant (MIC ≥ 4 μg/ml)
Streptococcus pneumoniae
clinical isolates revealed only 1 for which the quinolone resistance-determining regions of the
parC
,
parE
, and
gyrB
genes were genetically related to those of viridans group streptococci. Our findings support the occurrence of interspecies recombination of type II topoisomerase genes; however, its contribution to the emergence of quinolone resistance among pneumococci appears to have been minimal.
Initial Colonization, Nutrient Supply, and Fungal Activity on Leaves Decaying in Streams
Authors: Sridhar K. R. , Bärlocher Felix
Published: July 2002

Aquatic hyphomycetes dominate leaf decomposition in streams, and their biomass is an important component in the diet of leaf-eating invertebrates. After 2 weeks of exposure in a first-order stream, maple leaf disks had low levels of fungal biomass and species diversity. Spore production by aquatic hyphomycetes also was low. Subsets of these disks were left in the stream for another 3 weeks or incubated in defined mineral solutions with one of three levels of nitrate and phosphate. Stream disks lost mass, increased ergosterol levels and spore production, and were colonized by additional fungal species. External N and P significantly stimulated mass loss, ergosterol accumulation, and spore production of laboratory disks. On disks incubated without added N and P, ergosterol levels declined while conidium production continued, suggesting conversion of existing hyphal biomass to propagules. In all other treatments, approximately equal amounts of newly synthesized biomass were invested in hyphae and conidia. Net yield (fungal biomass per leaf mass lost) varied between 1% (in the laboratory, without added N or P) and 31% (decay in stream). In most treatments, the three aquatic hyphomycete species that dominated spore production during the first 2 weeks in the stream also produced the largest numbers of conidia in the following 3 weeks. Principal-component analysis suggested two divergent trends from the initial fungal community established after 2 weeks in the stream. One culminated in the community of the second phase of stream exposure, and the other culminated in the laboratory treatment with the highest levels of N and P. The results suggest that fungal production in streams, and, by extension, production of invertebrates and higher tropic levels, is stimulated by inorganic N and P.
Simple and Efficient Method for Heterologous Expression of Clostridial Proteins
Authors: Zdanovsky Alexey G. , Zdanovskaia Marina V.
Published: July 2002

Many clostridial proteins are poorly produced in
Escherichia coli
. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in
E. coli
and the quantities of the corresponding tRNAs in
E. coli
are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three
E. coli
genes,
ileX
,
argU
, and
leuW
, in
E. coli
; these genes encode tRNAs that are rarely used in
E. coli
(the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of
ileX
dramatically increased the level of production of most of the clostridial proteins tested, while amplification of
argU
had a moderate effect and amplification of
leuW
had no effect. Thus, amplification of certain tRNA genes for rare codons in
E. coli
improves the expression of clostridial genes in
E. coli
, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in
E. coli
.
Evidence for Detachment of Indigenous Bacteria from Aquifer Sediment in Response to Arrival of Injected Bacteria
Authors: Johnson W. P. , Zhang P. , Gardner P. M. , Fuller M. E. , DeFlaun M. F.
Published: July 2002

Two bacterial strains isolated from the aquifer underlying Oyster, Va., were recently injected into the aquifer and monitored using ferrographic capture, a high-resolution immunomagnetic technique. Injected cells were enumerated on the basis of a vital fluorescence stain, whereas total cell numbers (stained target cells plus unstained target and antigenically similar indigenous bacteria) were identified by cell outlines emanating from fluorophore-conjugated antibodies to the two target strains. The arrival of injected bacteria at the majority of monitored sampling ports was accompanied by simultaneous temporary increases in unstained cell counts that outnumbered the injected bacteria by 2- to 100-fold. The origin and mechanism of appearance of the unstained cells are considered.
Comparison of Extracellular Enzyme Activities and Community Composition of Attached and Free-Living Bacteria in Porous Medium Columns
Authors: Lehman R. Michael , O'Connell Seán P.
Published: July 2002

Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of β-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater β-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 × 10
−7
nmol cell
−1
h
−1
) than the free-living community (5.02 × 10
−8
nmol cell
−1
h
−1
). Conversely, the free-living community had a significantly higher cell-specific β-glucosidase activity (1.92 × 10
−6
nmol cell
−1
h
−1
) than the surface-associated community (6.08 × 10
−7
nmol cell
−1
h
−1
). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.
ALimulusAntilipopolysaccharide Factor-Derived Peptide Exhibits a New Immunological Activity with Potential Applicability in Infectious Diseases
Authors: Vallespi Maribel G. , Glaria Luis A. , Reyes Osvaldo , Garay Hilda E. , Ferrero Joel , Araña Manuel J.
Published: July 2002

Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of theLimulusantilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF31–52peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-γ) and IFN-α. Analysis of the effect of LALF31–52on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF31–52protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-α levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.
Slow Human Immunodeficiency Virus (HIV) Infectivity Correlated with Low HIV Coreceptor Levels
Authors: Bristow Cynthia L.
Published: July 2002

The absolute number of CD4
+
lymphocytes in blood is prognostic for disease progression, yet the cell surface density of CD4 receptors or chemokine receptors on a single cell has not previously been found to be predictive of human immunodeficiency virus (HIV) infectivity outcome. It has recently been shown that human leukocyte elastase (HLE) and its ligand α
1
proteinase inhibitor (α
1
PI; α
1
antitrypsin) act as HIV fusion cofactors. The present study shows that decreased HIV infectivity is significantly correlated with decreased cell surface density of HLE but not with decreased CD4 nor chemokine receptors. In vitro HIV infectivity outcome in this study was predicted by the surface density of HLE on mononuclear phagocytes but not on lymphocytes. The set point HLE surface density was in part determined by α
1
PI. Decreased circulating α
1
PI was correlated with increased cell surface HLE and with increased HIV infectivity. The correlation of HIV infectivity outcome with surface HLE and circulating α
1
PI supports the utility of these HIV cofactors in diagnostic analysis and therapeutic intervention.
Surface Immobilization Antigen of the Parasitic Ciliate
Ichthyophthirius multifiliis
Elicits Protective Immunity in Channel Catfish (
Ictalurus punctatus
)
Authors: Wang Xuting , Dickerson Harry W.
Published: July 2002

Channel catfish (
Ictalurus punctatus
) that survive infection with the parasitic ciliate
Ichthyophthirius multifiliis
acquire immunity to subsequent challenge and produce specific antibodies in serum that immobilize the parasite in vitro. Cellular surface protein antigens targeted by these antibodies are referred to as immobilization antigens (i-antigens). By using an immobilizing mouse monoclonal antibody as a ligand, the i-antigen of
I. multifiliis
isolate G5 was purified to homogeneity by immunoaffinity chromatography, and its immunogenicity was confirmed by inoculating rabbit and channel catfish to produce immobilizing antisera. To test the purified i-antigen as a subunit vaccine, channel catfish fingerlings were injected intraperitoneally (i.p.) with purified i-antigen at a dose of 10 μg/fish in complete Freund’s adjuvant on day 1, followed by a second i.p. injection of the same amount of i-antigen in incomplete Freund’s adjuvant on day 15. Negative control fish were immunized similarly with either bovine serum albumin (BSA) or an immobilization-irrelevant
I. multifiliis
protein. On day 84, the fish were challenged with live
I. multifiliis
G5 theronts at a dose of 15,000 cells per fish. Seventy-two percent of the fish immunized with i-antigen survived the challenge. All negative control fish died within 16 days of exposure. There was a significant difference in the median days to death between the negative control fish injected with BSA and the fish that died following vaccination with i-antigen. Fish injected with i-antigen developed high immobilizing antibody titers in serum. This is the first demonstration of a direct role for i-antigens in the elicitation of protective immunity, suggesting that these proteins by themselves serve as effective subunit vaccines against
I. multifiliis.
Mutants ofListeria monocytogenesDefective in In Vitro Invasion and Cell-to-Cell Spreading Still Invade and Proliferate in Hepatocytes of Neutropenic Mice
Authors: Appelberg Rui , Leal Irene S.
Published: July 2002

