bluuebunny/crossref_metadata_2025 huggingface dataset:
A Generic Pipelined Task Scheduling Algorithm for Fault-Tolerant Decentralized Control of a Segmented Telescope Testbed
Authors: Fallorina Salvador , Boussalis Helen , Liu Charles , Rad Khosrow , Dong Jane , Nasser Dani , Thienphrapa Paul
Published: July 2008

Control of complex structures requires high computational power to achieve real-time performance. Through decentralized techniques, a complex structure can be controlled by multiple lower-order local controllers, leading to reduced computational complexities. Furthermore, a decentralized approach can both simplify the development of parallel controllers and facilitate fault-tolerant designs. In our research, multiple digital signal processors are employed in a NASA-sponsored segmented telescope testbed to increase the throughput of control tasks. Although increased performance is realized when subsystems are statically mapped to specific processors for control, inefficiency arises if the number of subsystems M is not an integer multiple of the number of processors P (M > P) because (M mod P) processors are necessarily controlling more subsystems than others. Optimality is sacrificed because processors with lighter loads wait for processors with heavier loads. Furthermore this mechanism does not lend itself favorably towards fault tolerance because the failure of a single processor will result in the failure of its subsystem. This paper describes the design and implementation of a pipelined task mapping approach for the decentralized control of a segmented reflector telescope testbed. In our pipelined processing implementation only four of the six subsystems are processed in any given control cycle; the two unprocessed subsystems in each cycle propagate about the system in a round-robin fashion, so processors are never idle. Fault tolerance is facilitated because processors are no longer tied to specific subsystems. Instead, control computations are distributed dynamically such that the pipeline flow structure is maintained. The implementation of a watchdog technology is presented for detecting the possible processor failures. Experimental results are shown comparing the performance of the pipelined and straightforward approaches. The throughput of the system has also been estimated on a system with a larger number of processors. Such estimation shows the linearity of speedup achieved by using the pipelined approach.
Control of a Mobile Support Platform for Vehicle Detectors on Highway
Authors: Yu Qingcang , Eke Fidelis O. , Cheng Harry H. , Duane Jacob S. , Palen Joe A.
Published: July 2008

The California Department of Transportation has sponsored the development of a new family of out of pavement, laser based sensing devices for monitoring road vehicles on the highway. These devices are to be placed over highway traffic lanes, so that they can have an unobstructed view of vehicles moving along the highway. It is expected that there will be need for relatively frequent adjustment of the actual location of these devices over the freeway, so that they can be moved from one lane to another, or so the position over a given lane can be modified. Because of these constraints, a mobile support platform is planned for these devices. This paper presents a motion control strategy for such a mobile platform, and the necessary hardware to implement the control system. The ideas presented in the paper have been tested on a prototype mobile support platform.
Analysis and Evaluation for STEP-Based Electro-Mechanical Assemblies: An Integrated Fuzzy AHP Approach
Authors: Zha Xuan F. , Foufou Sebti , Sudarsan Rachuri , Sriram Ram D.
Published: July 2008

Assemblability analysis and evaluation play a key role in assembly design, assembly operation analysis and assembly planning. This paper proposes an integrated approach to assemblability analysis and evaluation for STEP (STandard for the Exchange of Product model data, officially ISO 10303) based electro-mechanical products. In the paper, two assembly representation models are first brought up and elaborated: the EXPRESS/XML schema based model and the NIST object-oriented UML-based Open Assembly Model (OAM). These two models are then integrated together; the OAM incorporates the EXPRESS/XML schema based assembly model to completely capture the detailed geometric information. Based on STEP, the proposed assembly evaluation approach uses the EXPRESS/XML schema based model as the information source, and the evaluation structure covers not only the assembly parts’ geometric and physical characteristics but also takes into account the assembly operation data necessary to assemble the parts. The feature of this approach is the linkage of the STEP product definition to the fuzzy analytic hierarchy process (AHP) for assembly evaluation. The proposed approach has the flexibility to be used in various assembly methods and different environments. The case study shows that the proposed approach is feasible.
Effects of a Scenario-Based Design Information Model on Design Activities
Authors: Kurakawa Kei , Tanaka Hiroshi
Published: July 2008

