bluuebunny/crossref_metadata_2025 huggingface dataset:
Buoyancy-Driven Flow in Two Interconnected Rooms: Effects of the Exterior Vent Location and Size
Authors: Tovar R. , Campo Garrido C. A. , Linden P. F. , Thomas L. P.
Published: April 2009

This paper describes scale-model laboratory experiments with salt-bath simulations, on the flow and stratification in two coupled rooms connected to the exterior by a single vent. The two rooms are connected by two openings one at high-level and one at low-level in the dividing wall. One “forced” room has a buoyancy source, while the outlet vent is in the “unforced” room and is placed in such a way that the buoyancy in the room can potentially drive an exchange flow through it. The buoyancy source is also a source of volume flux, such as cool air pumped into a room from an overhead duct, in which case the outlet is on the floor of the unforced room. We consider the effect of the size of the outlet vent on the resulting stratification in the two rooms. For a small vent only unidirectional flow occurs, and since no ambient fluid enters the rooms, the buoyancy in both rooms becomes uniform and approaches asymptotically to the buoyancy of the source. Above a critical vent size a bidirectional flow is driven through the vent by the buoyancy forces. In this case ambient fluid enters the rooms and a steady state is reached when the buoyancy flux through the outlet vent equals that of the buoyancy source. Both rooms remain stratified with two-layer weak stratification in this case, with a mean density that decreases as the size of the outlet increases. The implications for ventilation are discussed.
Characterization of a Thermosyphon Heat Exchanger for Solar Domestic Hot Water Systems
Authors: Cruickshank Cynthia A. , Harrison Stephen J.
Published: April 2009

This paper presents a simplified test method that was developed to allow preconfigured solar domestic hot water systems that use natural convection/thermosyphon heat exchangers to be characterized. The results of this test method produce performance coefficients for simple empirical expressions that describe the fluid flow and heat transfer in the heat-exchange loop. These empirically derived coefficients can be used as an input to a general simulation routine that allows overall system performance to be determined for various loads and climatic conditions. To illustrate the test procedure, results are presented for a typical heat exchanger under a range of operational conditions.
Study on Curriculum of Network Agile Manufacturing
Authors: Zhou Jiehan , Xiong Guangleng , Wu Bo , Yu Changrong
Published: April 2009

Network Agile Manufacturing (NAM) is a new manufacturing mode, which is adapted to the social development of 21st century. Manufacturing business based on NAM is characterized by virtual organization, accumulation innovation, interactive innovation, resource global deployment, data-process integrated management for Product Life Cycle (PLC). The implementation of NAM needs comprehensive technicians holding manufacturing, management, information technology. Realizing the importance of cultivating the qualified technicians, Tsinghua University in China is starting the project named Curriculum of NAM. An attempt has been made in this paper to review the main literature available on Curriculum of NAM with the objective to: (i) retrace manufacturing informatization progress, identify key strategy and techniques of NAM. Here, informatization means computerization to a great extend, (ii) study the fundamental technologies of impacting NAM, including PDM, ERP, and CSM, (iii) introduce the concept and framework of NAM, suggest some future research directions and (iv) summarize the basic curriculum of NAM by analyzing the keywords appearing in theses of Tsinghua University NAM’2001 symposium.
Embedding Real Engineering Decisions and Sometimes-Consequent Errors in a Small Design Project Toward Reflective Learning
Authors: Fujita Kikuo
Published: April 2009

This paper reports an intention and experiences in introducing a project-based design class. While project-based learning is a widely recognized framework for design education, its methodology is still under discussion. Department of Mechanical Engineering at Osaka University starts a new design class for junior students under the demands arisen for Japanese universities and by focusing a latest meaning of knowledge process. The class is characterized by the possibility of errors consequent on the students’ decisions in system-level design. It aims to make the students learn some design paradigms through reflection of events in live situation. For this direction, the students solve a peculiarly simplified but contextually rich design problem by executing all phases of the entire design process, produce hardware, use them, and do post-analysis in two months. The project’s mission and associated schedule are arranged for students to implicitly confront tradeoffs across engineering disciplines and the entire engineering process. After the class it is observed that the students began to learn the naiveness of individual knowledge in system-level design, how it occurs, how it can be avoided, and so forth from their own experiences. The paper is concluded with discussion on the prospects in this type of project-based design education.
Crossing the ’63 Riviera With a Concept Cielo: Capturing, Representing and Generating the Buick Brand
Authors: McCormack Jay P. , Cagan Jonathan , Vogel Craig M.
Published: April 2009

