bluuebunny/crossref_metadata_2025 huggingface dataset:
Forest Thinning Residues as a Potential Fuel Source
Authors: Brown Alexander L. , Jepsen Richard A.
Published: July 2010

Northern New Mexico forests are characterized predominantly by small (i.e. around 10 cm diameter), densely populated conifers. Land managers, both private and government, often thin the forests to reduce the risks from wildland fire. Thinned residues typically amount to approximately 20 to 50 tons per acre. With no obvious market use for these small thinning residues, they are presently either discarded on the ground, or burnt as waste. Through a small business assistance initiative, Sandia National Laboratories is helping to identify and promote process improvements. Several productive uses of the residues have been evaluated, and are presented. The concept of a mobile pyrolysis unit is presently being examined in more detail for several pragmatic reasons. It could remove a significant fraction of the mass as a dense liquid that could be shipped to a refinery for conversion to a fossil fuel additive or substitute. Also, it is a process that is sufficiently well self contained that it could be reasonably sized for a mobile system. Present issues with the concept are addressed, including yield, benefit, and cost.
Homogeneous Charge Catalytic Ignition of Water-Ethanol Blends in a CFR Engine
Authors: Anderson Edwin , Cyr Jason , Cordon Dan , Beyerlein Steve
Published: July 2010

The burning of water ethanol blends has the potential to reduce NOx, CO, and HC emissions while reducing the ethanol fermentation production cost of distillation and dehydration by utilization of these blends. The torch style ignition produced by the catalytic igniter allows for the operation and cold start of an SI engine on ethanol/water fuels up to a 50/50 blend. This paper describes the operating characteristics of a catalytic igniter in a modified Co-operative Fuels Research (CFR) engine. Performance data was evaluated using a high speed in-cylinder combustion pressure analyzer. Emission data for premixed ethanol/water was also compared to separate water and ethanol injection for concentrations of 0–30% water in ethanol. Emissions and performance results for both fuel preparation methods were compared with engine operation on 100% ethanol. Premixed ethanol/water displayed significantly lower NOx and CO emissions and somewhat higher hydrocarbon emissions than separate water and ethanol injection. At a compression ratio of 10:1, the catalytic igniter configuration studied in this work was able to control cycle to cycle pressure variation even at high water fractions.
Visualization and Parametric Study of Reaction Propagation in Meso-Scale Tubes
Authors: Wang Chan-Yu , Wang Jun-Kai , Wu Ming-Hsun
Published: July 2010

Reaction propagation of ethylene/oxygen and methane/oxygen mixtures in capillary tubes of 1 and 2 mm in diameters with initial pressure and temperature at ambient condition were experimentally visualized and analysed using high speed cinematography. Deflagrative flame was initiated in middle of the smooth tube, and the reaction fronts accelerated as they propagated towards the exits in the opposite directions. Lengths of the tubes investigated ranged from 0.4 to 1 m (one side), and deflagration-to-detonation transitions were observed for equivalence ratios between 0.5 and 3. The visible reaction front propagates at speeds approach Chapman-Jouguet speed for ethylene/oxygen mixture in the 1 mm and 2 mm tubes. An overshoot in propagation velocity was found during transition process. For leaner and richer mixtures beyond the detonation limits, steady deflagration wave propagation was observed. Reaction propagation in methane/oxygen mixture was also investigated. Several near-limit propagation modes were found.
Modeling and Applications of Electrochemical Machining Process
Authors: Tsuboi Ryo , Yamamoto Makoto
Published: July 2010

Electrochemical Machining (below ECM) is one of advanced machining technologies and has been developed and applied in highly specialized fields, such as aerospace, aeronautics, defense and medical industries. In recent years, ECM is used in other industries such as automobile and turbo-machinery because of the following advantages. That is, it has no tool wear, and it can machine difficult-to-cut metals and complex geometries with relatively high accuracy. However, ECM still has some problems to be overcome. The efficient tool-design procedure, electrolyte processing, disposal of metal hydroxide sludge are the typical issues. In order to solve these problems, a numerical simulation is considered to be a powerful tool. However, the numerical code that can satisfactorily predict the flow field and the machining process has not been developed because of the complex flow natures such as the three-dimensionality, hydrogen bubble/metal sludge generation (i.e. three-phase effect), temperature increase and flow separation. In present paper, summery of my PhD works is mentioned, about modeling and applications. Modeling for ECM process takes into account metal dissolution, electrolyte flow, void fraction distribution of hydrogen bubbles generated from the tool cathode, thermal, electric potential, and electric conductivity. Especially, two types of method are used for the coupling between gas- and liquid-phase in electrolyte. One is one-way coupling method; only the electrolyte flow affects the void fraction distribution of hydrogen bubbles. The other is two-way coupling method; considering the interaction between the electrolyte flow and the void fraction distribution. In the two-way coupling method, considering bulk density distribution in the electrolyte flow path due to hydrogen bubble, Low-Mach-Number approximation is used for simulations. For applications with our numerical code, simulations for machining 3-D compressor blade are performed. Blade geometry is successfully predicted and we can obtain some guideline of ECM process.
Experimental and Numerical Investigation of Spatial and Temporal Dispersion of Forced Fuel Oscillations
Authors: Chishty Wajid A. , Boulanger Joan , Yun Sangsig
Published: July 2010

