bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Central North American Prairie Management Based on Species Diversity, Life Form, and Individual Species Metrics
Authors: BRUDVIG LARS A. , MABRY CATHERINE M. , MILLER JAMES R. , WALKER TRACY A.
Published: January 2007

Abstract: Reintroduction of fire and grazing, alone or in combination, has increasingly been recognized as central to the restoration of North American mixed‐grass and tallgrass prairies. Although ecological studies of these systems are abundant, they have generally been observational, or if experimental, have focused on plant species diversity. Species diversity measures alone are not sufficient to inform management, which often has goals associated with life‐form groups and individual species. We examined the effects of prescribed fire, light cattle grazing, and a combination of fire and grazing on three vegetation components: species diversity, groups of species categorized by life‐form, and individual species. We evaluated how successful these three treatments were in achieving specific management goals for prairies in the Iowa Loess Hills (U.S.A.). The grazing treatment promoted the greatest overall species richness, whereas grazing and burning and grazing treatments resulted in the lowest cover by woody species. Burning alone best achieved the management goals of increasing the cover and diversity of native species and reducing exotic forb and (predominantly exotic) cool‐season grass cover. Species‐specific responses to treatments appeared idiosyncratic (i.e., within each treatment there existed a set of species attaining their highest frequency) and nearly half of uncommon species were present in only one treatment. Because all management goals were not achieved by any one treatment, we conclude that management in this region may need refining. We suggest that a mosaic of burning and grazing (alone and in combination) may provide the greatest landscape‐level species richness; however, this strategy would also likely promote the persistence of exotic species. Our results support the need to consider multiple measures, including species‐specific responses, when planning and evaluating management.
Processing and Properties of Nano‐Hydroxyapatite(n‐HAp)/Poly(Ethylene‐Co‐Acrylic Acid)(EAA) Composite Using a Phosphonic Acid Coupling Agent for Orthopedic Applications
Authors: Pramanik Nabakumar , Mohapatra Sasmita , Pramanik Panchanan , Bhargava Parag
Published: January 2007

A hydroxyapatite/poly(ethylene‐co‐acrylic acid) (HAp/EAA) nanocomposite has been synthesized by a solution‐based method. p‐Aminophenyl phosphonic acid has been used as a coupling agent in order to enhance the bonding between HAp and EAA, and hence to improve the mechanical properties of the composite. XRD study has indicated the development of compressive and tensile stresses in a nanocomposite due to thermal expansion mismatch between nano‐hydroxyapatite (n‐HAp) and EAA. Fourier‐transform infrared spectrometry (FTIR) and thermal analysis have shown the presence of strong interfacial bonding between n‐HAp and EAA. The surface roughness and the homogeneous dispersion of nanoparticles have been observed by field emission scanning electron microscopy (FESEM). A comparison of mechanical properties between phosphonic acid treated (cn‐HAp/EAA) and untreated (un‐HAp/EAA) nanocomposites has been made. The use of a phosphonic acid coupling agent promotes the uniform dispersion of n‐HAp in the polymer matrix with a strong nanoparticle‐polymer interfacial bonding, which provides a means of preparing a HAp/polymer nanocomposite for implant applications.
Use of Cube‐Corner Nano‐Indentation Crack Length Measurements to Estimate Residual Stresses Over Small Spatial Dimensions
Authors: Tandon Rajan , Buchheit Thomas E.
Published: January 2007

Cube‐corner indenters, by virtue of their acuity, possess a lowered threshold load for cracking. Shorter crack lengths allow the sampling of residual stresses in small spatial dimensions. We conducted cube‐corner indentation on tempered and annealed glasses. Indentation crack geometry was found to be “quarter‐penny.” A stress‐intensity factor for this geometry, and crack length decrements on tempered materials were used in a stress‐intensity superposition to provide reasonable estimates of residual stress. Stresses ∼100 MPa over a length scale of 10 μm, and 30 MPa over 20 μm were measured accurately, indicating that cube‐corner indentation is a promising tool for materials characterization.
Crystallization Maps for SiCO Amorphous Ceramics
Authors: Saha Atanu , Raj Rishi
Published: January 2007

