bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Gas Atmosphere and Temperature on the Respiration Rates of Whole and Sliced Mushrooms (Agaricus bisporus)— Implications for Film Permeability in Modified Atmosphere Packages
Authors: Cliffe‐Byrnes Valerie , O' Beirne David
Published: April 2007

ABSTRACT:  An open flowthrough respirometer was used to determine the respiration rates of whole and sliced mushrooms as a function of O2, CO2, and storage temperature. Respiration rates were measured under the following O2 concentrations: 20, 15, 10, 5, and 2% at 5 temperatures (4, 8, 10, 13, and 16 °C). The effects of 5 CO2 concentrations (0, 2, 5, 10, 15%) at two O2 levels (2 and 20%) were also examined at these temperatures. Mushroom respiration increased with temperature following an Arrhenius‐type relationship; activation energies and corresponding Q10 values were calculated. Lowering the oxygen concentration significantly decreased the respiration rate. The effects of reducing O2 levels were greater at higher storage temperatures. Both activation energies and Q10 values were lower under reduced O2 atmospheres compared with those in air. A Michaelis–Menten enzyme kinetics model was evaluated for describing the influence of gas concentrations on respiration rate. The effects of O2 fitted the O2 enzyme kinetics model well. CO2 exhibited small inhibitory effects on respiration of whole and sliced mushrooms, especially at low O2 concentrations and low temperatures. The enzyme model was used to predict respiration under an optimal modified atmosphere for mushrooms, and the required film O2 and CO2 permeabilities were defined for key pack design parameters.
Investigation of Dielectric Properties of Different Cake Formulations during Microwave and Infrared–Microwave Combination Baking
Authors: Sakiyan Ozge , Sumnu Gulum , Sahin Serpil , Meda Venkatesh
Published: April 2007

ABSTRACT:  Dielectric properties can be used to understand the behavior of food materials during microwave processing. Dielectric properties influence the level of interaction between food and high frequency electromagnetic energy. Dielectric properties are, therefore, important in the design of foods intended for microwave preparation. In this study, it was aimed to determine the variation of dielectric properties of different cake formulations during baking in microwave and infrared–microwave combination oven. In addition, the effects of formulation and temperature on dielectric properties of cake batter were examined. Dielectric constant and loss factor of cake samples were shown to be dependent on formulation, baking time, and temperature. The increase in baking time and temperature decreased dielectric constant and loss factor of all formulations. Fat content was shown to increase dielectric constant and loss factor of cakes.
Renal agenesis in an alpaca cria
Authors: Hardefeldt LY , Textor JA , Dart AJ
Published: April 2007

A 4‐day‐old alpaca cria presented for inappetence that responded to symptomatic treatment. The cria re‐presented with acute signs of inappetence and azotaemia. The azotaemia persisted despite intravenous fluid therapy. There was no right kidney on ultrasound and there appeared to be perirenal oedema around the left kidney. A diagnosis of right renal agenesis and acute renal failure of the left kidney was made. The cria failed to improve and was euthanased. Necropsy examination confirmed right renal agenesis and agenesis of the right ureter and right renal artery. A section of left kidney submitted for histological examination revealed diffuse, acute, marked tubular degeneration and nephrosis. The cause of the renal failure in the left kidney was not determined.
Acute tumour lysis syndrome in a dog with B‐Cell multicentric lymphoma
Authors: Mylonakis ME , Koutinas AF , Papaioannou N , Lekkas S
Published: April 2007

A 5‐year‐old, spayed female German Shepherd dog was admitted to hospital with marked generalised lymphadenomegaly and splenomegaly. A stage Va B‐cell multicentric lymphoma was diagnosed on clinical, cytological (lymph node, bone marrow), histological‐immunohistochemical (lymph node excision) and imaging grounds. Since no satisfactory remission was achieved using a multi‐drug chemotherapy protocol that included cyclophosphamide, vincristine, cytosine arabinoside, prednisolone, and subsequently supplemented by L‐asparaginase, it was replaced by another protocol combining vincristine, L‐asparaginase, prednisolone, cyclophosphamide and doxorubicin. Soon after the third weekly session of the second protocol, the clinical status of the animal deteriorated suddenly and severely, with a bleeding tendency, jaundice, hyperuricaemia, hyperphosphataemia, azotaemia, hyperbilirubinaemia and, presumptive disseminated intravascular coagulation. There was also complete regression of lymphadenomegaly. This report emphasises the clinicopathological features and the diagnostic peculiarities of the acute tumour lysis syndrome, which occurs uncommonly in dogs.
Comparison of sampling sites and detection methods forHaemophilus parasuis
Authors: Turni C , Blackall PJ
Published: April 2007

Objective To improve the isolation rate and identification procedures forHaemophilus parasuisfrom pig tissues.