Listeria monocytogenesmutants defective in theactAgene, theplcBgene, and theinlAandinlBgenes were less virulent when injected intravenously into BALB/c mice. The growth of these strains as well as of the virulent wild-type strains was increased by treating mice with a neutrophil-specific depleting monoclonal antibody, RB6-8C5. Histologic examination of the livers of the treated animals showed intrahepatocytic proliferation of the listeriae in all cases. Our data show that more than one pathway exists that allowsL. monocytogenesto invade parenchymal cells. One pathway most likely involves theactAandplcBgene products, and a second one probably involves the internalins.
Epitope Mapping of the Outer Membrane Protein P5-Homologous Fimbrin Adhesin of Nontypeable
Haemophilus influenzae
Authors: Novotny Laura A. , Jurcisek Joseph A. , Pichichero Michael E. , Bakaletz Lauren O.
Published: July 2002

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable
Haemophilus influenzae
(NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.
Inhibition of Vesicular Secretion in Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative of
Clostridium botulinum
Neurotoxin Type A
Authors: Chaddock John A. , Purkiss John R. , Friis Lorna M. , Broadbridge Janice D. , Duggan Michael J. , Fooks Sarah J. , Shone Clifford C. , Quinn Conrad P. , Foster Keith A.
Published: July 2002

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin from
Clostridium botulinum
(termed LH
N
/A) that retains catalytic activity can be prepared by proteolysis. The LH
N
/A, however, lacks the putative native binding domain (H
C
) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LH
N
/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LH
N
/A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the H
C
domain of
C. botulinum
neurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.
Role of Interleukin-18 (IL-18) during Lethal Shock: Decreased Lipopolysaccharide Sensitivity but Normal Superantigen Reaction in IL-18-Deficient Mice
Authors: Hochholzer Peter , Lipford Grayson B. , Wagner Hermann , Pfeffer Klaus , Heeg Klaus
Published: July 2002

Lethal shock can be associated with excessive secretion of cytokines such as tumor necrosis factor (TNF) and gamma interferon (IFN-γ). IFN-γ mediates macrophage activation and appears to be controlled by interleukin (IL)-12 and IL-18. To investigate the role of IL-18 in vivo, we generated IL-18-deficient mice by gene targeting. IL-18−/−mice showed decreased sensitivity towards lipopolysaccharide (LPS)-induced shock. LPS-induced IFN-γ production was abrogated, yet induction of IL-12 and TNF was not affected. Both wild-type and IL-18-deficient mice succumbed to LPS-induced lethal shock after sensitization withd-galactosamine. However, in marked contrast to LPS, the bacterial superantigenStaphylococcus aureusenterotoxin B (SEB) induced comparable serum levels of IFN-γ in IL-18+/+and IL-18−/−mice, accompanied by an upregulation of cell surface markers CD14, CD122 (IL-2Rβ), and CD132 (IL-2Rγ) on peritoneal macrophages. Moreover, SEB injection rendered IL-18-deficient mice sensitive for subsequent challenge with LPS. The degree of sensitization was comparable to that in wild-type controls with respect to lethality. However, LPS-induced TNF levels in serum were significantly reduced in SEB-sensitized IL-18-deficient mice. These results imply that IL-18 plays an important role in induction of IFN-γ and lethality in response to LPS.
A Recombinant Baculovirus-ExpressedPlasmodium falciparumReceptor-Binding Domain of Erythrocyte Binding Protein EBA-175 Biologically Mimics Native Protein
Authors: Liang Hong , Narum David L. , Fuhrmann Steven R. , Luu Tin , Sim B. Kim Lee
Published: July 2002

EBA-175 ofPlasmodium falciparumis a merozoite ligand that binds its receptor glycophorin A on erythrocytes during invasion. The ligand-receptor interaction is dependent on sialic acids as well as the protein backbone of glycophorin A. Region II (RII) of EBA-175 has been defined as the receptor-binding domain. RII is divided into regions F1 and F2, which contain duplicated cysteine motifs. We expressed RII in a baculovirus and show that RII binds erythrocytes with a specificity identical to that of the native protein. We found that, consistent with the binding of erythrocytes to COS cells expressing F2, recombinant baculovirus-expressed F2 bound erythrocytes. About 20% of all baculovirus-expressed RII is N-glycosylated, unlike nativeP. falciparumproteins that remain essentially unglycosylated. However, glycosylation of recombinant RII did not affect its immunogenicity. Antibodies raised against both glycosylated and unglycosylated baculovirus-expressed RII recognizedP. falciparumschizonts in immunofluorescence assays and also gave similar enzyme-linked immunosorbent assay titers. Furthermore, these antibodies have similar abilities to block native EBA-175 binding to erythrocytes. These results allow the development of RII as a vaccine candidate for preclinical assessment.
Identification of
ragAB
as a Temperature-Regulated Operon of
Porphyromonas gingivalis
W50 Using Differential Display of Randomly Primed RNA
Authors: Bonass William A. , Marsh Philip D. , Percival Rimondia S. , Aduse-Opoku Joseph , Hanley Shirley A. , Devine Deirdre A. , Curtis Michael A.
Published: July 2002