Amorphous silicon–oxycarbide (SiCO) can retain large mole fractions of carbon when it is made from controlled pyrolysis of silicon‐based polymers. The crystallization resistance of these ceramics, which is quite remarkable, varies with the carbon content. In high‐carbon materials, crystallization is usually accompanied by weight loss (resulting from the carbothermal reduction of silica), whereas phase separation can lead to crystallization without significant weight loss in the low‐carbon materials. A simple graphical method is developed to estimate the composition of the crystallized material, and the concomitant weight loss. The results are presented as maps for a quick estimate of crystallization and weight loss for any composition. Experiments with a medium–high carbon SiCO are used to quantify the degree of crystallization and the associated weight loss at 1300°C and at 1350°C; these results show that, in the case of medium high carbon content, crystallization begins with phase separation but becomes quickly dominated by weight loss.
Precipitation Coating of Monazite on Woven Ceramic Fibers: I. Feasibility
Authors: Fair Geoff E. , Hay Randall S. , Boakye Emmanuel E.
Published: January 2007

Monazite coatings were deposited on woven cloths and tows of Nextel™ 610 fibers by heterogeneous nucleation and growth using solution precursors. Initial experiments revealed two coating regimes in which monazite was either precipitated both in solution and onto the fiber surfaces or only onto the fiber surfaces depending on the precursor solution concentration and fiber surface area. In both cases, regions of tightly packed fibers within cloth were uncoated. Image analysis of coated fiber cross sections revealed a strong correlation between fiber separation and coating thickness, suggesting that the coating of tightly packed fibers was limited by transport of the reactants in solution to these areas. By adopting a coating procedure in which the tightly packed regions are saturated with reactants before precipitation, more uniform coatings of monazite were obtained throughout the cloth; however, the strength of as‐coated and heat‐treated fibers was degraded and remains problematic.
Addition of a Sr, K, Nb (SKN) Combination to PZT(53/47) for High Strain Applications
Authors: Donnelly Niall J. , Shrout Thomas R. , Randall Clive A.
Published: January 2007

A lead zirconate titanate composition incorporating the dopants Sr, K, and Nb (SKN) in the specific ratio 4:1:3 has been studied. In principle, the SKN should act as a donor dopant but since its addition reduced the grain size from 11.4 μm (for 1% SKN) to 1.5 μm (for 5% SKN), the overall effect was found to be more complicated. It was observed that the addition of SKN reduced the Curie temperature, by 16°C/mol (%) and broadened the dielectric peak. X‐ray measurements further suggested that the ceramic was a mixture of rhombohedral and tetragonal phases and that the room temperature c/a ratio of the tetragonal phase decreased with SKN addition. The piezoelectric coefficient d33, determined from high field unipolar drives, gave an optimum value of 779 pm/V for the 0.02 SKN compositions, which also exhibited a relatively high Curie temperature of 356°C. Competing effects of enhanced domain wall mobility from donor doping and reduced mobility due to smaller grain size may explain the observed compositional variation in the measured material properties. Materials based on this composition are attractive for high performance piezoelectric actuator applications such as fuel injection.
Synthesis and Characterization of Ce1−xGdxO2−δ Nanorods
Authors: Lee Jong Soo , Kim Sangtae
Published: January 2007

Single crystalline Ce1−xGdxO2−δ nanorods with x=0.05 and 0.1 as well as x=0 were synthesized by a sol–gel process without using any template and/or applying high pressure. The nanorods grow preferentially along the [110] direction regardless of the dopant concentration. The diameter and the length of the synthesized nanorods range from 10 to 50 nm and from 200 to 700 nm, respectively. The length of the nanorods decreases with increasing dopant concentration. The role of the surfactant as a structure‐directing agent is critical to the growth of Ce1−xGdxO2−δ nanorods.
Behavior of CAL72 osteoblast‐like cells cultured on zirconia ceramics with different surface topographies
Authors: Bächle Maria , Butz Frank , Hübner Ute , Bakalinis Emmanouil , Kohal Ralf J.
Published: January 2007

Objectives: Because of its inherent strength, biocompatibility, and tooth‐like color, zirconia ceramics have the potential to become an alternative to titanium as dental implant material. This study aimed at investigating the osteoblastic response to yttrium‐stabilized tetragonal zirconia polycrystal (Y‐TZP) with different surface topographies.