Design Thirteen sampling sites and up to three methods were used to confirm the presence ofH.parasuisin pigs after experimental challenge.

Procedure Colostrum‐deprived, naturally farrowed pigs were challenged intratracheally withH parasuisserovar 12 or 4. Samples taken during necropsy were either inoculated onto culture plates, processed directly for PCR or enriched prior to being processed for PCR. The recovery ofH parasuisfrom different sampling sites and using different sampling methods was compared for each serovar.

Results H parasuiswas recovered from several sample sites for all serovar 12 challenged pigs, while the trachea was the only positive site for all pigs following serovar 4 challenge. The method of solid medium culture of swabs, and confirmation of the identity of cultured bacteria by PCR, resulted in 38% and 14% more positive results on a site basis for serovars 12 and 4, retrospectively, than direct PCR on the swabs. This difference was significant in the serovar 12 challenge.

Conclusion Conventional culture proved to be more effective in detectingH parasuisthan direct PCR or PCR on enrichment broths. For subacute (serovar 4) infections, the most successful sites for culture or direct PCR were pleural fluid, peritoneal fibrin and fluid, lung and pericardial fluid. For acute (serovar 12) infections, the best sites were lung, heart blood, affected joints and brain. The methodologies and key sampling sites identified in this study will enable improved isolation ofH parasuisand aid the diagnosis of Glässer's disease.
Prognostic value of platelet indices as determined by ADVIA 120 in patients suspected of having disseminated intravascular coagulation
Authors: KIM H. K. , KIM J.‐E. , HAM C. K. , LEE D. S. , PARK S. , CHO H.‐I.
Published: April 2007

Recent technological advances have made it possible to record a variety of platelet indices using automated hematology analyzers. Disseminated intravascular coagulation (DIC) is associated with the dramatic hemostasis activation, with evidence of fibrin formation and platelet consumption. We investigated the prognostic significance of platelet indices as measured by ADVIA in 222 patients suspected of having DIC. The presence of overt DIC was defined using the scoring system of the International Society of Thrombosis and Haemostasis Subcommittee. Twenty‐eight day hospital mortality was used as a clinical prognosis parameter. Median platelet count and platelet‐crit (PCT) levels markedly decreased in nonsurvivors, whereas mean platelet volume (MPV), platelet component distribution width (PCDW) and platelet dry mass distribution width (PMDW) were significantly increased in nonsurvivors. In terms of ROC analysis, which was conducted to predict 28‐day mortality, areas under the receiver operating characteristic curve (AUC) were; 0.73 platelet count, 0.72 for PCT, 0.69 for PCDW, 0.65 for PMDW and 0.61 for MPV. The odds ratio of a reduced platelet count for the relative risk of 28‐day mortality was 5.249 (95% CI: 2.399–11.486), and the odds ratio for PCDW was 3.240 and for PMDW 3.262. Among these indices, platelet count, PCDW and PMDW were found to be more predictive of 28‐day hospital mortality. Our results suggest that these indices may provide prognostic information on hospital mortality in the patients suspected of having DIC.
A strategy of splitting individual high volume cord blood units into two half subunits prior to processing increases the recovery of cells and facilitates ex vivo expansion of the infused haematopoietic progenitor cells in adults
Authors: PAPASSAVAS A. C. , GIOKA V. , CHATZISTAMATIOU T. , KOKKINOS T. , ANAGNOSTAKIS I. , GECKA G. , REDOUKAS I. , PATERAKIS G. , STAVROPOULOS‐GIOKAS C.
Published: April 2007

This study was designed to maximize the recovery of the desirable cell populations contained in the cord blood (CB) freezing bag, in order to optimize donor selection for adolescents and adults. To evaluate this hypothesis, high volume CB units (CBUs) were categorized into three volume collection groups (120–139, 140–159 and ≥160 ml) and were randomly split before volume reduction into two half low volume CBUs; (a) and (b). Using the SEPAX Cell Processing System, all CBUs were standardized to 26 ml. In 128 high volume split CBUs, the WBC, mononuclear cell and CD34+ cell recoveries were significantly higher (P ≤ 0.05; 80%, 79.89% and 87.41% respectively) than those of the 106 high volume nonsplit CBUs (59.7%, 62.82% and 68.62% respectively), resulting in significantly higher (P ≤ 0.05) mean weights of the potential recipients. The strategy of splitting high volume CBUs into two half low volume CBUs improves the recovery of the cells and is an attractive option for ex vivo expansion, in order to facilitate the CB transplantation in adults who are not eligible for single CB transplantation because of limitations of cell dose.
Study on Embedding and Integration of Microsensors Into Metal Structures for Manufacturing Applications
Authors: Cheng Xudong , Datta Arindom , Choi Hongseok , Zhang Xugang , Li Xiaochun
Published: April 2007