Developing and maintaining a consistent brand statement is an important aspect of developing a successful product. However, maintaining that statement is difficult due in part to the inconsistent and often insufficient understanding of brand by marketing, engineering, and design. This paper presents shape grammars as a method for encoding the key elements of a brand into a repeatable language, which can be used to generate products consistent with the brand. A detailed investigation into the history of Buick styling reveals the brand characteristics of the front view of Buick vehicles, which are then captured in a shape grammar.
A Direct Synthesis Method for Early Stage Parametric Design of Large-Scale Mechanical Systems
Authors: Ge Ping , Lu Stephen C.-Y. , Bukkapatnam Satish T. S.
Published: April 2009

Design productivity plays a crucial role in the development of large-scale mechanical systems because of the involvement of a great amount of resources, long life cycle, and multi-disciplinary teams. At the early stage parametric design, the generation and evaluation of robust design alternatives are expected at the least expense. In this paper, a direct synthesis (DS) method is presented. With simultaneous consideration of negotiability, bi-directional robustness, and efficiency in a “Zoom-in” process, the DS can facilitate the quick synthesis of robust design alternatives without resorting to numerous iterations. The preliminary industrial application results of the Direct Synthesis method show its effectiveness for robustness enhancement and cost reduction at the early stage parametric design of a vehicle structural system.
Guidelines for Product Evolution Using Effort Flow Analysis: Results of an Empirical Study
Authors: Greer James L. , Wood John J. , Jensen Daniel D. , Wood Kristin L.
Published: April 2009

The results of an empirical product study aimed at deducing product evolution design guidelines are presented. The derived design guidelines support a directed product evolution methodology known as effort flow analysis. Effort flow analysis provides a systematic framework for identifying component combination opportunities leading to either rigid-body or compliant mechanisms in the domain of mechanical effort transmissions. Design guidelines, effort flow analysis, and the empirical study are discussed. A classified list of the derived guidelines is presented along with analysis of a sample product group from the study.
Improving Turbomachinery Design Process Management
Authors: Jarrett Jerome P. , Clarkson P. John , Dawes William N.
Published: April 2009

It is widely acknowledged that a company’s ability to aquire market share, and hence its profitability, is very closely linked to the speed with which it can produce a new design. Indeed, a study by the U.K. Department of Trade and Industry has shown that the critical factor which determines profitability is the timely delivery of the new product. Late entry to market or high production costs dramatically reduce profits whilst an overrun on development cost has little significant effect. This paper describes a method which aims to assist the designer in producing higher performance turbomachinery designs more quickly by accelerating the process by which they are produced. The adopted approach combines an enhanced version of the ‘Signposting’ design process management methodology [1] with industry-standard analysis codes and Computational Fluid Dynamics (CFD). It has been specifically configured to enable process-wide iteration, near instantaneous generation of guidance data for the designer and fully automatic data handling. A successful laboratory experiment based on the design of a large High Pressure Steam Turbine is described and this leads on to current work which incorporates the extension of the proven concept to a full industrial application for the design of Aeroengine Compressors with Rolls-Royce plc.
Incorporating Uncertainty in Diagnostic Analysis of Mechanical Systems
Authors: Mocko Gregory , Paasch Robert
Published: April 2009

The increase in complexity of modern mechanical systems can often lead to systems that are difficult to diagnose, and therefore require a great deal of time and money to return to a normal operating condition. Analyzing mechanical systems during the product development stages can lead to systems optimized in the area of diagnosability, and therefore to a reduction of life cycle costs for both consumers and manufacturers and an increase in the useable life of the system. A methodology for diagnostic evaluation of mechanical systems incorporating indication uncertainty is presented. First, Bayes formula is used in conjunction with information extracted from the Failure Modes and Effects Analysis (FMEA), Fault Tree Analysis (FTA), component reliability, and prior system knowledge to construct the Component-Indication Joint Probability Matrix (CIJPM). The CIJPM, which consists of joint probabilities of all mutually exclusive diagnostic events, provides a diagnostic model of the system. The Replacement Matrix is constructed by applying a predetermined replacement criterion to the CIJPM. Diagnosability metrics are extracted from a Replacement Probability Matrix, computed by multiplying the transpose of the Replacement Matrix by the CIJPM. These metrics are useful for comparing alternative designs and addressing diagnostic problems of the system, to the component and indication level. Additionally, the metrics can be used to predict cost associated with fault isolation over the life cycle of the system.
A Framework for Creating a Function-Based Design Tool for Failure Mode Identification
Authors: Arunajadai Srikesh G. , Stone Robert B. , Tumer Irem Y.
Published: April 2009