Methods: CAL72 osteoblast‐like cells were cultured on machined (TZP‐m), airborne particle abraded (TZP‐s), and airborne particle abraded and acid‐etched Y‐TZP (TZP‐sa) surfaces. Polystyrene and airborne particle abraded with large grit and acid‐etched (SLA) titanium served as a reference control. The surface topography was examined by scanning electron microscopy (SEM) and profilometry. At culture days 3, 6, and 12, cell proliferation, at day 12 cell morphology, and cell‐covered surface area were determined.

Results: The surface roughness of Y‐TZP was increased by airborne particle abrasion and additionally by acid etching. No statistically significant differences were found between average roughness (Ra) and maximum peak‐to‐valley height (Rp–v) values of airborne particle abraded and acid‐etched Y‐TZP and SLA titanium. Whereas the cell proliferation assay revealed statistically significant greater values at day 3 for surface‐treated Y‐TZP and polystyrene cultures as compared with machined Y‐TZP, no differences between the Y‐TZP groups, SLA titanium, and polystyrene were observed at culture days 6 and 12.

Conclusions: Cell morphology and cell‐covered surface area were not affected by the type of substrate. The results suggest that roughened Y‐TZP is an appropriate substrate for the proliferation and spreading of osteoblastic cells.
Morphogenesis of the peri‐implant mucosa: an experimental study in dogs
Authors: Berglundh Tord , Abrahamsson Ingemar , Welander Maria , Lang Niklaus P. , Lindhe Jan
Published: January 2007

Purpose: The objective of the present experiment was to study the morphogenesis of the mucosal attachment to implants made of c.p. titanium.

Material and methods: All mandibular premolars were extracted in 20 Labrador dogs. After a healing period of 3 months, four implants (ITI® Dental Implant System) were placed in the right and left sides of the mandible. A non‐submerged implant installation technique was used and the mucosal tissues were secured to the conical marginal portion of the implants with interrupted sutures. The sutures were removed after 2 weeks and a plaque control program including daily cleaning of the remaining teeth and the implants was initiated. The animals were sacrificed and biopsies were obtained at various intervals to provide healing periods extending from Day 0 (2 h) to 12 weeks. The mandibles were removed and placed in the fixative. The implant sites were dissected using a diamond saw and processed for histological analysis.

Results: Large numbers of neutrophils infiltrated and degraded the coagulum that occupied the compartment between the mucosa and the implant during the initial phase of healing. At 2 weeks after surgery, fibroblasts were the dominating cell population in the connective tissue interface but at 4 weeks the density of fibroblasts had decreased. Furthermore, the first signs of epithelial proliferation were observed in specimens representing 1–2 weeks of healing and a mature barrier epithelium occurred after 6–8 weeks of healing. The collagen fibers of the mucosa were organized after 4–6 weeks of healing.

Conclusion: It is suggested that the soft‐tissue attachment to implants placed using a non‐submerged installation procedure is properly established after several weeks following surgery.
Development and implementation of new educational concepts in a dental curriculum
Authors: Kersten H. W. , Vervoorn J. M. , Zijlstra A. E. , Blok B. Snyders , Van Eijden T. M. G. J.
Published: January 2007

Abstract  The Academic Centre for Dentistry Amsterdam introduced a fully renewed 5‐year dental curriculum in September 2003. In this article, the educational principles and didactic choices that form the basis of the curriculum development are presented and attention is given to the process of development and the implementation strategy that constitute such an important part of the success of introducing a new curriculum. Special characteristics of the new curriculum are the clinical training practice, professional conduct, the elective profiles and academic education. In clinical practice, groups of students from different levels run a group practice in which they learn to work together, delegate tasks, solve clinical problems and apply evidence‐based dentistry. In the new curriculum students learn to conduct themselves as professionals. In the third and in the fifth year, students choose an elective profile oriented, respectively, on research and on clinical knowledge and skills. Academic education is an important spearhead in this curriculum in which students not only learn why research is important to dentistry but also how dentists can use research to their own benefit. In development and implementation, a stepwise approach was used in which as many people as logistically possible were involved.
The introduction of a new assessment system in restorative dentistry: the undergraduate and patient experience
Authors: O'Sullivan Dominic , Hooper Susan , McNally Lisa , Jagger Daryll
Published: January 2007