One of the challenging issues that designers face during a conceptual design meeting is to maintain a variety of complex information. To tackle this issue, preliminary studies on design rationale have been conducted. The research approach in those studies is to construct a design information model and then implement a computer system based on the model. In this study, we pay attention to the importance of scenarios generated by designers during design meetings, and propose a scenario-based design information model that consists of three representations: the design solution refinement process representation, the design solution-centered structure representation, and the product scenario representation. We have implemented an experimental system for representing design ideas according to the scenario-based design information model, and conducted a design experiment to analyze how designers represent their ideas according the model and benefit from the represented ideas on the system.
N-Rep: A Neutral Feature Representation to Support Feature Mapping and Data Exchange Across Applications
Authors: Medichalam Madhu S. , Shah Jami J. , D’Souza Roshan
Published: July 2008

The proliferation of different feature based systems has made feature data exchange an important issue. Unlike geometry data exchange, where different representations use the same fundamental concepts; the most popular being B-Rep and CSG [Shah et al. 88], different feature representation schemes use different concepts to represent features corresponding to the application and domain. Therefore, feature data transfer between applications not only involves transfer of instance data but also transformation of feature concepts. This paper presents N-Rep, an application independent declarative language, for feature definition that includes topology, topological relationships, geometry, geometric relationships, parameters and parametric relationships. N-Rep has been designed to serve three roles, viz., (a) to generate feature recognition algorithms for recognizing features from geometry, (b) to generate feature producing procedures to be used in design by feature approaches, and (c) to serve as a neutral feature data exchange medium between representations.
Case Studies Using Immersive Virtual Assembly in Industry
Authors: Jayaram Uma , Jayaram Sankar , DeChenne Charles , Kim Young Jun , Palmer Craig , Mitsui Tatsuki
Published: July 2008

The Virtual Assembly Technology Consortium is a university/government/industry consortium that seeks to investigate the application of virtual assembly methods in mechanical system assembly processes. In this paper we report three categories of engineering case studies that have been developed by the consortium members over the past two years, describe the overall methodology, and then proceed to feature specific details of two key case studies. An engineering case study has been defined as an account of an engineering activity, event or problem containing some of the background and complexities actually encountered by an engineer, with the objective of providing a medium for learning. The objective of the case studies was to assist consortium members in demonstrating and validating the use of immersive virtual assembly technologies and tools in the simulation of factory floor manufacturing processes. What is of special significance is that instead of modeling simplified problems or perceived representative situations, the case studies were constructed from actual assembly floor projects and situations encountered at industry member sites and with considerable participation from industry engineers and manufacturing shop floor personnel. Based on the success of the case studies, the consortium members inferred that virtual assembly methods are poised to move out of the realm of special projects and test scenarios to deployment in the actual design and manufacturing cycle. However, in order to be truly accepted in industry, there are still issues to be addressed in terms of ease of use, portability of the applications, and preparation of the models for the evaluations. Thus, the case studies added a new dimension to the exploration and understanding of how this new technology could be of practical value in industry.
Fuzzy Logic Control for an Autonomous Underwater Crawler
Authors: Welling Douglas , Edwards Dean , Anderson Mike
Published: July 2008

Current underwater crawling vehicles could benefit by using rotating head sonar data to avoid collisions with obstacles. We have developed and optimized a fuzzy logic controller using software for simulation of an underwater environment. The optimization results show near an order of magnitude increase in performance over both straight line and lawnmower search patterns with relatively small changes in the system parameters. The fuzzy logic controller has the capability of navigating a crawler safely and quickly between mission specific points.
Modeling and Representation of Manufacturing Knowledge for DFM Systems
Authors: Zhao Zuozhi , Shah Jami
Published: July 2008

The manufacturing knowledge today spans a vast spectrum, from manufacturing process capability/constraint, precedence, algorithms/heuristics of performing feature recognition, process planning and manufacturing time/cost estimation, to Design for Manufacturing (DfM) tactics and strategies. In this paper, different types of manufacturing knowledge have been identified and the ways to represent and apply them are described. An information model is developed as the backbone to integrate other existing tools into the framework. A computational framework is presented to help the manufacturing knowledge engineers formulize their knowledge and store it into the computer, and help the designers systematically analyze the manufacturability of the design.
A Collision Contact Modeling Method for Rigid Bodies Represented as Triangle Meshes
Authors: Wu Qiang , Jayaram Sankar , Jayaram Uma
Published: July 2008