Porphyromonas gingivalis
is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of
P. gingivalis
has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island
ragAB
; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of
P. gingivalis
by changing its patterns of gene expression. In this study, RNA has been isolated from cells of
P. gingivalis
grown to steady state at temperatures of 37, 39, and 41°C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the
ragAB
locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of
ragAB
may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.
exl
, an Exchangeable Genetic Island in
Neisseria meningitidis
Authors: Kahler C. M. , Blum E. , Miller Y. K. , Ryan D. , Popovic T. , Stephens D. S.
Published: July 2002

The genetic structure and evolution of a novel exchangeable meningococcal genomic island was defined for the important human pathogen
Neisseria meningitidis
. In 125 meningococcal strains tested, one of three unrelated nucleotide sequences, designated
exl
(exchangeable locus), was found between a gene required for heme utilization,
hemO
, and
col
, encoding a putative
Escherichia coli
collagenase homologue. The 5′ boundary of each
exl
cassette was the stop codon of
hemO
, whereas the 3′ boundary was delineated by a 33-bp repeat containing neisserial uptake sequences located downstream of
col
. One of the three alternative
exl
cassettes contained the meningococcal hemoglobin receptor gene,
hmbR
(
exl3
). In other meningococcal strains,
hmbR
was absent from the genome and was replaced by either a nucleotide sequence containing a novel open reading frame,
exl2
, or a cassette containing
exl3
. The proteins encoded by
exl2
and
exl3
had no significant amino acid homology to HmbR but contained six motifs that are also present in the lipoprotein components of the lactoferrin (LbpB), transferrin (TbpB), and hemoglobin-haptoglobin (HpuA) uptake systems. To determine the evolutionary relationships among meningococci carrying
hmbR
,
exl2
, or
exl3
, isolates representing 92 electrophoretic types were examined.
hmbR
was found throughout the population structure of
N. meningitidis
(genetic distance, >0.425), whereas
exl2
and
exl3
were found in clonal groups at genetic distances of <0.2. The commensal neisserial species were identified as reservoirs for all of the
exl
cassettes found in meningococci. The structure of these cassettes and their correlation with clonal groups emphasize the extensive gene pool and frequent horizontal DNA transfer events that contribute to the evolution and virulence of
N. meningitidis.
Tumor Necrosis Factor Alpha Increases Human Cerebral Endothelial Cell Gb
3
and Sensitivity to Shiga Toxin
Authors: Eisenhauer Patricia B. , Chaturvedi Prasoon , Fine Richard E. , Ritchie Andrew J. , Pober Jordan S. , Cleary Thomas G. , Newburg David S.
Published: July 2002

Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic
Escherichia coli
strains that produce Shiga toxins. Globotriaosylceramide (Gb
3
) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-α) upregulates Gb
3
in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-α treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb
3
species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb
3
levels. Differential expression of Gb
3
species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.
YopB of
Yersinia enterocolitica
Is Essential for YopE Translocation
Authors: Nordfelth Roland , Wolf-Watz Hans
Published: July 2002

A previous study has shown that YopB of
Yersinia
spp. is essential for translocation of Yop effectors across the eucaryotic plasma membrane (M.-P. Sory and G. R. Cornelis, Mol. Microbiol. 14:583–594, 1994). However, this role was recently challenged (V. T. Lee and O. Schneewind, Mol. Microbiol. 31:1619–1629, 1999). Using protease protection and digitonin extraction, we reconfirm that YopB of
Yersinia enterocolitica
is essential for the translocation of YopE into HeLa cell monolayers.