Abstract:  In 2000, the University of Bristol Dental School Division of Restorative Dentistry carried out a review of the assessment methods used within the undergraduate programme. Following this review, a number of key recommendations were made and the system of formative assessment within the division was changed. Audits were conducted immediately prior to the introduction of the new system and 2 years after it had been introduced, the results of these audits are presented. There was no change in the number of failed appointments between the initial audit and the second audit. There was a reduction in the number of patients treated by more than one student from 25% to 14% (approximating to 3% if student withdrawal from the course is taken into account). The length of time taken to complete treatment reduced between the two audits with more than half of all patients having their treatment completed within 3 months of their initial examination in the second audit. Ninety‐five percent of treatment plans were completed in the second audit compared with only 62% in the first audit. In the second audit, the outstanding 5% of cases were signed off as incomplete for satisfactory reasons by the member of supervisory staff (e.g. patients who had moved away or were discharged due to poor attendance). Student comments were mostly positive, the small number of negative comments related to some students being confused by the paperwork in the new system. The overall process has been an extremely positive experience which has resulted in an assessment system with increased clarity which appears to have reduced the stress levels reported by the undergraduate students on the programme and improved the quality of patient care within restorative dentistry.
CD Rainbows
Authors: Ouseph P. J.
Published: January 2007

Several papers have been published on the use of a CD as a grating for undergraduate laboratories and/or for high school and college class demonstrations.1–7 Four years ago The Physics Teacher had a spectacular cover picture showing emission spectrum as viewed through a CD with no coating.8 That picture gave the impetus to develop a system that can project the spectra of light falling on a CD on a white board or a screen. Such an arrangement would be more suitable to show diffraction spectra to a large class.
An Inquiry-based Course Using “Physics?” in Cartoons and Movies
Authors: Rogers Michael
Published: January 2007

Books, cartoons, movies, and video games provide engaging opportunities to get both science and nonscience students excited about physics. An easy way to use these media in one's classroom is to have students view clips and identify unusual events, odd physics, or list things that violate our understanding of the physics that governs our universe.1,2 These activities provide a lesson or two of material, but how does one create an entire course on examining the physics in books, cartoons, movies, and video games? Other approaches attempt to reconcile events in various media with our understanding of physics3–8 or use cartoons themselves to help explain physics topics.9
Bactericidal Activity and Resistance Development Profiling of Dalbavancin
Authors: Goldstein Beth P. , Draghi Deborah C. , Sheehan Daniel J. , Hogan Patricia , Sahm Daniel F.
Published: January 2007

Dalbavancin, a semisynthetic lipoglycopeptide being developed for the treatment of skin and skin structure infections (SSSIs), has a half-life of 5 to 7 days in humans and offers promise for a convenient weekly dosing regimen. We studied the in vitro bactericidal activity of dalbavancin against target organisms, using the concentrations that are maintained in human blood with the proposed dosage regimen. Dalbavancin minimal bactericidal concentrations (MBCs) were ≤0.5 μg/ml for eight staphylococcal isolates; and for six of these strains, including one vancomycin-intermediate
Staphylococcus aureus
(VISA) isolate, the MBCs were equal to or within 1 doubling dilution of the MIC. Dalbavancin MICs for all three
Streptococcus pyogenes
strains were 0.008 μg/ml, as were the MBCs for two of the isolates. In time-kill studies conducted with a different set of seven strains (two methicillin-susceptible
S. aureus
isolates, three methicillin-resistant
S. aureus
isolates, one VISA isolate, and one
S. pyogenes
isolate), all strains exhibited a ≥3-log
10
decrease in their viable counts when they were exposed to ≥1 μg/ml of dalbavancin for 24 h. Resistance development studies by both direct selection (resistance frequency, <10
−10
) and serial passage failed to produce stable mutants with decreased susceptibility to dalbavancin. These observations suggest that dalbavancin will be an effective choice for the management of patients with SSSIs.
Population Pharmacokinetic Model for Gatifloxacin in Pediatric Patients
Authors: Rubino Christopher M. , Capparelli Edmund V. , Bradley John S. , Blumer Jeffrey L. , Kearns Gregory L. , Reed Michael , Jacobs Richard F. , Cirincione Brenda , Grasela Dennis M.
Published: January 2007