In this paper, a collision contact modeling method for virtual reality environments is presented. Models in virtual environments are typically represented using triangle meshes. Most traditional collision contact simulations require the surface information of rigid bodies. This makes traditional collision contact simulations inapplicable for most virtual environments. This paper presents methods to model the behavior of colliding objects in virtual environments using triangle mesh representations of the objects. RAPID™ is used to obtain the approximate collision contact information between individual triangle meshes. Impulse calculations are combined with the collision detection to simulate post-collision behavior of objects. A three-point method is introduced to calculate the final resting place of objects or calculate unstable equilibrium and further dynamics of objects. These methods have been integrated with the Virtual Assembly Design Environment (VADE) to enhance VADE’s existing dynamics simulation capabilities. A series of test cases are presented and analyzed to validate these new methods.
The Open Assembly Model for the Exchange of Assembly and Tolerance Information: Overview and Example
Authors: Baysal M. M. , Roy U. , Sudarsan R. , Sriram R. D. , Lyons K. W.
Published: July 2008

In early design phases an effective information exchange among CAD (Computer Aided Design) tools depends on a standardized representation for the product data in all PLM (Product Lifecycle Management) tools. The NIST Core Product Model (CPM) and its extension are proposed to provide the required base-level product model that is open, non-proprietary, generic, extensible, independent of any one product development process and capable of capturing the full engineering context commonly shared in product development [1,2]. The Open Assembly Model (OAM) Model extends CPM to provide a standard representation and exchange protocol for assembly. The assembly information model emphasizes the nature and information requirements for part features and assembly relationships. The model includes both assembly as a concept and assembly as a data structure. For the latter it uses the model data structures of ISO 10303, informally known as the Standard for the Exchange of Product model data (STEP)[3]. The objective of the paper is to show how the OAM can be used to realize seamless integration of product information, with an emphasis on assembly, throughout all phases of a product design. A gearbox design example is used to illustrate the process.
A Novel Approach to Preserving Geometric Constraints in Curve/Surface MRA Modeling
Authors: Yin Jinxiang , Chen Guanlong
Published: July 2008

MRA editing destroys the geometric constraints of curves/surfaces, a high efficient algorithm called wavelet signal separation to preserve geometric constraints during wavelet transformation in MRA modeling is proposed in this paper. The algorithm divides the B-spline control points into those associated and those unassociated with the geometric constraints. Through preserving the signal information associated with the constraint control points, the geometric constraints can be directly preserved after wavelet transformation. The detailed mathematics for this approach is presented. Different examples are included to demonstrate the power of the approach.
Design and Deformation of CAD Surface Models With Haptics
Authors: Liu X. , Dodds G. , McCartney J. , Hinds B. K.
Published: July 2008

With traditional two-dimensional based interfaces, many CAD surface models, such as automobile bodies and ship hulls, are difficult to design and edit due to their 3D nature. This paper discusses the haptic-based deformation for the design of CAD surface models. With haptic devices (force feedback interfaces) designers can, in virtual space, touch a native B-rep CAD model, and use their tactile senses to manipulate it by pushing, pulling and dragging its surfaces in a natural 3D environment. The paper presents shape control functions. By using the shape functions, designers can directly manipulate and deform a selected region of a surface to the desired shape, and generate complex geometry with simple operations. Force feedback gives designers the greatest flexibility for the design of complex surfaces.
Designing Design Processes in Product Lifecycle Management: Research Issues and Strategies
Authors: Panchal Jitesh H. , Ferna´ndez Marco Gero , Paredis Christiaan J. J. , Allen Janet K. , Mistree Farrokh
Published: July 2008

Product Lifecycle Management (PLM) promises to further a holistic consideration of product design, emphasizing integration, interoperability, and sustainability throughout a product’s lifecycle. Thus far, efforts have focused on addressing lifecycle concerns from a product-centric perspective by exploiting the reusability and scalability of existing products through product platform and product family design. Not much attention has been paid to leveraging the design process and its design in addressing lifecycle considerations, however. In striving for sustainability, it is the design process that should be considered to constitute an engineering enterprise’s primary resource commitment. In this paper, an overview of the challenges inherent in designing design processes is provided. These challenges are subsequently illustrated with regard to several design scenarios of varying complexity, using an example involving the design of Linear Cellular Alloys. A distinction is made between product related requirements/goals and design process related requirements/goals. Requirements, research issues, and strategies for addressing the diverse needs of modeling design processes from a decision-centric perspective are established. Finally, key elements for enabling the integrated design of products and their underlying design processes in a systematic fashion are provided, motivating the extension of PLM to include the lifecycle considerations of design processes, thereby moving towards Design Process Lifecycle Management (DPLM).
Tool Sequence Selection for 2.5-D Pockets With Uneven Stock
Authors: D’Souza Roshan M.
Published: July 2008