Real time monitoring, diagnosis, and control of numerous manufacturing processes is of critical importance in reducing operation costs, improving product quality, and shortening response time. Current sensors used in manufacturing are normally unable to provide measurements with desired spatial and temporal resolution at critical locations in metal tooling structures that operate in hostile environments (e.g., elevated temperatures and severe strains). Microsensors are expected to offer tremendous benefits for real time sensing in manufacturing processes. Rapid tooling, a layered manufacturing process, could allow microsensors to be placed at any critical location in metal tooling structures. However, a viable approach is needed to effectively integrate microsensors into metal structures during the process. In this study, a novel batch production of metal embedded microsensor units was realized by transferring thin-film sensors from silicon wafers directly into nickel substrates through standard microfabrication and electroplating techniques. Ultrasonic metal welding (USMW) was studied to obtain optimized process parameters and then used to integrate nickel embedded thin-film thermocouple (TFTC) units into copper workpieces. The embedded TFTCs successfully survived the welding tests, validating that USMW is a viable method to integrate microsensors to metallic tool materials. Moreover, the embedded microsensors were also able to measure the transient temperature in situ at 50μm directly beneath the welding interface during welding. The transient temperatures measured by the metal embedded TFTCs provide strong evidence that the heat generation is not critical for weld formation during USMW. Metal embedded microsensors yield great potential to improve fundamental understanding of numerous manufacturing processes by providing in situ sensing data with high spatial and temporal resolution at critical locations.
A Uniform Pressure Electromagnetic Actuator for Forming Flat Sheets
Authors: Kamal Manish , Daehn Glenn S.
Published: April 2007

High velocity electromagnetic forming can lead to better formability along with additional benefits. The spatial distribution of forming pressure in electromagnetic forming can be controlled by the configuration of the actuator. A new type of actuator is discussed which gives a uniform pressure distribution in forming. It also provides a mechanically robust design and has a high efficiency for flat sheet forming. A simplified analysis of the actuator is presented that helps in the design of the system. Examples of uses of the actuator are then presented, specifically with regards to forming shapes and surface embossing. Some practical challenges in the design of the actuator are also addressed. This paper emphasizes the approaches and engineering calculations required to effectively use this actuator.
Experiments on Remelting and Solidification of Molten Metal Droplets Deposited in Vertical Columns
Authors: Fang M. , Chandra S. , Park C. B.
Published: April 2007

Experiments were done to determine conditions under which vertical columns could be built by metal droplets landing sequentially on top of each other. Molten tin droplets (0.6mm diameter) were deposited using a pneumatic droplet generator on an aluminum substrate. The primary parameters varied in experiments were those found to most affect bonding between droplets: droplet temperature (250-345°C), substrate temperature (60-200°C), and deposition rate (1-15Hz). At lower deposition rates the substrate cooled down too much to induce remelting whereas at higher rates the tip of the column remained liquid and surface tension forces pulled it into a spherical mass. Assuming one-dimensional conductive heat transfer in a column a simple analytical model was developed to calculate the temperature at the tips of the column. It predicts that deposition frequency should be decreased as column height increases to hold the tip temperature constant. Droplet coalescence was best achieved when the tip temperature of a column was maintained at the melting point of the metal. Columns fabricated following the deposition frequency predicted by the model show good bonding between droplets and uniform diameter.
Modeling and Simulation of Shrinkage During the Picture Tube Panel Forming Process
Authors: Zhou Huamin , Xi GuoDong , Li Dequn
Published: April 2007