In lean premixed combustors of gas turbine engines, fuel-air mixing is considered vital for controlling pollutant emissions as well as combustion instability. Enhancement in mixing may be obtained by modulating the fuel flow rate. Modulation of fuel flow is also a useful technique to actively control combustion instabilities arising from the pressure oscillations in the combustor and thrust augmenters. Effectiveness of the forced oscillations depends on the level of dispersion present in the system. Knowledge of dispersion levels is also important in determining the degree of mixing and therefore, the effectiveness of a premixer. This paper presents the experimental efforts undertaken to study the spatial and temporal dispersion of fuel flow rate oscillation introduced at the premixer inlet. Effects of oscillation amplitude and frequency are investigated at different bulk flow rates and at various locations in the premixer. Also presented is a review of the in-house numerical work done towards this end, using three computational methods. Results show that the degree of dispersion in fuel flow rate oscillations depends on modulation amplitude and frequency as well as advective velocity of the bulk flow.
Design of Statically Balanced Six-Degree-of-Freedom Parallel Mechanisms Based on Tensegrity System
Authors: Shekarforoush S. M. Mehdi , Eghtesad Mohammad , Farid Mehrdad
Published: July 2010

A parallel mechanism that is based on tensegrity system is studied in this article. Tensegrity systems are a suitable alternative for conventional engineering systems like mechanisms for some application. In this article, tensegrity mechanisms are classified into tensegrity mechanism with passive and active compliant components. Based on this classification, two types of six-degree-of-freedom parallel mechanism are proposed and kinematics and static of them are solved. The first type is the 6–6 tensegrity mechanism with passive compliant components and the second type is the 6-3 tensegrity mechanism with active compliant components.
The Numerical Simulation and the Structure Optimization of the Tiny-Oil Ignition Burner
Authors: Fu Zhongguang , Wang Mingchen , Zhou Chenn Qian , Peng Guosheng , Yang Guoqiang
Published: July 2010

The pulverized coal burner is widely used in industry, especially in electricity industry. Stable combustion of the burner is very important to conserve energy and minimize environmental impact. Generally, most of gas or oil will be consumed when the burner is ignited. Meanwhile, the operation of the dust catcher will be affected when oil is burned in the furnace which leads to more emission of solid particle into the air. Tiny-oil ignition burner can be a solution to the above problems. The structure of burner should be different for fuel and each different application. It is a challenge to optimize the structure of burner. Three-dimensional CFD simulations were performed to optimize the design of a tiny-oil ignition burner. The temperature distributions along the central line of the tiny-oil ignition burner show that the structure of the oil combustion chamber and the first combustion chamber are worthy of optimization based on the type of Chinese coal.
Modeling of Mechanical and Electrical Behavior of Piezoelectric Fiber Composites
Authors: Liu Y. G. , Li J. S. , Li L. , Shen X.
Published: July 2010

As a new actuators piezoelectric fiber composites is like a sandwich comprised of interdigitated electrodes, polymer and piezoceramic fibers. A series of piezoelectric unit’s equations about longitudinal length extension vibration mode are set up. According to the dynamic FEM equations for piezoelectric actuator, which are established by means of Lagrange function and Hamilton principle, a series of finite models are built. The performance of mechanics and electrics is analyzed in detail. The distribution of stain, stress and electrostatic field inside the PFC actuators is given by FEM. Influence of geometry parameters and material properties on the lengthways strain and stress are studied. In addition, effect of space and width of IDE and affiliated polymer on the electrostatic field are invested. The results show that PFC actuators perform well when the distance between IDE and fibers is zero and conductive polymer is used as affiliated polymer.
CBR-Based Process Planning for Special-Type Spring Coiling
Authors: Li Jishun , Ku Xiangchen , Xue Yujun , Jia Xianzhao
Published: July 2010