This paper describes an algorithm to select the cheapest tool sequence for machining 2.5-D pockets using the milling process when the stock is uneven (non cylindrical). Uneven stock is generated when multiple setups are used to machine a prismatic part. Even though the pockets have flat bottom faces, the amount of material to be removed will vary along the depth of the pocket. This research has developed algorithms for finding accessible areas for tools, and pocket decomposition when the stock is uneven. Finally, it is shown that tool sequence selection problem can be formulated as the shortest path problem in a single-source, single-sink directed acyclic graph.
Genotyping as a Tool for Antibiotic Resistance Surveillance of
Neisseria gonorrhoeae
in New Caledonia: Evidence of a Novel Genotype Associated with Reduced Penicillin Susceptibility
Authors: Vernel-Pauillac Frédérique , Nandi Sobhan , Nicholas Robert A. , Goarant Cyrille
Published: July 2008

Antibiotic resistance in
Neisseria gonorrhoeae
continues to be a major concern in public health. Resistance of
N. gonorrhoeae
bacteria to penicillin G is widespread in most developed countries, which has necessitated a change to newer drugs for treatment of gonococcal infections. Recent reports indicate that resistance to these newer drugs is increasing, highlighting the need for accurate therapeutic recommendations. In some countries or communities, however,
N. gonorrhoeae
isolates are still susceptible to penicillin, so the use of this antibiotic for single-dose treatments of medically under-resourced patients is beneficial. In order to evaluate the adequacy and sustainability of this treatment approach, we explored the presence and prevalence of chromosomally mediated resistance determinants in
N. gonorrhoeae
isolates collected from 2005 to 2007 in New Caledonia. We developed two new real-time PCR assays targeting the
penB
and
mtrR
determinants, to be used together with a previously described duplex assay targeting the
penA
and
ponA
determinants. The results of this study provided evidence that neither the most-common
mtrR
determinants nor the most-resistance-associated
penB
alleles are currently circulating in New Caledonia, suggesting that penicillin should still be considered a valuable treatment strategy. Additionally, using our genotyping assay, we observed an unexpected
penB
genotype at a relatively high frequency that was associated with a decreased susceptibility to penicillin (average MIC, 0.15 μg/ml). Sequencing revealed that this genotype corresponded to an A102S mutation in the
penB
gene. The molecular tools developed in this study can be used successfully for prospective epidemiological monitoring and surveillance of penicillin susceptibility.
Evaluation of Daptomycin Pharmacodynamics and Resistance at Various Dosage Regimens against
Staphylococcus aureus
Isolates with Reduced Susceptibilities to Daptomycin in an In Vitro Pharmacodynamic Model with Simulated Endocardial Vegetations
Authors: Rose Warren E. , Leonard Steven N. , Rybak Michael J.
Published: July 2008

The need to investigate novel dosing regimens and combinations is essential in combating poor treatment outcomes for
Staphylococcus aureus
bacteremia and endocarditis. We evaluated the impact of simulated standard- and high-dose daptomycin in combination with gentamicin or rifampin against daptomycin-susceptible and nonsusceptible matched strains of
S. aureus.
These strains were collected from the daptomycin bacteremia and endocarditis clinical trial and consisted of three susceptible strains (MIC, 0.25 mg/liter) and four nonsusceptible isolates (MICs, 2 to 4 mg/liter). Daptomycin regimens of 6 and 10 mg/kg of body weight daily alone and in combination with gentamicin at 5 mg/kg daily or rifampin at 300 mg every 8 h were evaluated using an in vitro model with simulated endocardial vegetations over 96 h. Rapid bactericidal activity, identified by time to 99.9% kill, was displayed in all regimens with the daptomycin-susceptible strains. Concentration-dependent activity was noted by more-rapid killing with the 10-mg/kg/day dose. The addition of gentamicin improved activity in the majority of susceptible isolates. Daptomycin 6-mg/kg/day monotherapy displayed bactericidal activity for only one of the nonsusceptible isolates and for only two isolates with increased doses of 10 mg/kg/day. Combination regimens demonstrated improvement with some but not all nonsusceptible isolates. Three isolates developed a reduction in daptomycin susceptibility with 6-mg/kg/day monotherapy, but this was suppressed with both combination therapy and high-dose daptomycin. These results suggest that high-dose daptomycin therapy and combination therapy may be reasonable treatment options for susceptible isolates; however, more investigations are needed to confirm the variability of these regimens with nonsusceptible isolates.
Intracellular Time Course, Pharmacokinetics, and Biodistribution of Isoniazid and Rifabutin following Pulmonary Delivery of Inhalable Microparticles to Mice
Authors: Verma Rahul Kumar , Kaur Jatinder , Kumar Kaushlendra , Yadav Awadh Bihari , Misra Amit
Published: July 2008