Knowledge of potential failure modes during design is critical for prevention of failures. Currently industries use procedures such as Failure Modes and Effects Analysis (FMEA), Fault Tree analysis, or Failure Modes, Effects and Criticality analysis (FMECA), as well as knowledge and experience, to determine potential failure modes. When new products are being developed there is often a lack of sufficient knowledge of potential failure mode and/or a lack of sufficient experience to identify all failure modes. This gives rise to a situation in which engineers are unable to extract maximum benefits from the above procedures. In this work we report on a new failure identification scheme and integrate it with a function-based failure identification methodology, which would act as a storehouse of information and experience, providing useful information about the potential failure modes for the design under consideration, as well as enhancing the usefulness of procedures like FMEA. As an example, the method is applied to 41 products and the benefits are illustrated.
Functional Modeling, Reverse Engineering, and Design Reuse
Authors: Gietka Paul , Verma Manish , Wood William H.
Published: April 2009

Experience is a tremendous asset for any designer. To leverage the experience of many designers, a general methodology for case-based functional design is required. Function-based design is a natural foundation for this methodology because its goal is to structure the solution space and support concept generation. Gaining access to experience about how functions combine will help designers to explore more, better design concepts. This experience is gleaned by reverse engineering existing products and storing and indexing the information gained. This work studies the preliminary steps in matching functional information derived from reverse engineering to that generated in the design process. A language of function developed for reverse engineering is tested in the context of design.
Viewing Product Development as a Decision Production System
Authors: Herrmann Jeffrey W. , Schmidt Linda C.
Published: April 2009

Product development includes many different types of decision-making by engineers and managers. Design decisions determine the product form and specify the manufacturing processes to be used. Development decisions control the progress of product development projects by specifying which activities should happen, their sequence, and who should perform them. This paper introduces the concept of a decision production system to describe a product development organization as a system of decision-makers who use and create information to develop a product. This perspective does not advocate any particular type of product development process. Instead, it looks at the organization in which the product development process exists and considers the decision-makers as a manufacturing system that can be viewed separately from the organization structure.
Implementation of DFE in the Daily Design Work: An Approach Derived From Surveys
Authors: Ernzer Marc , Gru¨ner Chris , Birkhofer Herbert
Published: April 2009

In this paper, three surveys were carried out at the Darmstadt University of Technology to reveal the problems regarding the implementation of DFE. It furthermore attempts to bridge the gap between approaches mainly proposed by researches from university and the current practice in industry. The first survey gives a good overview about current DFE practice in companies with certified environmental management systems (EMS). The second survey summarizes the general experience of consultants from university and industry, who are well-versed in implementing methods in industry. The third and last survey was carried out at ‘environmental champions’ and inspects the differences between the individual applications. With these three surveys as a background, an approach for successful implementation of DFE will be derived. Finally, the concept is divided into different levels of implementation, based on the prerequisites of the company and the effort the company is willing to invest. Therefore, the concept can be used by large companies, as well as by small- and medium-sized companies.
Kinematic Analysis of the Antikythera Gear Mechanism by Means of Graph Theory
Authors: Pennestri` Ettore , Valentini Pier Paolo , Petti Ferdinando
Published: April 2009

The Antikythera mechanism is one of the most extraordinary examples of the degree of sophistication reached by the Hellenistic culture in the field of mechanism design. This calendar mechanism not only witness the degree of astronomic knowledge of the greeks, but also their capability to conceive and build marvelous mechanical devices. The mechanism is the first documented example of differential gear arrangement in western culture. The paper gives first a description of the kinematic structure of the mechanism in terms by means of a graph, then a full kinematic analysis of this complex gear train is performed. The results of our kinematic analysis, only partially coincident with those obtained by De Solla Price, match very closely the discoveries made by the Greek astronomer Hipparcus about the duration of the lunar months. There is also a conjecture on the kinematic structure and gear dimensions of some of the missing parts. In the Appendix is reported the listing of the Maple program developed for the kinematic analysis.
The Culture of Design and Production in Abbasid Samarra
Authors: Al-Harithy Howayda Nawwaf
Published: April 2009

The paper discusses the culture of design produced by a particular patronage pattern of city building; the case study is that of Samarra in the 9th century. It investigates the process through which mechanical mass production of art transforms the Late Antique visual forms into new vocabulary that acquires universal popularity and new cultural significance.
Design for Cultural Difference
Authors: Smaili Ahmad
Published: April 2009