The accurate prediction of residual stresses and shrinkage of the formed picture tube panel is important to achieve high quality and precision of the final product. In this paper, the numerical simulation of shrinkage of molded panels was carried out in consideration of the residual stresses accumulated during both the packing and free-to-contract stages. For residual stress analysis, a thermorheologically simple viscoelastic material model was introduced to consider the stresses relaxation effect and to describe the mechanical behavior according to the temperature change. The deformation of molded parts induced by the residual stresses was calculated based on the theory of shells, represented as an assembly of flat elements formed by combining the constant strain and the discrete Kirchhoff triangular elements. The simulation results of the developed program were side verified by comparing the dimensional accuracy of the panels produced by the molds, which compensated for a uniform part shrinkage or the predicted ununiform part shrinkage.
Stability-Based Spindle Design Optimization
Authors: Gagnol Vincent , Bouzgarrou Belhassen C. , Ray Pascal , Barra Christian
Published: April 2007

Prediction of stable cutting regions is a critical requirement for high-speed milling operations. These predictions are generally made using frequency-response measurements of the tool-holder-spindle set obtained from a nonrotating spindle. However, significant changes in system dynamics occur during high-speed rotation. In this paper, a dynamic high-speed spindle-bearing system model is elaborated on the basis of rotor dynamics prediction and readjusted on the basis of experimental modal identification. The dependency of dynamic behavior on speed range is then investigated and determined with accuracy. Dedicated experiments are carried out in order to confirm model results. They show that dynamic effects due to high rotational speed and elastic deformations, such as gyroscopic coupling and spin softening, have a significant influence on spindle behavior. By integrating the modeled speed-dependent spindle transfer function in the chatter vibration stability approach of Altintas and Budak (1995, CIRPS Ann, 44(1), pp. 357–362), a new dynamic stability lobe diagram is predicted. Significant changes are observed in the stability limits constructed using the proposed approach and allow accurate prediction of cutting conditions to be established. Finally, optimization studies are performed on spindle design parameters in order to obtain a chatter vibration-free cutting operation at the desired speed and depth of cut for a given cutter.
The Problem with Determining Atomic Structure at the Nanoscale
Authors: Billinge Simon J. L. , Levin Igor
Published: April 2007

Emerging complex functional materials often have atomic order limited to the nanoscale. Examples include nanoparticles, species encapsulated in mesoporous hosts, and bulk crystals with intrinsic nanoscale order. The powerful methods that we have for solving the atomic structure of bulk crystals fail for such materials. Currently, no broadly applicable, quantitative, and robust methods exist to replace crystallography at the nanoscale. We provide an overview of various classes of nanostructured materials and review the methods that are currently used to study their structure. We suggest that successful solutions to these nanostructure problems will involve interactions among researchers from materials science, physics, chemistry, computer science, and applied mathematics, working within a “complex modeling” paradigm that combines theory and experiment in a self-consistent computational framework.
Paleocene-Eocene Thermal Maximum and the Opening of the Northeast Atlantic
Authors: Storey Michael , Duncan Robert A. , Swisher Carl C.
Published: April 2007

The Paleocene-Eocene thermal maximum (PETM) has been attributed to a sudden release of carbon dioxide and/or methane.
40
Ar/
39
Ar age determinations show that the Danish Ash-17 deposit, which overlies the PETM by about 450,000 years in the Atlantic, and the Skraenterne Formation Tuff, representing the end of 1 ± 0.5 million years of massive volcanism in East Greenland, are coeval. The relative age of Danish Ash-17 thus places the PETM onset after the beginning of massive flood basalt volcanism at 56.1 ± 0.4 million years ago but within error of the estimated continental breakup time of 55.5 ± 0.3 million years ago, marked by the eruption of mid-ocean ridge basalt–like flows. These correlations support the view that the PETM was triggered by greenhouse gas release during magma interaction with basin-filling carbon-rich sedimentary rocks proximal to the embryonic plate boundary between Greenland and Europe.
NOV (CCN3) Functions as a Regulator of Human Hematopoietic Stem or Progenitor Cells
Authors: Gupta Rajeev , Hong Dengli , Iborra Francisco , Sarno Samantha , Enver Tariq
Published: April 2007

Clinically successful hematopoietic cell transplantation is dependent on hematopoietic stem and progenitor cells. Here we identify the matricellular protein Nephroblastoma Overexpressed (Nov, CCN3) as being essential for their functional integrity. Nov expression is restricted to the primitive (CD34) compartments of umbilical vein cord blood, and its knockdown in these cells by lentivirus-mediated RNA interference abrogates their function in vitro and in vivo. Conversely, forced expression of Nov and addition of recombinant Nov protein both enhance primitive stem and/or progenitor activity. Taken together, our results identify Nov (CCN3) as a regulator of human hematopoietic stem or progenitor cells.
Fast Routing in Road Networks with Transit Nodes
Authors: Bast Holger , Funke Stefan , Sanders Peter , Schultes Dominik
Published: April 2007