Intracellular concentrations of isoniazid and rifabutin resulting from administration of inhalable microparticles of these drugs to phorbol-differentiated THP-1 cells and the pharmacokinetics and biodistribution of these drugs upon inhalation of microparticles or intravenous administration of free drugs to mice were investigated. In cultured cells, both microparticles and dissolved drugs established peak concentrations of isoniazid (∼1.4 and 1.1 μg/10
6
cells) and rifabutin (∼2 μg/ml and ∼1.4 μg/10
6
cells) within 10 min. Microparticles maintained the intracellular concentration of isoniazid for 24 h and rifabutin for 96 h, whereas dissolved drugs did not. The following pharmacokinetic parameters were calculated using WinNonlin from samples obtained after inhalation using an in-house apparatus (figures in parentheses refer to parameters obtained after intravenous administration of an equivalent amount, i.e., 100 μg of either drug, to parallel groups): isoniazid, serum half-life (
t
1/2
) = 18.63 ± 5.89 h (3.91 ± 1.06 h), maximum concentration in serum (
C
max
) = 2.37 ± 0.23 μg·ml
−1
(3.24 ± 0.57 μg·ml
−1
), area under the concentration-time curve from 0 to 24 h (AUC
0-24
) = 55.34 ± 13.72 μg/ml
−1
h
−1
(16.64 ± 1.80 μg/ml
−1
h
−1
), and clearance (CL) = 63.90 ± 13.32 ml·h
−1
(4.43 ± 1.85 ml·h
−1
); rifabutin,
t
1/2
= 119.49 ± 29.62 h (20.18 ± 4.02 h),
C
max
= 1.59 ± 0.01 μg·ml
−1
(3.47 ± 0.33 μg·ml
−1
), AUC
0-96
= 109.35 ± 14.78 μg/ml
−1
h
−1
(90.82 ± 7.46 μg/ml
−1
h
−1
), and CL = 11.68 ± 7.00 ml·h
−1
(1.03 ± 0.11 ml·h
−1
). Drug targeting to the lungs in general and alveolar macrophages in particular was observed. It was concluded that inhaled microparticles can reduce dose frequency and improve the pharmacologic index of the drug combination.
Efficacy of Pyrvinium Pamoate against
Cryptosporidium parvum
Infection In Vitro and in a Neonatal Mouse Model
Authors: Downey Autumn S. , Chong Curtis R. , Graczyk Thaddeus K. , Sullivan David J.
Published: July 2008

No effective approved drug therapy exists for
Cryptosporidium
infection of immunocompromised patients. Here we investigated the nonabsorbed anthelmintic drug pyrvinium pamoate for inhibition of the growth of the intestinal protozoan parasite
Cryptosporidium parvum
. The concentration of pyrvinium that effected 50% growth inhibition in human enterocytic HCT-8 cells by a quantitative alkaline phosphatase immunoassay was 354 nM. For comparison, in the same assay, 50% growth inhibition was obtained with 711 μM paromomycin or 27 μM chloroquine. We used a neonatal mouse model to measure the anti-
Cryptosporidium
activity of pyrvinium pamoate in vivo. Beginning 3 days after infection, pyrvinium at 5 or 12.5 mg/kg of body weight/day was administered to the treatment group mice for 4 or 6 consecutive days. Nine days after infection, the mice were sacrificed, and drug efficacy was determined by comparing the numbers of oocysts in the fecal smears of treated versus untreated mice. The intensities of trophozoite infection in the ileocecal intestinal regions were also compared using hematoxylin-and-eosin-stained histological slides. We observed a >90% reduction in infection intensity in pyrvinium-treated mice relative to that in untreated controls, along with a substantial reduction in tissue pathology. Based on these results, pyrvinium pamoate is a potential drug candidate for the treatment of cryptosporidiosis in both immunocompetent and immunocompromised individuals.
Lung Microdialysis Study of Levofloxacin in Rats following Intravenous Infusion at Steady State
Authors: Marchand Sandrine , Frasca Denis , Dahyot-Fizelier Claire , Breheret Céline , Mimoz Olivier , Couet William
Published: July 2008