Design, the cross-fertilization of science and art, is a basic function of all species that occupy a masterfully orchestrated and designed ecosystem in which man is but one. On the other hand, culture with its complex mix is the expression of what a group of people creates — arts, beliefs customs, institutions, products and thought — at a particular time within the context of the natural environment. Design and culture therefore are intimately linked and undoubtedly influence each other. This suggests that designers, with their problem solving skills and keen interest to preserve nature and advance quality of life are capable of reshaping culture in a positive way. This paper is not intended to provide specific answers on how to achieve that, but it highlights some aspects of the design-culture interface and asserts that designers, armed with good will and respect for all have under their disposal a strong force by which they can help fashion a peaceful world. The paper also addresses possible things designers can do to influence that objective.
A Systematic Approach to the Reconstruction of Ancient Chinese Escapement Regulators
Authors: Yan Hong-Sen , Lin Tsung-Yi
Published: April 2009

It is generally believed that the first escapement regulator invented is the waterwheel steelyard-clepsydra device made in ancient China by Su Song during the Northern Song Dynasty (960~1,127 AD). The device, which was a unique feature of the ancient Chinese escapement regulators, was used in Su’s famous clock tower, Shui Yun Yi Xiang Tai (Water-Powered Armillary Sphere and Celestial Globe). Evidence found in certain historical literature, however, suggests that the astronomical clocks made before the Northern Song Dynasty were also equipped with escapement regulators. But due to insufficient literature on the specific design of the devices and the fact that none of the earlier escapement regulators have been recovered, it has been very difficult to recreate the original mechanism design. Therefore, in view of this problem, we wish to present a reconstruction design procedure for the ancient machinery in this paper. By combining the innovative mechanism design methodology with the mechanical evolution and variation theory, we can systematically recreate all feasible and appropriate designs that are consistent with the science theories and techniques of the subject time period. In this paper, the waterwheel steelyard-clepsydra device made by Su Song was adopted as the original design for the reconstruction of ancient Chinese escapement regulators. Utilizing the procedure proposed in this paper, the reconstruction designs we recreated included 12 six-bar and eight-joint waterwheel steelyard-clepsydra devices, among which 10 were with four-bar linkages and 2 were with rope-and-pulley mechanisms. These results can be further used in the study of ancient Chinese mechanical clocks, especially in reconstruction research.
A Study of the Effects of Culture on Refrigerator Design: Towards Design for Culture
Authors: Ellsworth Kyle , Magleby Spencer , Todd Robert
Published: April 2009

In designing products, the needs and values of customers in foreign countries differ as influenced by their respective cultures. The authors present a new aspect to be considered in designing: Design for Culture. A case study is presented of the effects culture has on the design of refrigerators in regions of the world including United States, Europe, Japan, and Developing Countries such as Brazil.
Electrical levels of defect investigation of ZrO2 thin film by spectroscopic ellipsometry
Authors: Chen Y. R. , Zhou P. , Li J. , Chen L. Y.
Published: April 2009

The electrical levels of defects of high-k dielectric ZrO2 films deposited with different oxygen fluxes have been investigated using x-ray diffraction, x-ray photoelectron spectroscopy, and spectroscopic ellipsometry. Various defects with the formation of interstitial oxygen and oxygen vacancies existed in monoclinic ZrO2 films. The optical properties of the films are strongly influenced by configuration changes in defects. Parameters were extracted from spectroscopic ellipsometry raw data by Lorentz oscillator model fitting that indicate the probabilities and transition energies for different charged oxygen interstitial and vacancy defects. These correspond to the electron affinity energies calculated by density functional theory and assigned to electrons trapped at different charged defects from the bottom of the conduction band. A nondestructive and noncontact solution is suggested for detecting the defect states and energy level of high-k film.
Strengthening Materials by Engineering Coherent Internal Boundaries at the Nanoscale
Authors: Lu K. , Lu L. , Suresh S.
Published: April 2009

Strengthening materials traditionally involves the controlled creation of internal defects and boundaries so as to obstruct dislocation motion. Such strategies invariably compromise ductility, the ability of the material to deform, stretch, or change shape permanently without breaking. Here, we outline an approach to optimize strength and ductility by identifying three essential structural characteristics for boundaries: coherency with surrounding matrix, thermal and mechanical stability, and smallest feature size finer than 100 nanometers. We assess current understanding of strengthening and propose a methodology for engineering coherent, nanoscale internal boundaries, specifically those involving nanoscale twin boundaries. Additionally, we discuss perspectives on strengthening and preserving ductility, along with potential applications for improving failure tolerance, electrical conductivity, and resistance to electromigration.
DNA Binding Site Sequence Directs Glucocorticoid Receptor Structure and Activity
Authors: Meijsing Sebastiaan H. , Pufall Miles A. , So Alex Y. , Bates Darren L. , Chen Lin , Yamamoto Keith R.
Published: April 2009