The broad spectrum of antimicrobial activity, oral bioavailability, extensive tissue distribution, and once-daily intravenous or oral dosing of gatifloxacin, an expanded-spectrum 8-methoxy fluoroquinolone, make it a potentially useful agent for the treatment of pediatric infections. A population pharmacokinetic model was developed to describe the pharmacokinetics of gatifloxacin in children. Data for analysis were obtained from a single-dose safety/pharmacokinetic study utilizing intensive blood sampling in patients aged 6 months to 16 years. Each subject received a single oral dose of gatifloxacin as a suspension, at doses of 5, 10, or 15 mg/kg of body weight. A total of 845 samples were obtained from 82 patients. A one-compartment model with first-order absorption and elimination was the most appropriate to describe the gatifloxacin concentrations. Covariate analysis using forward selection and backward elimination found that apparent clearance was related to body surface area, and apparent volume of distribution was related to body weight. No effect of age on drug clearance could be identified once clearance was corrected for body surface area. Based on pharmacokinetic simulations, the 10-mg/kg (maximum, 400 mg) once-daily dose of gatifloxacin is expected to provide drug exposure similar to that in healthy adults. The population pharmacokinetic model described herein will be used for Bayesian analyses of sparse pharmacokinetic sampling in phase II/III clinical trials and for Monte Carlo simulation experiments. The success of this strategy provides a model for future pediatric drug development programs.
Mosaic Structure of p1658/97, a 125-Kilobase Plasmid Harboring an Active Amplicon with the Extended-Spectrum β-Lactamase Gene
bla
SHV-5
Authors: Zienkiewicz M. , Kern-Zdanowicz I. , Gołȩbiewski M. , Żyliñska J. , Mieczkowski P. , Gniadkowski M. , Bardowski J. , Cegłowski P.
Published: January 2007

Escherichia coli
isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum β-lactamase (ESBL) of the SHV type. The β-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64,
Providencia rettgeri
genomic island R391,
Vibrio cholerae
STX transposon,
Klebsiella pneumoniae
or
E. coli
chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in
E. coli
. The presence of an active partition system (
sopABC
locus) and two postsegregational killing systems (
pemIK
and
hok/sok
) indicates that the plasmid should be stably maintained in
E. coli
populations. The conjugative transfer is ensured by the operons of the
tra
and
trb
genes. We also demonstrate that the plasmidic segment undergoing amplification contains the
bla
SHV-5
gene and is homologous to a 7.9-kb fragment of the
K. pneumoniae
chromosome. The amplicon displays the structure of a composite transposon of type I.
Gamma Interferon Loaded onto Albumin Nanoparticles: In Vitro and In Vivo Activities against
Brucella abortus
Authors: Segura S. , Gamazo C. , Irache J. M. , Espuelas S.
Published: January 2007

The aim of this study was to evaluate the activity of gamma interferon (IFN-γ) when it was either adsorbed onto or loaded into albumin nanoparticles.
Brucella abortus
-infected macrophages and infected BALB/c mice were selected as the models for testing of the therapeutic potentials of these cytokine delivery systems, in view of the well-established role of IFN-γ-activated macrophages for the control of
Brucella
sp. infections. Whereas the encapsulation of IFN-γ inside the matrix of nanoparticles completely abrogated its activity, adsorbed IFN-γ increased by 0.75 log unit the bactericidal effect induced by RAW macrophages activated with free IFN-γ, along with a higher level of production of nitric oxide. In infected BALB/c-mice, IFN-γ adsorbed onto nanoparticles was also more active than free cytokine in reducing the number of bacteria in the spleens, and the effect was mediated by an increased ratio of IFN-γ-secreting (Th1) to interleukin-4-secreting (Th2) cells. Overall, albumin nanoparticles would be suitable as carriers that target IFN-γ to macrophages and, thus, potentiate their therapeutic activity.
Evolution of TEM-Type Enzymes: Biochemical and Genetic Characterization of Two New Complex Mutant TEM Enzymes, TEM-151 and TEM-152, from a Single Patient
Authors: Robin Frédéric , Delmas Julien , Schweitzer Cédric , Tournilhac Olivier , Lesens Olivier , Chanal Catherine , Bonnet Richard
Published: January 2007