Lung microdialysis has been used with rats to investigate antibiotic distribution after single-dose administration. However, conducting such experiments after intravenous infusion at steady state would constitute a more convenient alternative, which was evaluated here, using levofloxacin (LVX) as a test compound. Microdialysis probes were inserted in blood and muscle, used as a comparator, between 9:00 a.m. and 11:00 a.m. Intravenous LVX infusion was started 6 h later and maintained until the end of the experiment at a rate of 1.0 mg·h
−1
. Lung microdialysis probes were inserted on the morning of the next day. Rats were kept anesthetized during dialysate collection. In vivo probe recoveries were estimated by retrodialysis using a calibrator method, with ciprofloxacin (CIP) as the calibrator. LVX and CIP were analyzed in dialysates by high-performance liquid chromatography. The steady-state tissue-to-blood unbound-drug concentration ratios were 1.00 ± 0.15 in muscle tissues and 1.06 ± 0.40 in lungs, suggesting passive distribution of LVX in tissue. Although providing no information on rate of distribution, microdialysis investigations following drug infusion at steady state appear to be an interesting approach for characterization of antibiotic distribution in rat lungs.
Mechanisms of Human Immunodeficiency Virus Type 1 Concerted Integration Related to Strand Transfer Inhibition and Drug Resistance
Authors: Zahm Jacob A. , Bera Sibes , Pandey Krishan K. , Vora Ajaykumar , Stillmock Kara , Hazuda Daria , Grandgenett Duane P.
Published: July 2008

The “strand transfer inhibitors” of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3′ OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3′-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.
Convergent Evolutionary Analysis Identifies Significant Mutations in Drug Resistance Targets of
Mycobacterium tuberculosis
Authors: Hazbón Manzour Hernando , Motiwala Alifiya S. , Cavatore Magali , Brimacombe Michael , Whittam Thomas S. , Alland David
Published: July 2008

Mycobacterium tuberculosis
adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of
M. tuberculosis
were related phylogenetically and tested for the distribution of SNPs in putative drug resistance genes. Drug resistance-associated versus non-drug-resistance-associated SNPs in putative drug resistance genes were compared for associations with single versus multiple-branch outcomes using the chi-square and Fisher exact tests. All 286 (100%) isolates containing isoniazid (INH) resistance-associated SNPs had multibranch distributions, suggestive of multiple ancestry and convergent evolution. In contrast, all 327 (100%) isolates containing non-drug-resistance-associated SNPs were monophyletic and thus showed no evidence of convergent evolution (
P
< 0.001). Convergence testing was then applied to SNPs at position 481 of the
iniA
(Rv0342) gene and position 306 of the
embB
gene, both potential drug resistance targets for INH and/or ethambutol. Mutant
embB306
alleles showed multibranch distributions, suggestive of convergent evolution; however, all 44
iniA
(
H481Q
) mutations were monophyletic. In conclusion, this study validates convergence analysis as a tool for identifying mutations that cause INH resistance and explores mutations in other genes. Our results suggest that
embB306
mutations are likely to confer drug resistance, while
iniA
(
H481Q
) mutations are not. This approach may be applied on a genome-wide scale to identify SNPs that impact antibiotic resistance and other types of biological fitness.
Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents
Authors: Vaara Martti , Fox John , Loidl Günther , Siikanen Osmo , Apajalahti Juha , Hansen Frank , Frimodt-Møller Niels , Nagai Junya , Takano Mikihisa , Vaara Timo
Published: July 2008

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-
d
-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of
Escherichia coli
were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of
Enterobacteriaceae
and against
Acinetobacter baumannii
. At subinhibitory concentrations, it dramatically sensitized
A. baumannii
to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against
Pseudomonas aeruginosa
. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.
Antifungal Resistance of
Candida glabrata
Vaginal Isolates and Development of a Quantitative Reverse Transcription-PCR-Based Azole Susceptibility Assay
Authors: Gygax Scott E. , Vermitsky John-Paul , Chadwick Sean G. , Self Matthew J. , Zimmerman Jessica A. , Mordechai Eli , Adelson Martin E. , Trama Jason P.
Published: July 2008