When coiling a special-type spring in CNC coiler, the setup jobs of the coiler, including the type selection and position decision of various forming tools, cam layout, angle calculation of camshaft, are achieved by manual work using a trial-and-error method. This method seriously affects the spring production efficiency. In order to solve this problem, the coiling process planning system for spring based on CBR (case based reasoning) is developed in this paper. The system mainly comprises a case library that holds the details of coiling process plan. Similar cases are retrieved and adapted when a new spring is manufactured. The process of finding the most similar case is divided into three stages. Some key technologies of the system, including the information model of the spring coiling process and the structure of the case library, are also revealed in this paper. The result indicates that this system reduces the setup time and enhances the production efficiency of spring.
The Effect of Mahua (Madhuca Longfolia) Oil With Dioxane Fuel Blends on Diesel Engine and Studies on Combustion and Emission Characteristics
Authors: Ashok K. , Alagumurthi N. , Saravanan C. G.
Published: July 2010

An organic compound, Dioxane, is blended to reduce the viscosity of raw vegetable oil (Mahua). A dilute blend was prepared by mixing with raw vegetable oil (Mahua) and 10% dioxane in volume basis. Tests were conducted on a single cylinder, water cooled, DI diesel engine coupled with the eddy current dynamometer. Emissions like HC, NOX, etc., were measured by using gas analyzer and smoke density was measured by using smoke meter. The cylinder pressure, heat release rate were measured by combustion analyzer. From the experimental investigation, it was observed that operating at a blend ratio of 10% diesel-80% mahua oil-10% Dioxane significantly reduced the HC and NOx emissions when compared to diesel fuel. It was also observed, the variation of break thermal efficiency is almost same to that of diesel fuel. Hence, it can be concluded that raw vegetable oil (mahua) with Dioxane blend could partially replace the diesel, as a fuel.
An Examination of the Alternative Methods Used by the Investment Casting Industry to Produce Sacrificial Patterns: Have All of the Available Options Been Considered?
Authors: Winter Daniel T.
Published: July 2010

The investment casting (IC) process has remained relatively unchanged throughout recorded history [1]. Indeed, there have been refinements to the individual processes required to produce castings; pattern making, the ceramic materials used to create the molds, foundry equipment, and post-cast processing of the castings. All have undergone numerous improvements. Yet, with the innovations that IC foundries have enjoyed, the casting process still requires a one-to-one ratio to produce an IC component from a sacrificial master pattern [2]. It is the alternative technologies used to produce the sacrificial master pattern that are the focus of this paper. New product development and introduction (NPDI) of an IC component requires the expertise of several engineering disciplines. Communications between the various engineering disciplines are critical to assure that cost effective decisions during product design and process development are made. With that in mind, several of the options available to the engineering teams responsible for NPDI are explored in the subsequent text. Rapid prototype (RP) technologies have played a crucial role during NPDI. The patterns produced using the various forms of RP technologies have allowed the IC industry to be considered by design engineers as a viable manufacturing option for their products. A more recent development in the United States is rapid tooling (RT). RT is an additive process, using metal to build the individual layers of the cavity block or the cavity insert. The finished component can then be used as a wax pattern cavity. Engineering teams should also consider a combination of RT and traditional wax pattern tooling. The complex pattern cavities are produced using RT while the more simple components are produced using traditional tooling. For IC foundries to be competitive all available pattern making options must be considered. Multi-disciplined engineering teams will play an important role choosing the technologies discussed. With careful consideration, the engineering teams responsible for NPDI can ensure the future of the IC industry.
Numerical Modeling of Pulverized Charcoal and Hot Oxygen Co-Injection in a Blast Furnace Tuyere
Authors: Wu Bin , Roesel Tom , Zhou Chenn Q.
Published: July 2010

Pulverized Coal Injection (PCI) is a technique that has been applied to blast furnaces over the last several decades. Due to the increased fuel economy seen in furnaces utilizing pulverized coal technology, several companies are in the process of developing a new hot oxygen injection technology in a charcoal based blast furnace. Computational Fluid Dynamics (CFD) has been employed to obtain a better understanding of this new technique. In the current study, a 3D multiphase reacting CFD model had been developed to evaluate the utilization of a very high temperature, high velocity stream of oxygen in a PCI tuyere. With the use of hot oxygen, both the injection and combustion rates of the charcoal fines in the blast furnace tuyere have been dramatically increased and contribute to the reduction of the lump charcoal consumption rate of the furnace. Parametric studies of different oxygen and PCI lance locations have also been conducted. The simulation results provide insight into optimization of the lance configuration to achieve maximum combustion performance and avoid overheating of the tuyere.
A Generic Formulaic Characterization of the Time to Reverse Engineer the Tolerances of a Product
Authors: Curtis Shane K. , Harston Stephen P. , Mattson Christopher A.
Published: July 2010