When you drive to somewhere far away, you will leave your current location via one of only a few important traffic junctions. Starting from this informal observation, we developed an algorithmic approach, transit node routing, that allows us to reduce quickest path queries in road networks to a small number of table lookups. For road maps of Western Europe and the United States, our best query times improved over the best previously published figures by two orders of magnitude. This is also more than one million times faster than the best known algorithm for general networks.
Revisiting Carbon Flux Through the Ocean's Twilight Zone
Authors: Buesseler Ken O. , Lamborg Carl H. , Boyd Philip W. , Lam Phoebe J. , Trull Thomas W. , Bidigare Robert R. , Bishop James K. B. , Casciotti Karen L. , Dehairs Frank , Elskens Marc , Honda Makio , Karl David M. , Siegel David A. , Silver Mary W. , Steinberg Deborah K. , Valdes Jim , Van Mooy Benjamin , Wilson Stephanie
Published: April 2007

The oceanic biological pump drives sequestration of carbon dioxide in the deep sea via sinking particles. Rapid biological consumption and remineralization of carbon in the âtwilight zoneâ (depths between the euphotic zone and 1000 meters) reduce the efficiency of sequestration. By using neutrally buoyant sediment traps to sample this chronically understudied realm, we measured a transfer efficiency of sinking particulate organic carbon between 150 and 500 meters of 20 and 50% at two contrasting sites. This large variability in transfer efficiency is poorly represented in biogeochemical models. If applied globally, this is equivalent to a difference in carbon sequestration of more than 3 petagrams of carbon per year.
Distinct Pathways of Antigen Uptake and Intracellular Routing in CD4 and CD8 T Cell Activation
Authors: Burgdorf Sven , Kautz Andreas , Böhnert Volker , Knolle Percy A. , Kurts Christian
Published: April 2007

The mechanisms that allow antigen-presenting cells (APCs) to selectively present extracellular antigen to CD8
+
effector T cells (cross-presentation) or to CD4
+
T helper cells are not fully resolved. We demonstrated that APCs use distinct endocytosis mechanisms to simultaneously introduce soluble antigen into separate intracellular compartments, which were dedicated to presentation to CD8
+
or CD4
+
T cells. Specifically, the mannose receptor supplied an early endosomal compartment distinct from lysosomes, which was committed to cross-presentation. These findings imply that antigen does not require intracellular diversion to access the cross-presentation pathway, because it can enter the pathway already during endocytosis.
Modeling the Initiation and Progression of Human Acute Leukemia in Mice
Authors: Barabé Frédéric , Kennedy James A. , Hope Kristin J. , Dick John E.
Published: April 2007

Our understanding of leukemia development and progression has been hampered by the lack of in vivo models in which disease is initiated from primary human hematopoietic cells. We showed that upon transplantation into immunodeficient mice, primitive human hematopoietic cells expressing a
mixed-lineage leukemia
(
MLL
) fusion gene generated myeloid or lymphoid acute leukemias, with features that recapitulated human diseases. Analysis of serially transplanted mice revealed that the disease is sustained by leukemia-initiating cells (L-ICs) that have evolved over time from a primitive cell type with a germline immunoglobulin heavy chain (IgH) gene configuration to a cell type containing rearranged IgH genes. The L-ICs retained both myeloid and lymphoid lineage potential and remained responsive to microenvironmental cues. The properties of these cells provide a biological basis for several clinical hallmarks of
MLL
leukemias.
Environment-Induced Sudden Death of Entanglement
Authors: Almeida M. P. , de Melo F. , Hor-Meyll M. , Salles A. , Walborn S. P. , Ribeiro P. H. Souto , Davidovich L.
Published: April 2007

We demonstrate the difference between local, single-particle dynamics and global dynamics of entangled quantum systems coupled to independent environments. Using an all-optical experimental setup, we showed that, even when the environment-induced decay of each system is asymptotic, quantum entanglement may suddenly disappear. This “sudden death” constitutes yet another distinct and counterintuitive trait of entanglement.
PCR-Based Strategy To Detect and Identify Species of
Phaeoacremonium
Causing Grapevine Diseases
Authors: Aroca Angeles , Raposo Rosa
Published: April 2007