A multiplex quantitative reverse transcription-PCR assay was developed to detect azole resistance in
Candida glabrata
, an important opportunistic pathogen that develops resistance rapidly. Resistance was defined as a ≥3-fold increase in
CDR1
expression by this assay, which proved to be 100% sensitive and 95% specific in comparison to the gold standard broth microdilution assay.
Functional Cloning and Characterization of the Multidrug Efflux Pumps NorM from
Neisseria gonorrhoeae
and YdhE from
Escherichia coli
Authors: Long Feng , Rouquette-Loughlin Corinne , Shafer William M. , Yu Edward W.
Published: July 2008

Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of
Neisseria gonorrhoeae
and the YdhE protein of
Escherichia coli
have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro.
E. coli
strains expressing
norM
or
ydhE
showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in
E. coli
, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na
+
-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region.
Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of
Yersinia pseudotuberculosis
by Macrophages
Authors: Zhang Yue , Murtha James , Roberts Margaret A. , Siegel Richard M. , Bliska James B.
Published: July 2008

Yersinia pseudotuberculosis
uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and
Y. pseudotuberculosis
can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV
+
wild-type, and
yop
mutant
Y. pseudotuberculosis
strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV
+
Y. pseudotuberculosis
. Interestingly, macrophages restricted survival of pYV
+
but not pYV-cured or
ΔyopB Y. pseudotuberculosis
within phagosomes: only a small fraction of the pYV
+
bacteria internalized replicated by 24 h. In addition, ∼20% of macrophages infected with wild-type pYV
+
Y. pseudotuberculosis
died of apoptosis after 20 h. Analysis of
yop
mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV
+
Y. pseudotuberculosis
with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of
Y. pseudotuberculosis
suggests new roles for the T3SS in
Yersinia
pathogenesis.
Stages of Meningococcal Sepsis Simulated In Vitro, with Emphasis on Complement and Toll-Like Receptor Activation
Authors: Hellerud Bernt Christian , Stenvik Jørgen , Espevik Terje , Lambris John D. , Mollnes Tom Eirik , Brandtzaeg Petter
Published: July 2008

The clinical presentation of meningococcal disease is closely related to the number of meningococci in the circulation. This study aimed to examine the activation of the innate immune system after being exposed to increasing and clinically relevant concentrations of meningococci. We incubated representativeNeisseria meningitidisserogroup B (ST-32) and serogroup C (ST-11) strains and a lipopolysaccharide (LPS)-deficient mutant (the 44/76lpxAmutant) in human serum and whole blood and measured complement activation and cytokine secretion and the effect of blocking these systems. HEK293 cells transfected with Toll-like receptors (TLRs) were examined for activation of NF-κB. The threshold for cytokine secretion and activation of NF-κB was 103to 104meningococci/ml. LPS was the sole inflammation-inducing molecule at concentrations up to 105to 106meningococci/ml. The activation was dependent on TLR4-MD2-CD14. Complement contributed to the inflammatory response at ≥105to 106meningococci/ml, and complement activation increased exponentially at ≥107bacteria/ml. Non-LPS components initiated TLR2-mediated activation at ≥107bacteria/ml. As the bacterial concentration exceeded 107/ml, TLR4 and TLR2 were increasingly activated, independent of CD14. In this model mimicking human disease, the inflammatory response toN. meningitidiswas closely associated with the bacterial concentration. Therapeutically, CD14 inhibition alone was most efficient at a low bacterial concentration, whereas addition of a complement inhibitor may be beneficial when the bacterial load increases.
Bacterial Protein Secretion Is Required for Priming of CD8+T Cells Specific for theMycobacterium tuberculosisAntigen CFP10
Authors: Woodworth Joshua S. , Fortune Sarah M. , Behar Samuel M.
Published: July 2008

Mycobacterium tuberculosisinfection elicits antigen-specific CD8+T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted byM. tuberculosis, and they are immunodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10 secretion byM. tuberculosisis required for the priming of CD8+T cells in vivo. Our results reveal an explicit dependence upon the bacterial secretion of the CFP10 antigen for the induction of antigen-specific CD8+T cells in vivo. By using well-definedM. tuberculosismutants and carefully controlling for virulence, we show that ESX-1 function is required for the priming of CD8+T cells specific for CFP10. CD4+and CD8+T-cell responses to mycobacterial antigens secreted independently of ESX-1 were unaffected, suggesting that ESX-1-dependent phagosomal escape is not required for CD8+T-cell priming during infection. We propose that the overrepresentation of secreted proteins as dominant targets of the CD8+T-cell response duringM. tuberculosisinfection is a consequence of their preferential sampling by the MHC-I pathway. The implications of these findings should be considered in all models of antigen presentation duringM. tuberculosisinfection and in vaccine development.
Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation
Authors: Overhage Joerg , Campisano Andrea , Bains Manjeet , Torfs Ellen C. W. , Rehm Bernd H. A. , Hancock Robert E. W.
Published: July 2008