Reverse engineering is the process of extracting information about a product from the product itself. An estimate of the barrier and time to extract information from any product is useful for the original designer and those reverse engineering, as both are affected by reverse engineering activities. The authors have previously presented a set of metrics and parameters to estimate the barrier and time for product reverse engineering. This work has laid the foundation for the developments of the current paper, which address the issue of tolerance extraction during reverse engineering. Under the developments presented herein, measurement and statistical analysis of the variation between multiple samples of a product are required to reverse engineer its tolerances. When reconstruction is the reason reverse engineering activities are carried out, this level of reverse engineering can be critical, as tolerances ensure that products function properly and consistently. In this paper, we introduce metrics that (i) characterize how the flow of information from a product during reverse engineering changes as additional product samples are evaluated, and (ii) estimate the total barrier and time to reverse engineer the tolerances of a product. Additionally, a simple example is introduced to illustrate how to use the newly developed metrics and to serve as empirical validation.
Resistance Spot Welding (RSW) Evaluation of Uncoated 1.7 mm Direct Strip Production Complex 700B (DSPC) Steel for Automotive Body Structural Applications
Authors: Koganti Ramakrishna , Maatz Don
Published: July 2010

There has been a substantial increase in the use of advanced high strength steel (AHSS) in automotive structures in the last few years. The usage of these materials is projected to grow significantly in the next 5–10 years with the introduction of new safety and fuel economy regulations. AHSS are gaining popularity due to their superior mechanical properties and use in parts for weight savings potential, as compared to mild steels. These new materials pose significant manufacturing challenges, particularly for welding and stamping. Proper understanding of the weldability of these materials is critical for successful application on future vehicle programs. Due to the high strength nature of AHSS materials, higher weld forces and longer weld times are often needed to weld these advanced steels. In this paper, the weld current lobes, mechanical properties (shear tension and coach peel), metallographic cross-section and microhardness profile of uncoated Direct Strip Production Complex 700B (DSPC 700B) 1.7 mm steel welded to itself in a two-metal stackup are discussed. Weld lobes were developed with Medium Frequency Direct Current (MFDC) equipment, ISO-type B20 tips, weld force of 5.78 kN and hold time of 5 cycles. The weld times were varied at 19, 24 and 29 cycles, with each producing current ranges at or below 4.0 kA. Tensile shear and coach peel samples were made at weld time of 19 cycles, with samples showing average loads of 18.38 kN and 3.14 kN respectively. Also, microhardness assessment using metallographic cross-sections were analyzed at three different weld cycles (19, 24 and 29 cycles). Similar heat affected zones (HAZ) and weld zones were observed for three different weld cycles.
Gas Metal Arc Welding (GMAW) Process Optimization of 1.5 mm Uncoated Dual Phase 780 (DP780) Joint for Automotive Body Structural Applications
Authors: Koganti Ramakrishna , Jiang Cindy , Karas Chris
Published: July 2010

With the increasing demand for safety, energy saving and emission reduction, Advanced High Strength Steels (AHSS) have become very attractive steels for automobile makers. The usage of AHSS steels is projected to grow significantly in the next 5–10 years with new safety and fuel economy regulations. These new steels have significant manufacturing challenges, particularly for welding and stamping. Welding of AHSS remains one of the technical challenges in the successful application of AHSS in automobile structures due to heat-affected zone (HAZ) at the weld joint. In this study Gas Metal Arc Welding (GMAW) of a lap joint configuration consisting of 1.5 mm uncoated DP780 to itself was investigated. The objective of the study was to understand the wire feed rate (WFR) and torch (or robot) speed (TS) influence on lap joint tensile strength (static and fatigue). A two factor, two level, full factorial design of experiment (DOE) was conducted to understand the wire feed and torch speed influence on tensile and fatigue strength of the welded joints. In order to understand the curvature effect, center point was also considered in the experiment. Based on the statistical analysis both factors are significant on static tensile strength and two way interaction between wire feed rate and torch speed was also significant on static tensile strength. Wire feed rate was the common significant factor on all three fatigue load conditions (1200 lbf, 1500 lbf and 1900 lbf). Metallurgical properties of the lap joints were evaluated using optical microscopy. Significant hardness drop of 25% was observed at the HAZ. To understand the influence of weld parameters and weld geometry on mechanical properties, correlation analysis was conducted among weld heat input parameters, weld geometry and mechanical properties (both static and fatigue loads).
Resistance Spot Welding Evaluation of Complex Phase 780 (CP780) Steel for Automotive Body Structural Applications
Authors: Koganti Ramakrishna , Orsette Charles
Published: July 2010