Species of
Phaeoacremonium
(especially
Phaeoacremonium aleophilum
) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants.
Phaeoacremonium
species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of
Phaeoacremonium
that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect
Phaeoacremonium
directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of
Phaeoacremonium
species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for
Phaeoacremonium viticola
and
Phaeoacremonium angustius
, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate
P. angustius
from
P. viticola
, respectively. An easy and fast protocol was developed to detect and identify species of
Phaeoacremonium
in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of
Phaeoacremonium
spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.
Fruit-Specific Expression of the Human Immunodeficiency Virus Type 1 Tat Gene in Tomato Plants and Its Immunogenic Potential in Mice
Authors: Ramírez Yuri Jorge Peña , Tasciotti Ennio , Gutierrez-Ortega Abel , Torres Alberto J. Donayre , Flores María Teresa Olivera , Giacca Mauro , Lim Miguel Ángel Gómez
Published: April 2007

The human immunodeficiency virus type 1 (HIV-1) Tat protein is considered a potential candidate vaccine antigen. In an effort to design a strategy for noninvasive vaccination against HIV-1, we developed transgenic tomatoes expressing the Tat protein. Two independent plants testing positive in transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against Tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant, as in all cases the fruit exhibited underdeveloped reproductive structures and no seeds. Nine groups of 10 pathogen-free BALB/c male mice were primed either orally, intraperitoneally, or intramuscularly with 10 mg of tomato fruit extract derived from transgenic or wild-type plants and with 10 μg of Tat86 recombinant protein. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also demonstrated that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development.
Evaluation of the LIAISON Chemiluminescence Immunoassay for Diagnosis of Syphilis
Authors: Knight Carrie S. , Crum Mary A. , Hardy Robert W.
Published: April 2007

We report the results of an evaluation of the LIAISON
Treponema pallidum
-specific assay, a one-step sandwich chemiluminescence immunoassay (CLIA), as a screening test and as a confirmatory test for the diagnosis of syphilis. The assay was compared with the CAPTIA Syphilis-G enzyme immunoassay (EIA) and with a testing algorithm that also included the rapid plasma reagin (RPR) and
T. pallidum
particle agglutination (PA) assays. As a screening test, the CLIA showed levels of agreement with the EIA and with the algorithm, respectively, of 94.1 and 100% for 51 samples from patients with primary or secondary syphilis, 93.2 and 98.7% for 999 samples sent to the laboratory for routine syphilis testing, 84.5 and 94.0% for 200 samples from human immunodeficiency virus-positive patients, 98.0 and 100% for 200 samples from pregnant patients, and 94.3 and 98.3% for 992 samples from apparently healthy adults. As a confirmatory test, the CLIA showed 99% agreement with the EIA for 204 RPR-positive samples. After resolution with further
T. pallidum
PA testing and the discarding of one sample of insufficient quantity, there was 100% agreement for the remaining 203 samples. For the total group of 2,645 samples, the overall relative sensitivity was 95.8% and the relative specificity was 99.1%. We conclude that the LIAISON CLIA demonstrated excellent sensitivity and specificity when evaluated as a confirmatory test and as a screening test for syphilis among various patient populations, including specific populations with reportedly increased rates of false-positive nontreponemal test results.
Milk and Serum J5-Specific Antibody Responses, Milk Production Change, and Clinical Effects following Intramammary
Escherichia coli
Challenge for J5 Vaccinate and Control Cows
Authors: Wilson David J. , Mallard Bonnie A. , Burton Jeanne L. , Schukken Ynte H. , Gröhn Yrjo T.
Published: April 2007

Holstein dairy cows (four J5 vaccinates and four controls) selected for no recorded intramammary disease and low somatic cell count (SCC) during the previous lactation were challenged by intramammary infusion of
Escherichia coli
. Vaccination with J5 was at 8 weeks and again 4 weeks before the anticipated calving date. Cows were challenged at 8 to 16 days in milk (DIM). Shedding of
E. coli
in milk was significantly higher among controls than vaccinates (no shedding) from 6 h to 21 h postchallenge. From 21 h to 132 h postchallenge, SCC in challenged quarters of controls (5,429,000/ml) was significantly higher than that of vaccinates (490,000/ml). On the day after challenge, milk production in control cows was 8 kg less, while vaccinates gained 0.5 kg, a significant difference. In serum immediately prior to challenge, J5-specific immunoglobulin G1 (IgG1) was significantly higher, IgG2 was nearly significantly higher, and IgM was the same in J5 vaccinates relative to controls. Vaccinates had proportionally more IgG2 in serum postcalving and in the first 12 h following challenge and less IgG2 in milk 24 h after challenge than the controls, approaching statistical significance. The ratio of J5-specific IgG1 and IgG2 combined compared to IgM was significantly higher in vaccinates than in controls in prechallenge serum (ratios of 15.8 and 3.2, respectively) and milk (5.0 and 1.3, respectively). Cows with higher IgM titers in milk 12 h postchallenge produced significantly less milk. Vaccination with J5 was significantly associated with higher production of J5-specific IgG1 and IgG2 in early lactation, reduced SCC, faster clearance of
E. coli
from milk, and less milk production loss following intramammary challenge.
Biofilm Production by
Candida
Species and Inadequate Antifungal Therapy as Predictors of Mortality for Patients with Candidemia
Authors: Tumbarello Mario , Posteraro Brunella , Trecarichi Enrico Maria , Fiori Barbara , Rossi Marianna , Porta Rosaria , de Gaetano Donati Katleen , La Sorda Marilena , Spanu Teresa , Fadda Giovanni , Cauda Roberto , Sanguinetti Maurizio
Published: April 2007