The ability to form biofilms is a critical factor in chronic infections by
Pseudomonas aeruginosa
and has made this bacterium a model organism with respect to biofilm formation. This study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37. In addition to its key role in modulating the innate immune response and weak antimicrobial activity, LL-37 potently inhibited the formation of bacterial biofilms in vitro. This occurred at the very low and physiologically meaningful concentration of 0.5 μg/ml, far below that required to kill or inhibit growth (MIC = 64 μg/ml). LL-37 also affected existing, pregrown
P. aeruginosa
biofilms. Similar results were obtained using the bovine neutrophil peptide indolicidin, but no inhibitory effect on biofilm formation was detected using subinhibitory concentrations of the mouse peptide CRAMP, which shares 67% identity with LL-37, polymyxin B, or the bovine bactenecin homolog Bac2A. Using microarrays and follow-up studies, we were able to demonstrate that LL-37 affected biofilm formation by decreasing the attachment of bacterial cells, stimulating twitching motility, and influencing two major quorum sensing systems (Las and Rhl), leading to the downregulation of genes essential for biofilm development.
A Functional Collagen Adhesin Gene,
acm
, in Clinical Isolates of
Enterococcus faecium
Correlates with the Recent Success of This Emerging Nosocomial Pathogen
Authors: Nallapareddy Sreedhar R. , Singh Kavindra V. , Okhuysen Pablo C. , Murray Barbara E.
Published: July 2008

Enterococcus faecium
recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some
E. faecium
strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90
E. faecium
isolates (99%
acm
+
) and found that the Acm protein was detected predominantly in clinically derived isolates, while the
acm
gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (
P
≤ 0.0003) was also demonstrated, and levels of adherence were highly correlated (
r
= 0.879) with the amount of cell surface Acm detected by whole-cell enzyme-linked immunosorbent assay and flow cytometry. Thirty-seven of 41 sera from patients with
E. faecium
infections showed reactivity with recombinant Acm, while only 4 of 30 community and hospitalized patient control group sera reacted (
P
≤ 0.0003); importantly, antibodies to Acm were present in all 14
E. faecium
endocarditis patient sera. Although pulsed-field gel electrophoresis indicated that multiple strains expressed collagen adherence, multilocus sequence typing demonstrated that the majority of collagen-adhering isolates, as well as 16 of 17 endocarditis isolates, are part of the hospital-associated
E. faecium
genogroup referred to as clonal complex 17 (CC17), which has emerged globally. Taken together, our findings support the hypothesis that Acm has contributed to the emergence of
E. faecium
and CC17 in nosocomial infections.
Contribution of the Collagen Adhesin Acm to Pathogenesis of
Enterococcus faecium
in Experimental Endocarditis
Authors: Nallapareddy Sreedhar R. , Singh Kavindra V. , Murray Barbara E.
Published: July 2008

Enterococcus faecium
is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating
E. faecium
virulence determinants. Our previous studies showed that some clinical
E. faecium
isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic
acm
deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Δ
acm
::
cat
is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (
P
< 0.0001) and for valve colonization 1 and 3 hours after infection (
P
≤ 0.0002), making Acm the first factor shown to be important for
E. faecium
pathogenesis. In contrast, no mortality differences were observed in a mouse peritonitis model. While 5 of 17 endocarditis isolates were Acm nonproducers and failed to adhere to collagen in vitro, all had an intact, highly conserved
acm
locus. Highly reduced
acm
mRNA levels (≥50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that
acm
transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in
E. faecium
collagen adherence by affinity-purified anti-Acm antibodies from
E. faecium
endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.
The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative
Escherichia coli
Mediates Binding to Extracellular Matrix Proteins
Authors: Farfan Mauricio J. , Inman Keith G. , Nataro James P.
Published: July 2008

Enteroaggregative
Escherichia coli
(EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene,
aafA
. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of
aafA
. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.