Two clinical isolates of
Escherichia coli
, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related.
E. coli
recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel
bla
TEM
genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel β-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (
k
cat
, 5 and 16 s
−1
, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 μM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.
EthA, a Common Activator of Thiocarbamide-Containing Drugs Acting on Different Mycobacterial Targets
Authors: Dover Lynn G. , Alahari Anuradha , Gratraud Paul , Gomes Jessica M. , Bhowruth Veemal , Reynolds Robert C. , Besra Gurdyal S. , Kremer Laurent
Published: January 2007

Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of
Mycobacterium bovis
BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [
14
C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.
Reduced Susceptibility of
Haemophilus influenzae
to the Peptide Deformylase Inhibitor LBM415 Can Result from Target Protein Overexpression Due to Amplified Chromosomal
def
Gene Copy Number
Authors: Dean Charles R. , Narayan Shubha , Richards Joel , Daigle Denis M. , Esterow Stacy , Leeds Jennifer A. , Kamp Heather , Puyang Xiaoling , Wiedmann Brigitte , Mueller Dieter , Voshol Hans , van Oostrum Jan , Wall Daniel , Koehn James , Dzink-Fox JoAnn , Ryder Neil S.
Published: January 2007

Previous genetic analysis of
Haemophilus influenzae
revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene
acrR
and in the
fmt
gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 μg/ml versus 4 μg/ml against the parent strain NB65044) that lacks mutations in the
acrR
or
fmt
structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in
def
transcript titer. No mutations were found in the region upstream of
def
that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing
def
had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the
def
gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.
In Planta Horizontal Transfer of a Major Pathogenicity Effector Gene
Authors: El Yacoubi B. , Brunings A. M. , Yuan Q. , Shankar S. , Gabriel D. W.
Published: January 2007

Xanthomonas citripv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus,X. citripv. aurantifolii, arose more recently in South America. Genomes ofX. citripv. aurantifolii strains carry two DNA fragments that hybridize topthA, anX. citripv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed thatX. citripv. aurantifolii strain B69 carried one functionalpthAhomolog, designatedpthB, that was required to cause cankers on citrus. GenepthBwas found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenicX. citripv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating thatpthBconferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.
Effect of Superoxide Dismutase Gene Inactivation on Virulence of
Pseudomonas aeruginosa
PAO1 toward the Silkworm,
Bombyx mori
Authors: Iiyama Kazuhiro , Chieda Yuuka , Lee Jae Man , Kusakabe Takahiro , Yasunaga-Aoki Chisa , Shimizu Susumu
Published: January 2007

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm,
Bombyx mori
, mutants of
Pseudomonas aeruginosa
PAO1 lacking manganese-SOD (PAO1
sodM
), iron-SOD (PAO1
sodB
), or both (PAO1
sodMB
) were generated. The mutants were injected into the hemocoel of
B. mori
. The virulence decreased in the order PAO1 = PAO1
sodM
> PAO1
sodB
> PAO1
sodMB
. In particular, PAO1
sodMB
was avirulent at a dose of 10
5
cells or less. The
sod
double mutant PAO1
sodMB
was then complemented with either pSodM or pSodB in
trans
. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of
P. aeruginosa
against
B. mori
. The results of growth in
B. mori
hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in
sod
mutants. In conclusion, the SODs are required for full virulence of
P. aeruginosa
against
B. mori
and Fe-SOD is more important than Mn-SOD in the infection process.
Autotransporter-Encoding Sequences Are Phylogenetically Distributed amongEscherichia coliClinical Isolates and Reference Strains
Authors: Restieri Concetta , Garriss Geneviève , Locas Marie-Claude , Dozois Charles M.
Published: January 2007

Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified amongEscherichia coliassociated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity ofE. colistrains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491E. coliisolates from human urinary tract infections, diarrheagenicE. coli, and avian pathogenicE. coli(APEC) andE. colireference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and thatagn43K12was the most common and widely distributed allele.agn43allelic variants were also phylogenetically distributed. Sequences encodingespC,espP, andsepAandagn43allelesEDL933andRS218were significantly associated with diarrheagenicE. colistrains compared to other groups.tshwas highly associated with APEC strains, whereassatwas absent from APEC.vat,sat, andpicwere associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis ofE. colistrains from clinical, food, and environmental sources.
Evolutionary Relationships of “CandidatusRiesia spp.,” EndosymbioticEnterobacteriaceaeLiving within Hematophagous Primate Lice
Authors: Allen Julie M. , Reed David L. , Perotti M. Alejandra , Braig Henk R.
Published: January 2007