Nosocomial
Candida
bloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a
Candida
bloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by
Candida albicans
, 64 (21.7%) by
Candida parapsilosis
, 28 (9.5%) by
Candida tropicalis
, and 26 (8.8%) by
Candida glabrata
. When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of
C. tropicalis
(20 of 28 patients [71.4%]), followed by
C. glabrata
(6 of 26 [23.1%]),
C. albicans
(38 of 168 [22.6%]), and
C. parapsilosis
(14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 1.09 to 5.10;
P
= 0.03), infection with overall biofilm-forming
Candida
species (OR, 2.33; 95% CI, 1.26 to 4.30;
P
= 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15;
P
< 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming
Candida
species studied, only infections caused by
C. albicans
(
P
< 0.001) and
C. parapsilosis
(
P
= 0.003) correlated with increased mortality. Together with well-established factors,
Candida
biofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.
Three-Year Longitudinal Study of Genotypes of
Mycobacterium tuberculosis
Isolates in Tuscany, Italy
Authors: Lari Nicoletta , Rindi Laura , Bonanni Daniela , Rastogi Nalin , Sola Christophe , Tortoli Enrico , Garzelli Carlo
Published: April 2007

The genetic diversity of 829 strains of
Mycobacterium tuberculosis
isolated during a 3-year period in Tuscany, Italy, a country with a low prevalence of tuberculosis, from 480 Italian-born and 349 foreign-born patients was determined by spoligotyping. The predominant spoligotype families were T (30.2% of isolates), Haarlem (19.9%), and the Latino-American and Mediterranean family (LAM) (11.2%); the remaining isolates were distributed among the Beijing (6.5%), S (4.2%), East Africa-India (EAI) (3.0%), Bovis (2.3%), Central Asia (CAS) (2.1%), Africanum (1.3%), and X (1.2%) families or were undefined (2.7%) or orphan (14.1%) isolates. Isolates of the families T, Haarlem, Bovis, and X were distributed among Italian- and foreign-born patients almost proportionally to the patients' numbers. Isolates of the LAM family were prevalent in foreign-born people (13.5%, versus 9.6% in Italian-born patients). Isolates of the S family were found almost exclusively in Italian-born patients, while strains of families EAI and CAS were isolated almost exclusively from foreign-born patients; Africanum isolates were all from African-born patients. The isolates of the Beijing family showed a trend to a steady increase during the survey. The prevalence of Beijing strains was 11.7% among foreign-born people and 2.7% among Italian-born patients. The Beijing strains were typed by the standardized IS
6110
restriction fragment length polymorphism assay, which yielded a total of 38 distinct IS
6110
patterns; 21 isolates (39.6%) occurred in six distinct clusters; of these, three contained two isolates and the other three contained four, five and six isolates, thus demonstrating that Beijing strains caused several tuberculosis outbreaks in the region. These findings indicate that transmission of Beijing strains between immigrants and the autochthonous population has occurred frequently and suggests an ongoing active transmission of the Beijing genotype in the region.
Inhibition of the Alpha/Beta Interferon Response by Mouse Hepatitis Virus at Multiple Levels
Authors: Roth-Cross Jessica K. , Martínez-Sobrido Luis , Scott Erin P. , García-Sastre Adolfo , Weiss Susan R.
Published: April 2007