Genes are not simply turned on or off, but instead their expression is fine-tuned to meet the needs of a cell. How genes are modulated so precisely is not well understood. The glucocorticoid receptor (GR) regulates target genes by associating with specific DNA binding sites, the sequences of which differ between genes. Traditionally, these binding sites have been viewed only as docking sites. Using structural, biochemical, and cell-based assays, we show that GR binding sequences, differing by as little as a single base pair, differentially affect GR conformation and regulatory activity. We therefore propose that DNA is a sequence-specific allosteric ligand of GR that tailors the activity of the receptor toward specific target genes.
Protection of
C. elegans
from Anoxia by HYL-2 Ceramide Synthase
Authors: Menuz Vincent , Howell Kate S. , Gentina Sébastien , Epstein Sharon , Riezman Isabelle , Fornallaz-Mulhauser Monique , Hengartner Michael O. , Gomez Marie , Riezman Howard , Martinou Jean-Claude
Published: April 2009

Oxygen deprivation is rapidly deleterious for most organisms. However,
Caenorhabditis elegans
has developed the ability to survive anoxia for at least 48 hours. Mutations in the DAF-2/DAF-16 insulin-like signaling pathway promote such survival. We describe a pathway involving the HYL-2 ceramide synthase that acts independently of DAF-2. Loss of the ceramide synthase gene
hyl-2
results in increased sensitivity of
C. elegans
to anoxia.
C. elegans
has two ceramide synthases,
hyl-1
and
hyl-2
, that participate in ceramide biogenesis and affect its ability to survive anoxic conditions. In contrast to
hyl-2(lf)
mutants,
hyl-1(lf)
mutants are more resistant to anoxia than normal animals. HYL-1 and HYL-2 have complementary specificities for fatty acyl chains. These data indicate that specific ceramides produced by HYL-2 confer resistance to anoxia.
Origin and Radiation of the Earliest Vascular Land Plants
Authors: Steemans Philippe , Hérissé Alain Le , Melvin John , Miller Merrell A. , Paris Florentin , Verniers Jacques , Wellman Charles H.
Published: April 2009

Colonization of the land by plants most likely occurred in a stepwise fashion starting in the Mid-Ordovician. The earliest flora of bryophyte-like plants appears to have been cosmopolitan and dominated the planet, relatively unchanged, for some 30 million years. It is represented by fossilized dispersed cryptospores and fragmentary plant remains. In the Early Silurian, cryptospore abundance and diversity diminished abruptly as trilete spores appeared, became abundant, and underwent rapid diversification. This change coincides approximately with the appearance of vascular plant megafossils and probably represents the origin and adaptive radiation of vascular plants. We have obtained a diverse trilete spore occurrence from the Late Ordovician that suggests that vascular plants originated and diversified earlier than previously hypothesized, in Gondwana, before migrating elsewhere and secondarily diversifying.
Growing Up Connected
Authors: Preece Jennifer
Published: April 2009

Born Digital
. Understanding the First Generation of Digital Natives.
By John Palfrey and Urs Gasser
. Basic Books, New York, 2008. 383 pp. $25.95, C$27.95. ISBN 9780465005154.

Palfrey and Gasser explore what growing up in a digital world may mean for children and society.
Sharing, Trading, Creating Culture
Authors: Benkler Yochai
Published: April 2009

Remix
. Making Art and Commerce Thrive in the Hybrid Economy.
By Lawrence Lessig
. Penguin Press, New York, 2008. 351 pp. $25.95, C$28.50. ISBN 9781594201721.