The usage of advanced high strength steel (AHSS) in automotive body structures is projected to grow significantly in the next 5–10 years with the introduction of new safety and fuel economy regulations. This is due to their superior mechanical properties and weight savings potential. These new materials pose significant manufacturing challenges, particularly for welding and stamping. Due to the high strength nature of AHSS materials, higher weld forces and longer weld times are often needed to weld these advanced steels. In this paper, the weldability of Complex Phase 780 (CP780) is discussed in terms of weld current lobes, mechanical properties (shear and cross tension), metallographic cross-section and micro-hardness profile of 1.4 mm electro-galvanized (EG) steel welded to itself in a two-metal stack-up Weld lobes and a full factorial Design of Experiment (DOE) was conducted.
Laser Hybrid Welding of Uncoated Boron Steel for Automotive Body Construction
Authors: Koganti Ramakrishna , Joaquin Armando , Kretzschmar Thomas , Bratt Craig , Stiles Eric
Published: July 2010

The automotive industry is in constant pursuit of alternative materials and processes to address the ever changing needs of their customer and the environment. This paper presents findings from a study using a laser hybrid process (laser with Gas Metal Arc Welding) to join uncoated boron steel. In order to understand the affect from laser hybrid joining process, bead on plate experiments were conducted using 1.0 mm, 1.5 mm and 2.0 mm thick coupons. Further, one lap joint configuration was also investigated using the 1.5 mm thick coupons. Based on the test results, a significant reduction in tensile strength was observed at the Heat Affected Zone (HAZ).
Structural Optimization Techniques for Developing Efficient Lightweight Vehicles and Components
Authors: Mohan Iyengar Raj , Laxman Srinivasan , Morgans Shawn , Koganti Ramakrishna
Published: July 2010

Developing automotive vehicles and components to achieve light-weight designs and to meet design targets on structural stiffness, modal frequencies, durability, and crashworthiness, can no longer be driven by a “trial-and-error” strategy. Structural optimization tools provide the necessary analyses during the initial stages of vehicle development to arrive at the most efficient and effective designs. In this paper, we illustrate the importance of topological and gage optimization in achieving mass reduction without compromising on the structural requirements through two design examples.
Domain dynamics in epitaxial Pb(Zr0.2Ti0.8)O3 films studied by piezoelectric force microscopy
Authors: Klein Levente J. , Dubourdieu Catherine , Frank Martin M. , Hoffman Jason , Reiner James W. , Ahn Charles H.
Published: July 2010

Ferroelastic 90° a-domain dynamics in epitaxial Pb(Zr0.2Ti0.8)O3 (lead zirconium titanate, PZT) films is investigated using lateral and vertical piezoelectric force microscopies. The PZT film consists of out-of-plane polarized c-domains separated by a cross-hatch network of in-plane polarized a-domains. When an electric field is applied across the PZT film, the a-domains are rearranged on the nanoscale. Depending on the direction of the electric field, this results either in larger or more fragmented c-domains. Both lateral and vertical piezoelectric force signals demonstrate the importance of ferroelastic a-domain movement since their dynamics will determine the final polarization retention time and stability of ferroelectric capacitors.
Impact of metal etch residues on etch species density and uniformity
Authors: Dictus Dries , Shamiryan Denis , Paraschiv Vasile , Boullart Werner , De Gendt Stefan , Vinckier Chris
Published: July 2010

Uniformity and wafer-to-wafer reproducibility of plasma etch processes are often related to the conditioning of the plasma etch chamber walls. For advanced complementary metal-oxide semiconductor fabrication, numerous metals are used which might deposit on the chamber walls during etch processes and as these metals are not always straightforward to remove, process instabilities can occur. This happens because recombination of atomic species on the chamber walls determines to a certain degree the plasma composition. Therefore, in this article, the impact of metal etch residues, especially titanium and tantalum residues, on plasma composition and uniformity is studied. The chamber walls are analyzed by x-ray photoelectron spectroscopy analysis of so-called floating samples and the densities of Cl, Br, O and F in Cl2, HBr, O2, and SF6 plasmas are monitored by optical emission spectroscopy. Plasma uniformity is checked by measuring etch rates across 300 mm silicon wafers. It is found that chlorine and bromine have similar recombination probabilities on the metals than on anodized aluminum. Fluorine and oxygen recombination, however, is strongly influenced by the presence of metal residues. Accordingly, for fluorine and oxygen based plasmas, metal residues showed to have an impact on the plasma uniformity.
Alcohol Takes Its Toll on the Brain
Authors: Minzenberg Michael J.
Published: July 2010

Alcohol abuse activates inflammatory responses via Toll-like receptors in the brain, potentially leading to irreversible cognitive changes.
Science Translational Medicine
Podcast: 7 July 2010
Authors: Broder Samuel , VanHook Annalisa M.
Published: July 2010