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body lousePediculus humanusand provide new characterizations of the P-endosymbionts fromPediculus schaeffifrom chimpanzees andPthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “CandidatusArsenophonus insecticola.” We propose that two additional species be added to the genus “CandidatusRiesia.” The new species proposed within “CandidatusRiesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated forBuchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.
Carbon Conversion Efficiency and Limits of Productive Bacterial Degradation of Methyl
tert
-Butyl Ether and Related Compounds
Authors: Müller Roland H. , Rohwerder Thore , Harms Hauke
Published: January 2007

The utilization of the fuel oxygenate methyl
tert
-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the Y
ATP
concept. Experiments were conducted to derive realistic maintenance coefficients and
K

s

values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g
−1
, which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient
m

s

and its associated biomass decay term
b
, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations,
S
min
, below which growth would not further be supported.
S
min
strongly depended on the maximum growth rate
μ
ma

x

, and
b
and was directly correlated with the half maximum rate-associated substrate concentration
K

s

, meaning that any effect impacting this parameter would also change
S
min
. The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase
K

s

and
S
min
for MTBE.
A 368-Base-Pair
cis
-Acting
HWP1
Promoter Region, HCR, of
Candida albicans
Confers Hypha-Specific Gene Regulation and Binds Architectural Transcription Factors Nhp6 and Gcf1p
Authors: Kim Samin , Wolyniak Michael J. , Staab Janet F. , Sundstrom Paula
Published: January 2007

To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene
HWP1
of
Candida albicans
, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the
HWP1
control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous
ENO1
promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in
Saccharomyces cerevisiae
, and another with weak similarity to an HMG-like condensation factor from
Physarum polycephalum
implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in
C. albicans
and suggest a new model for hypha-specific gene regulation.
Genome-Wide Analysis of Cellular Response to Bacterial Genotoxin CdtB in Yeast
Authors: Kitagawa Takao , Hoshida Hisashi , Akada Rinji
Published: January 2007

The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in the diploid by homologous recombination but not by other repair mechanisms. Our present results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.
In Vitro Induction of Immunoglobulin A (IgA)- and IgM-Secreting Plasma Blasts by Cholera Toxin Depends on T-Cell Help and Is Mediated by CD154 Up-Regulation and Inhibition of Gamma Interferon Synthesis
Authors: Arce Sergio , Nawar Hesham F. , Muehlinghaus Gwendolin , Russell Michael W. , Connell Terry D.
Published: January 2007

Cholera toxin (CT) and the type II heat-labile enterotoxins (LT-IIa and LT-IIb) are potent immunological adjuvants which are hypothesized to enhance the production of antibody (Ab)-secreting cells, although their mechanisms of action are not fully understood. The treatment of splenic cells with concanavalin A (ConA) plus CT enhanced the production of immunoglobulin A (IgA) and IgM by dividing cells that expressed high levels of major histocompatibility complex class II (MHC-II), CD19, and CD138 and low levels of B220 a phenotype characteristic of plasma blasts. LT-IIa or LT-IIb moderately enhanced IgA and IgM production without enhancing plasma blast differentiation. CT up-regulated CD25, CD69, CD80, CD86, and MHC-II in isolated B cells but failed to induce proliferation or differentiation. The treatment of unfractionated splenic cells with ConA plus CT induced B-cell proliferation and differentiation, but the elimination of CD4+T cells inhibited this effect. CT treatment of ConA-activated CD4+T cells up-regulated CD134 and CD154, whereas the blockage of CD40-CD154 interactions inhibited the induction of plasma blasts and Ig synthesis. The treatment of unfractionated splenic cells with CT, LT-IIa, or LT-IIb enhanced the production of interleukin-6 (IL-6) and IL-10, whereas the production of gamma interferon was inhibited in both CD4+and CD8+T cells mostly by CT. Thus, major regulatory effects of CT on lymphocytes are likely exerted early during the induction of immune responses when B and T cells initially encounter antigen. Neither LT-IIa or LT-IIb had these effects, indicating that type II enterotoxins augment Ab responses by other mechanisms.
Pneumococcal Capsular Polysaccharide Vaccine-Mediated Protection against Serotype 3Streptococcus pneumoniaein Immunodeficient Mice
Authors: Tian Haijun , Groner Avi , Boes Marianne , Pirofski Liise-anne
Published: January 2007