Arguing that current copyright law harms most everyone who creates, sells, or enjoys art (very broadly defined), Lessig presents an alternative approach, "read-write" culture.
Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal
Authors: Zaia John A. , Li Xiuli , Franck Anne E. , Wu Xiwei , Thao Lia , Gallez-Hawkins Ghislaine
Published: April 2009

The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.
Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists
Authors: Toka Felix N. , Nfon Charles K. , Dawson Harry , Golde William T.
Published: April 2009

The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells in swine by TLR7 and TLR8 agonists. These agonists activated porcine NK cells by increasing gamma interferon (IFN-γ) expression and perforin storage. The activation of porcine NK cells was mediated by accessory cells, since their depletion resulted in reduced cytotoxicity toward target cells. Accessory cells were stimulated to produce interleukin 12 (IL-12), IL-15, IL-18, and IFN-α after treatment with TLR7 or TLR8 agonists. Neutralization of these cytokines reduced but did not completely inhibit the induction of NK cell cytotoxicity. Direct stimulation of NK cells with TLR7 or TLR8 agonists resulted in minimal cytotoxicity but levels of IFN-γ equivalent to those detected in the presence of accessory cells. Porcine NK cells express both TLR7 and TLR8 mRNAs, and treatment with these TLR agonists induced higher mRNA expression levels of TRAIL and IL-15Rα, which may contribute to the activity of NK cells. These data indicate that TLR7 and TLR8 agonists indirectly or directly activate porcine NK cells but that optimum levels of activation require cytokine secretion by accessory cells activated by these compounds. Interestingly, NK cells activated by TLR7 or TLR8 agonists were cytotoxic against foot-and-mouth disease virus (FMDV)-infected cells in vitro, indicating that these TLR agonists may be beneficial as adjuvants to stimulate the innate immunity against FMDV.
Comparative Performance of a Novel Herpes Simplex Virus Type 2-Specific Enzyme-Linked Immunosorbent Assay Using a Targeted Chain Oligopeptide, Peptide 55
Authors: Al-Sulaiman A. M. , Vallely P. J. , Klapper P. E.
Published: April 2009

Herpes simplex virus (HSV) glycoprotein G (gG2) has been used as the basis of many serological assays for the detection of HSV type 2 (HSV-2)-specific antibodies. In the present study, an enzyme-linked immunosorbent assay (ELISA), the Pathozyme Viro HSV-2 immunoglobulin G (IgG) ELISA (Omega Diagnostics, Alva, United Kingdom), based on an immunodominant epitope of gG2 presented in a branched-chain format (peptide 55), was compared with two commercially available gG2-specific assays, the Bioelisa HSV-2 IgG assay (Biokit, S.A., Barcelona, Spain) and the HerpesSelect HSV-2 IgG assay (Focus Diagnostics, Cypress, CA). A panel of 218 well-characterized serum samples was tested. Thirty-one samples were determined to be HSV-2 IgG antibody positive and 164 samples were determined to be negative with all three kits. The levels of concordance between the tests were 95.9% between the Omega and HerpeSelect assays, 90.8% between the Omega and Bioelisa assays, and 94.5% between the HerpeSelect and Bioelisa assays. Twenty-three samples gave discordant results. Western blot results showed that of these, the results for 77% were correctly identified by the Omega assay, the results for 68% were correctly identified by the HerpeSelect assay, and the results for 13.6% were correctly identified by the Bioelisa assay. Although there was a high level of agreement between the results obtained by the three assays and no false-positive results were detected by any of the three kits, confirmation of the results for samples with discordant results by Western blotting suggested that the peptide 55-based Omega assay is the most sensitive and specific assay among the assays evaluated.
BrucellaCapt versus Classical Tests in the Serological Diagnosis and Management of Human Brucellosis
Authors: Casanova Aurora , Ariza Javier , Rubio Manuel , Masuet Cristina , Díaz Ramón
Published: April 2009