A conversation with Samuel Broder about early discoveries in HIV-1/AIDS research and strategies for successful translational research in the future.
Multicenter Study for Defining the Breakpoint for Rifampin Resistance in
Neisseria meningitidis
by
rpoB
Sequencing
Authors: Taha Muhamed-Kheir , Thulin Hedberg Sara , Szatanik Marek , Hong Eva , Ruckly Corinne , Abad Raquel , Bertrand Sophie , Carion Francoise , Claus Heike , Corso Alejandra , Enríquez Rocío , Heuberger Sigrid , Hryniewicz Waleria , Jolley Keith A. , Kriz Paula , Mollerach Marta , Musilek Martin , Neri Arianna , Olcén Per , Pana Marina , Skoczynska Anna , Sorhouet Pereira Cecilia , Stefanelli Paola , Tzanakaki Georgina , Unemo Magnus , Vázquez Julio A. , Vogel Ulrich , Wasko Izabela
Published: July 2010

Identification of clinical isolates of
Neisseria meningitidis
that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (
n
= 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of ≥0.25 mg/liter (
n
= 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of <0.25 mg/liter were also examined (
n
= 231). Typing of isolates was performed, and a 660-bp DNA fragment of the
rpoB
gene was sequenced. Sequences differing by at least one nucleotide were defined as unique
rpoB
alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed
rpoB
alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of ≤1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of ≤1 mg/liter. The
rpoB
allele sequence and isolate data have been incorporated into the PubMLST
Neisseria
database (
http://pubmlst.org/neisseria/
). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.
Characterization of Dengue Virus Resistance to Brequinar in Cell Culture
Authors: Qing Min , Zou Gang , Wang Qing-Yin , Xu Hao Ying , Dong Hongping , Yuan Zhiming , Shi Pei-Yong
Published: July 2010

Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for
de novo
pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.
Screen for Inducers of Autolysis in
Bacillus subtilis
Authors: Falk Shaun P. , Noah James W. , Weisblum Bernard
Published: July 2010

We describe a primary high-throughput screen that uses the reporter strain
Bacillus subtilis
BAU-102 to identify antibiotics that induce autolysis. The screen measures autolysis in terms of the incipient release of recombinant
Escherichia coli
β-galactosidase (β-Gal) from the periplasmic space of
B. subtilis
owing to a loss of integrity of the cell wall. In a model screen, β-Gal release values for 79 members of a library consisting of antibiotics and related compounds were collected, sorted, and plotted as a function of rank. Inducers of autolysis, which included compounds that inhibit cell wall synthesis and those that do not, were readily differentiated from other members of the library on the basis of their elevated β-galactosidase release responses. The results of the BAU-102 model screen called attention to the antibacterial activity of drugs normally used in other applications, describable as “repurposed.” Thus, the screen independently identified the potential antibacterial properties of the antifungal drug miconazole and of the antileishmaniasis drug miltefosine. Daptomycin-induced release of β-Gal was also detected and occurred in a Ca
2+
-dependent manner.
Unpredictable Effects of Rifampin as an Adjunctive Agent in Elimination of Rifampin-Susceptible and -Resistant
Staphylococcus aureus
Strains Grown in Biofilms
Authors: Croes Sander , Beisser Patrick S. , Neef Cees , Bruggeman Cathrien A. , Stobberingh Ellen E.
Published: July 2010

The use of rifampin as an adjunct in biofilm-associated infections is based on the ability to penetrate into biofilms and a presumed activity against dormant bacteria. Yet, its efficacy remains contradictory, and rifampin-resistant strains frequently emerge during therapy. Therefore, the efficacy against rifampin-susceptible and isogenic rifampin-resistant methicillin-susceptible
Staphylococcus aureus
(MSSA) strains was evaluated. Biofilms were generated under static conditions using MSSA with various genetic backgrounds. Oxacillin alone or with rifampin at various concentrations was subsequently added, and after 24 h biomass and viable cell counts were determined. Upon rifampin addition, interstrain variations in viable count change, ranging from a tendency toward antagonism to synergy, were observed among all strains tested, irrespective of the genetic background of the strain. Similar variations were observed in changes in biomass. The decrease in viable count upon rifampin addition was negatively correlated to formation of large amounts of biomass, since strains embedded by more biomass showed a diminished reduction in viable count. Rifampin (1 μg/ml) as adjunct to oxacillin achieved greater reductions in biomass produced by most rifampin-susceptible isolates, ranging from 17 to 54%, compared to 4% for oxacillin alone. In contrast, rifampin had no additional value in reduction of biomass of isogenic rifampin-resistant mutants. At subinhibitory concentrations of rifampin (0.008 μg/ml), none of the strains tested yielded an extra reduction in biomass that was ≥40%. In conclusion, the effects of rifampin as adjunct on biomass and viable count were unpredictable, and the use of rifampin against biofilm containing rifampin-resistant strains seems unwarranted.
Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to
Toxoplasma gondii
Authors: Fox Barbara A. , Bzik David J.
Published: July 2010