Pneumococcal capsular polysaccharide (PPS) vaccines are less immunogenic in immunocompromised than immunocompetent individuals. However, neither the efficacy of PPS vaccines in immunocompromised individuals nor the host cellular subsets required for vaccine efficacy against pneumococcal disease have been directly investigated. In this study, we vaccinated CD4-deficient (CD4−/−), CD8-deficient (CD8−/−), and secretory immunoglobulin M-deficient (sIgM−/−) mice and wild-type C57BL/6 (Wt) mice with a conjugate of PPS of serotype 3 and tetanus toxoid (PPS3-TT) and determined the antibody response and efficacy of vaccination against systemic and pulmonary challenge with serotype 3 pneumococcus in immunized and control mice. Our results showed that the isotype and predominant IgG subclass of the PPS3 response differed between immunodeficient mouse strains and between immunodeficient and Wt mice, with CD8−/−mice having the most robust response. Vaccination protected Wt, CD4−/−, and sIgM−/−mice from death resulting from both systemic and pulmonary challenge, whereas CD8−/−mice were protected only from systemic and not from pulmonary challenge. Passive vaccination with PPS3-TT-induced sera from Wt, CD4−/−, CD8−/−, and sIgM−/−mice protected naïve Wt mice from death due to pulmonary challenge; however, CD8−/−mice were not protected by sera from Wt or CD8−/−mice. Our findings suggest that PPS-based vaccines can be effective in the setting of CD4 T-cell deficiency but that CD8 T cells could be required for vaccine-mediated protection against pulmonary challenge with serotype 3 pneumococcus.
Kgp and RgpB, but Not RgpA, Are Important for
Porphyromonas gingivalis
Virulence in the Murine Periodontitis Model
Authors: Pathirana Rishi D. , O'Brien-Simpson Neil M. , Brammar Gail C. , Slakeski Nada , Reynolds Eric C.
Published: January 2007

The contributions of three proteinase genes (
rgpA
,
rgpB
, and
kgp
) to the virulence of
Porphyromonas gingivalis
W50 were investigated in the murine periodontitis model. Mice were orally inoculated with eight doses (1 × 10
10
cells per dose) of
rgpA
,
rgpB
,
kgp
,
rgpA rgpB
, or
rgpA rgpB kgp
isogenic mutants, and the level of alveolar bone loss, immune response induced, and number of bacterial cells per half maxilla were compared with those of animals inoculated with wild-type
P. gingivalis
. The
kgp
,
rgpB
,
rgpA rgpB
, and
rgpA rgpB kgp
isogenic mutants induced significantly (
P
< 0.05) less bone loss than the
rgpA
isogenic mutant and the wild type did, and the virulence of the
rgpA
isogenic mutant and the wild type were not significantly different. Mice inoculated with the wild type or the
rgpA
isogenic mutant exhibited significantly (
P
< 0.01) more
P. gingivalis
cells per half maxilla than mice inoculated with
rgpB
,
kgp
,
rgpA rgpB
, and
rgpA rgpB kgp
isogenic mutants or nonchallenged mice did, as determined using real-time PCR. A significant positive correlation was found between the number of
P. gingivalis
cells detected per half maxilla and the amount of alveolar bone loss induced. Enzyme-linked immunosorbent assay results showed that each isogenic mutant and the wild type induced a predominant
P. gingivalis
antigen-specific immunoglobulin G3 (IgG3) response. Furthermore, the
kgp
and
rgpA rgpB kgp
isogenic mutants induced significantly (
P
< 0.05) lower IgG3 antibody responses than the responses induced by the wild type or the
rgpA
,
rgpB
, and
rgpA rgpB
isogenic mutants. The results suggest that the order in which the proteinases contribute to the virulence of
P. gingivalis
in the murine periodontitis model is Kgp ≥ RgpB
≫
RgpA.