The BrucellaCapt test is an immunocapture agglutination test suggested as a possible substitute for the Coombs test in the diagnosis of human brucellosis. Here it is compared with classical tests using 321 samples from 48 patients with brucellosis (6.9 ± 1.7 samples per patient), including 20 patients with focal disease and 8 patients with a total of 9 relapse episodes (mean follow-up, 18 months). The BrucellaCapt test was used according to the manufacturer's instructions, and we also used a variant of the BrucellaCapt test in which the microtiter plates were not coated with antibodies against total human immunoglobulin (BCAPV). The correlation between the BrucellaCapt and BCAPV tests was 0.982 (
P
< 0.001), with 260 coincident pairs of titers (81%). The areas under the receiver operating characteristic curve for the BrucellaCapt and BCAPV tests with respect to the Coombs test were 0.969 and 0.960, respectively. Upon admission, the BrucellaCapt, BCAPV, and Coombs tests and the microagglutination test (MAT) were positive for all cases: titers were 1/2,560 by the BrucellaCapt test, 1/2,560 by the BCAPV test, 1/1,280 by the Coombs test, and 1/320 by the MAT. The decreases in the BrucellaCapt and BCAPV titers over time were pronounced in comparison with the Coombs titers. Cumulative probabilities of persistence 12 months after therapy were as follows: 80% by the BrucellaCapt test, 80% by the BCAPV test, 87% by the Coombs test, and 35% by the MAT. Serological changes during relapse were detected in seven cases (88%) by the Coombs test, in five cases by the BrucellaCapt and BCAPV tests, and in three cases by the MAT. The BrucellaCapt test is a sensitive, specific, and simple test for routine use in human brucellosis. Similar results were obtained with the BCAPV test. However, in some cases of relapse and chronic forms of the disease, the slight changes observed in low-affinity antibodies alone are better detected by the Coombs test.
New Real-Time PCR-Based Method for
Kingella kingae
DNA Detection: Application to Samples Collected from 89 Children with Acute Arthritis
Authors: Ilharreborde Brice , Bidet Philippe , Lorrot Mathie , Even Julien , Mariani-Kurkdjian Patricia , Liguori Sandrine , Vitoux Christine , Lefevre Yann , Doit Catherine , Fitoussi Franck , Penneçot Georges , Bingen Edouard , Mazda Keyvan , Bonacorsi Stéphane
Published: April 2009

Inoculation of blood culture vials with joint fluid samples has revealed the important pathogenic role of
Kingella kingae
in pediatric arthritis. However, recent studies based on broad-range 16S ribosomal DNA PCR and real-time PCR without a probe suggest that conventional methods remain suboptimal. We developed a new real-time PCR method with a probe that is highly specific for
K. kingae
and applied it to joint fluid samples collected from 89 children with suspected arthritis admitted to our institution during a 2-year period. Real-time PCR was also applied to blood samples obtained before surgery and to joint drainage fluid samples obtained during several days after surgery. Thirty-six (40%) of the 89 cases of suspected septic arthritis had positive culture
. Staphylococcus aureus
was the main isolate (
n
= 19/36, 53%), followed by
K. kingae
(
n
= 7/36, 19%). Specific real-time PCR identified
K. kingae
in 24 of the 53 culture-negative cases. Thus,
K. kingae
was present in 31 (52%) of the 60 documented cases, making it the leading pathogen. Real-time PCR on all 15 blood DNA extracts from patients with
K. kingae
infection was negative, demonstrating that joint fluid positivity did not result from DNA circulating in blood. Real-time PCR amplification of drainage fluid samples showed that the pathogen could be detected for up to 6 days after antibiotic initiation.
K. kingae
real-time PCR applied to DNA extracted from joint fluid samples, but not from blood samples, markedly improved the etiological diagnosis of septic arthritis in children. Retrospective diagnosis is feasible for up to 6 days after treatment initiation.
Development and Characterization of a Genotype 4 Hepatitis E Virus Cell Culture System Using a HE-JF5/15F Strain Recovered from a Fulminant Hepatitis Patient
Authors: Tanaka Toshinori , Takahashi Masaharu , Takahashi Hideyuki , Ichiyama Koji , Hoshino Yu , Nagashima Shigeo , Mizuo Hitoshi , Okamoto Hiroaki
Published: April 2009

We developed an efficient cell culture system for genotype 4 hepatitis E virus using the HE-JF5/15F strain recovered from a fulminant hepatitis patient. The sixth-passage virus in the culture supernatant reached 1.5 × 10
8
copies/ml at 10 days postinoculation and possessed 10 nucleotide mutations with four amino acid changes.
Clinical Significance and Phylogenetic Relationship of Novel Australian
Pneumocystis jirovecii
Genotypes
Authors: van Hal Sebastiaan J. , Gilgado Felix , Doyle Tom , Barratt Joel , Stark Damien , Meyer Wieland , Harkness Jock
Published: April 2009