The orotidine-5′-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the
de novo
pyrimidine biosynthesis pathway, was deleted using
Toxoplasma gondii
KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting ΔOMPDC, ΔUP, and ΔOMPDC ΔUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ΔUP knockout strain was replication competent and virulent. In contrast, the ΔOMPDC and ΔOMPDC ΔUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ΔOMPDC strain but not the ΔOMPDC ΔUP strain was also partially rescued
in vitro
with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ΔOMPDC and ΔOMPDC ΔUP knockout strains exhibited extreme attenuation in murine virulence (∼8 logs). Genetic complementation of the ΔOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated ΔOMPDC strain or the ΔOMPDC ΔUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ΔOMPDC and ΔOMPDC ΔUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.
Immunization with a Combination of Integral Chlamydial Antigens and a Defined Secreted Protein Induces Robust Immunity against Genital Chlamydial Challenge
Authors: Li Weidang , Murthy Ashlesh K. , Guentzel M. Neal , Chambers James P. , Forsthuber Thomas G. , Seshu J. , Zhong Guangming , Arulanandam Bernard P.
Published: July 2010

We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF; a secreted chlamydial protein) in inducing antigen-specific CD4+T cell/gamma interferon (IFN-γ)-mediated but not antibody-mediated chlamydial clearance and reduction of upper genital tract (UGT) pathological sequelae. Since chlamydial integral antigens may induce neutralizing antibody protection, we further evaluated induction of protective immunity using a combination of rCPAF and UV-inactivated chlamydial elementary bodies (UV-EB) against vaginal chlamydial challenge in comparison to immunization with the individual components or live EB. The rCPAF-UV-EB immunization induced a significantly enhanced anti-UV-EB cellular and antibody response and a reduced anti-CPAF cellular and antibody response, compared to immunization with the respective individual components. Moreover, vaccination with UV-EB and rCPAF-UV-EB induced serum antibodies that neutralized chlamydial infectivity. The rCPAF-UV-EB immunization resulted in a significant reduction of vaginal chlamydial shedding and induced earlier bacterial clearance than vaccination of mice with the individual components. Importantly, the UGT sequelae were significantly reduced in mice immunized with rCPAF or rCPAF-UV-EB, but not in those immunized with UV-EB alone, and approached the levels of protection induced by live EB. These results collectively suggest that a combination of neutralizing antibodies induced by integral chlamydial antigens and cell-mediated responses induced by secreted proteins such as CPAF induces optimal protective immunity against genital chlamydial infections.
Activation of the CpxRA System by Deletion of
cpxA
Impairs the Ability of
Haemophilus ducreyi
To Infect Humans
Authors: Spinola Stanley M. , Fortney Kate R. , Baker Beth , Janowicz Diane M. , Zwickl Beth , Katz Barry P. , Blick Robert J. , Munson Robert S.
Published: July 2010

Haemophilus ducreyi
must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the
H. ducreyi
genome and negatively regulates the
lspB-lspA2
operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame
H. ducreyi cpxA
deletion mutant, 35000HPΔ
cpxA
. In human inoculation experiments, 35000HPΔ
cpxA
formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the
lspB-lspA2
promoter was downregulated in 35000HPΔ
cpxA
, confirming that CpxR was activated. Deletion of
cpxA
downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in
trans
restored parental phenotypes. 35000HPΔ
cpxA
is the first
H. ducreyi
mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.
The Lung Is an Important Site for Priming CD4 T-Cell-Mediated Protective Immunity against Gastrointestinal Helminth Parasites
Authors: Harvie Marina , Camberis Mali , Tang Shiau-Choot , Delahunt Brett , Paul William , Le Gros Graham
Published: July 2010

The rodent hookwormNippostrongylus brasiliensistypically infects its host by penetrating the skin and rapidly migrating to the lungs and gut. Following primary infection, immunocompetent mice become highly protected from reinfection withN. brasiliensis, with the numbers of worms gaining access to the lungs and gut being reduced by up to 90%. We used green fluorescent protein/interleukin-4 (IL-4) reporter mice and truncated infection studies to identify both the tissue site and mechanism(s) by which the host protects itself from reinfection withN. brasiliensis. Strikingly, we demonstrated that the lung is an important site for priming immune protection. Furthermore, a lung-initiated, CD4 T-cell-dependent, and IL-4- and STAT6-dependent response was sufficient to confer protection against reinfection. In conclusion, vaccination strategies which seek to break the cycle of reinfection and egg production by helminths such as hookworms can include strategies which directly stimulate Th2 responses in the lung.
Anisakis pegreffii-Induced Airway Hyperresponsiveness Is Mediated by Gamma Interferon in the Absence of Interleukin-4 Receptor Alpha Responsiveness
Authors: Kirstein Frank , Horsnell William G. C. , Nieuwenhuizen Natalie , Ryffel Bernhard , Lopata Andreas L. , Brombacher Frank
Published: July 2010