Pneumocystis jirovecii
is an important opportunistic pathogen in immunocompromised patients. Molecular typing is employed to study this pathogen, as no culture system exists. No Australian
P. jirovecii
strains have been previously studied. Direct sequencing, targeting the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, the mitochondrial large-subunit rRNA (mt LSU rRNA), and the dihydropteroate synthase (DHPS) gene, was performed on 68 Australian samples, collected between 2001 and 2007. Seven novel Australian ITS haplotypes (a composite of the ITS1 and ITS2 regions) were identified (SYD1m, SYD1g, Isyd2, Esyd3, Osyd4, Ag, and Hc). A dendrogram of published ITS haplotypes revealed that of the seven novel haplotypes, three (SYD1m, SYD1g, and Osyd4) are closely related to the haplotype Eg. Applying statistical parsimony, an Australian haplotype network was constructed which identified Eg as the ancestral haplotype, with two unresolved loops encountered. This suggests that the ITS lacks the resolution required for evolutionary analysis. Only two mt LSU rRNA genotypes were detected, with genotype 1 predominating. Mutant DHPS genotypes were present in 13% (8/60) of the samples. The novel haplotype Isyd2 was associated with less severe disease than the other Australian haplotypes. In contrast, patients with mutant DHPS genotypes were more likely to have severe disease, require invasive ventilation, and have a poor outcome than patients with wild-type DHPS genotypes. In conclusion, genetic clinical correlates continue to be found for
Pneumocystis
pneumonia; however, they remain controversial and warrant further study.
Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant
Staphylococcus aureus
Bacteremia: Trends over 11 Years
Authors: Musta Adina C. , Riederer Kathleen , Shemes Stephen , Chase Patrick , Jose Jinson , Johnson Leonard B. , Khatib Riad
Published: April 2009

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate
Staphylococcus aureus
(hVISA) isolates are increasing among methicillin (meticillin)-resistant
Staphylococcus aureus
(MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (
P
< 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively;
P
= 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%;
P
= 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette
mec
(SCC
mec
) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCC
mec
type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.
Intracellular Localization of Hepatitis Delta Virus Proteins in the Presence and Absence of Viral RNA Accumulation
Authors: Han Ziying , Alves Carolina , Gudima Severin , Taylor John
Published: April 2009

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (δAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that δAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, δAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of δAg in the presence and absence of replicating and nonreplicating HDV RNAs. When δAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both δAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, δAg moved to the nucleoplasm, but nucleolin was unchanged. When δAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of δAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of δAg altered at specific locations considered to be essential for protein function. These studies provide evidence that δAg does not interact directly with either Pol II or nucleolin and that forms of δAg which support replication are also capable of prior nucleolar transit.
Role of the Lymphoreticular System in Prion Neuroinvasion from the Oral and Nasal Mucosa
Authors: Bessen Richard A. , Martinka Scott , Kelly Jessica , Gonzalez Daniel
Published: April 2009

Prion neuroinvasion from peripheral tissues involves agent replication in the lymphoreticular system (LRS) prior to entry into the nervous system. This study investigated the role of the LRS in prion neuroinvasion from the oral and nasal mucosa in wild-type and immunodeficient mice and in hamsters infected with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. Following inoculation at neural sites, all hosts were susceptible to prion disease and had evidence of prion infection in the brain, but infection of the LRS was found only in scrapie-infected wild-type mice and HY TME-infected hamsters. In the LRS replication-deficient models, prion neuroinvasion was not observed following intraperitoneal or oral inoculation. However, immunodeficient mice, which have impaired follicular dendritic cells, were susceptible to scrapie following intratongue and intranasal inoculation despite the absence of PrP
Sc
in the tongue or the nasal cavity. For DY TME, hamsters were susceptible following intratongue but not intranasal inoculation and PrP
Sc
was limited to nerve fibers of the tongue. These findings indicate that neuroinvasion from the tongue and nasal cavity can be independent of LRS infection but neuroinvasion was partially dependent on the strain of the prion agent and/or the host species. The paucity of PrP
Sc
deposition in the oral and nasal mucosa from LRS replication-deficient hosts following neuroinvasion from these tissues suggests an infection of nerve fibers that is below the threshold of PrP
Sc
detection and/or the transport of the prion agent along cranial nerves without agent replication.
Enumeration of an Extremely High Particle-to-PFU Ratio for Varicella-Zoster Virus
Authors: Carpenter John E. , Henderson Ernesto P. , Grose Charles
Published: April 2009

Varicella-zoster virus (VZV) is renowned for its low titers. Yet investigations to explore the low infectivity are hampered by the fact that the VZV particle-to-PFU ratio has never been determined with precision. Herein, we accomplish that task by applying newer imaging technology. More than 300 images were taken of VZV-infected cells on 4 different samples at high magnification. We enumerated the total number of viral particles within 25 cm2of the infected monolayer at 415 million. Based on these numbers, the VZV particle:PFU ratio was approximately 40,000:1 for a cell-free inoculum.
Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging
Authors: Garbitt-Hirst Rachel , Kenney Scott P. , Parent Leslie J.
Published: April 2009

The packaging of retroviral genomic RNA (gRNA) requires
cis
-acting elements within the RNA and
trans
-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.