Infection with the fish parasiteAnisakisfollowing exposure to contaminated fish can lead to allergic reactions in humans. The present study examined the immunological mechanisms underlying the development of allergic airway inflammation in mice after different routes of sensitization toAnisakis. Wild-type and interleukin-4 receptor alpha (IL-4Rα)-deficient BALB/c mice were sensitized intraperitoneally with live or heat-killedAnisakislarvae or by intranasal administration of anAnisakisextract and were subsequently challenged intranasally with anAnisakisextract. Both routes of sensitization induced IL-4Rα-dependent allergic airway responses, whereas allergen-specific antibody responses developed only when mice were sensitized intraperitoneally. Intranasal sensitization induced airway hyperresponsiveness (AHR) in wild-type mice only, showing that AHR was IL-4/IL-13 dependent. Unexpectedly, infection withAnisakislarvae induced AHR in both wild-type and IL-4Rα-deficient mice. IL-4Rα-independent AHR was mediated by gamma interferon (IFN-γ), as evidenced by the fact thatin vivoneutralization of IFN-γ abrogated AHR. Together, these results demonstrate that both infection with larvae and inhalational exposure toAnisakisproteins are potent routes of allergic sensitization toAnisakis, explaining food- and work-related allergies in humans. Importantly for diagnosis, allergic airway inflammation can be independent of detectableAnisakis-specific antibodies. Moreover, depending on the route of sensitization, AHR can be induced either by IL-4/IL-13 or by IFN-γ.
Indirect Pathogenicity of
Haemophilus influenzae
and
Moraxella catarrhalis
in Polymicrobial Otitis Media Occurs via Interspecies Quorum Signaling
Authors: Armbruster Chelsie E. , Hong Wenzhou , Pang Bing , Weimer Kristin E. D. , Juneau Richard A. , Turner James , Swords W. Edward
Published: July 2010

Otitis media (OM) is among the leading diseases of childhood and is caused by opportunists that reside within the nasopharynx, such as
Haemophilus influenzae
and
Moraxella catarrhalis
. As with most airway infections, it is now clear that OM infections involve multiple organisms. This study addresses the hypothesis that polymicrobial infection alters the course, severity, and/or treatability of OM disease. The results clearly show that coinfection with
H.
 
influenzae
and
M. catarrhalis
promotes the increased resistance of biofilms to antibiotics and host clearance. Using
H. influenzae
mutants with known biofilm defects, these phenotypes were shown to relate to biofilm maturation and autoinducer-2 (AI-2) quorum signaling. In support of the latter mechanism, chemically synthesized AI-2 (dihydroxypentanedione [DPD]) promoted increased
M. catarrhalis
biofilm formation and resistance to antibiotics. In the chinchilla infection model of OM, polymicrobial infection promoted
M. catarrhalis
persistence beyond the levels seen in animals infected with
M. catarrhalis
alone. Notably, no such enhancement of
M. catarrhalis
persistence was observed in animals infected with
M.
 
catarrhalis
and a quorum signaling-deficient
H. influenzae luxS
mutant strain. We thus conclude that
H. influenzae
promotes
M. catarrhalis
persistence within polymicrobial biofilms via interspecies quorum signaling. AI-2 may therefore represent an ideal target for disruption of chronic polymicrobial infections. Moreover, these results strongly imply that successful vaccination against the unencapsulated
H. influenzae
strains that cause airway infections may also significantly impact chronic
M. catarrhalis
disease by removing a reservoir of the AI-2 signal that promotes
M. catarrhalis
persistence within biofilm.

IMPORTANCE
Otitis media (OM) is one of the most common childhood infections and is a leading reason for antibiotic prescriptions to children. Chronic and recurrent OM involves persistence of bacteria within biofilm communities, a state in which they are highly resistant to immune clearance and antibiotic treatment. While it is clear that most of these infections involve multiple species, the vast majority of knowledge about OM infections has been derived from work involving single bacterial species. There is a pressing need for better understanding of the impact of polymicrobial infection on the course, severity, and treatability of OM disease. In this study, we show that communication between bacterial species promotes bacterial persistence and resistance to antibiotics, which are important considerations in the diagnosis, prevention, and treatment of OM. Moreover, the results of this study indicate that successful preventive measures against
H. influenzae
could reduce the levels of disease caused by
M. catarrhalis
.