Mouse hepatitis virus (MHV) was used as a model to study the interaction of coronaviruses with the alpha/beta interferon (IFN-α/β) response. While MHV strain A59 appeared to induce IFN-β gene transcription and low levels of nuclear translocation of the IFN-β transcription factor interferon regulatory factor 3 (IRF-3), MHV did not induce IFN-β protein production during the course of infection in L2 mouse fibroblast cells. In addition, MHV was able to significantly decrease the level of IFN-β protein induced by both Newcastle disease virus (NDV) and Sendai virus infections, without targeting it for proteasomal degradation and without altering the nuclear translocation of IRF-3 or IFN-β mRNA production or stability. These results indicate that MHV infection causes an inhibition of IFN-β production at a posttranscriptional level, without altering RNA or protein stability. In contrast, MHV induced IFN-β mRNA and protein production in the brains of infected animals, suggesting that the inhibitory mechanisms observed in vitro are not enough to prevent IFN-α/β production in vivo. Furthermore, MHV replication is highly resistant to IFN-α/β action, as indicated by unimpaired MHV replication in L2 cells pretreated with IFN-β. However, when L2 cells were coinfected with MHV and NDV in the presence of IFN-β, NDV, but not MHV, replication was inhibited. Thus, rather than disarming the antiviral activity induced by IFN-β pretreatment completely, MHV may be inherently resistant to some aspects of the antiviral state induced by IFN-β. These findings show that MHV employs unique strategies to circumvent the IFN-α/β response at multiple steps.
Human Cytomegalovirus UL84 Interacts with an RNA Stem-Loop Sequence Found within the RNA/DNA Hybrid Region of
ori
Lyt
Authors: Colletti Kelly S. , Smallenburg Kate E. , Xu Yiyang , Pari Gregory S.
Published: April 2007

Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex
cis
-acting
ori
Lyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within
ori
Lyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to
ori
Lyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within
ori
Lyt.
Viral Alteration of Cellular Translational Machinery Increases Defective Ribosomal Products
Authors: Berglund Peter , Finzi Diana , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2007

Here we show that cells expressing genes inserted into Semliki Forest virus (SFV) vectors generate a large fraction of defective ribosomal products (DRiPs) due to frequent initiation on downstream Met residues. In monopolizing the host cell translational machinery, SFV reduces levels of translation eukaryotic initiation factor 4E (eIF4E), diminishes phosphorylation of ribosome subunit S6, and phosphorylates translation initiation factor eIF2α. We show that the last event is required for SFV mistranslation of inserted genes. Downstream initiation is suppressed by fusing inserted genes with the open reading frame encoding the SFV capsid, demonstrating that one function of the capsid element is to enable ribosomes to initiate translation in the proper location. These results show that in modifying translation, viral vectors can unpredictably increase the generation of truncated polypeptides and thereby the DRiP fraction of inserted gene products, which can potentially affect their yield, therapeutic efficacy, and immunogenicity.
Diverse Herpes Simplex Virus Type 1 Thymidine Kinase Mutants in Individual Human Neurons and Ganglia
Authors: Wang Kening , Mahalingam Gowtham , Hoover Susan E. , Mont Erik K. , Holland Steven M. , Cohen Jeffrey I. , Straus Stephen E.
Published: April 2007

Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations oftkmutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSVtkmutants in animal neurons is impaired. Here we report the detection of mutated HSVtksequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1tkopen reading frames from ganglia were amplified by PCR, cloned, and sequenced.tkmutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1tkmutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSVtkmutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected withtkmutant virus alone. These data suggest that diversetkmutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.
Interaction of the C-Terminal Domains of Sendai Virus N and P Proteins: Comparison of Polymerase-Nucleocapsid Interactions within the Paramyxovirus Family
Authors: Houben Klaartje , Marion Dominique , Tarbouriech Nicolas , Ruigrok Rob W. H. , Blanchard Laurence
Published: April 2007

Interaction of the C-terminal domains of Sendai virus (SeV) P and N proteins is crucial for RNA synthesis by correctly positioning the polymerase complex (L+P) onto the nucleocapsid (N/RNA). To better understand this mechanism within the paramyxovirus family, we have studied the complex formed by the SeV C-terminal domains of P (PX) and N (N
TAIL
) proteins by solution nuclear magnetic resonance spectroscopy. We have characterized SeV N
TAIL
, which belongs to the class of intrinsically disordered proteins, and precisely defined the binding regions within this latter domain and within PX. SeV N
TAIL
binds with residues 472 to 493, which have a helical propensity (residues 477 to 491) to the surface created by helices α2 and α3 of PX with a 1:1 stoichiometry, as was also found for measles virus (MV). The binding interface is dominated by charged residues, and the dissociation constant was determined to be 57 ± 18 μM under conditions of the experiment (i.e., in 0.5 M NaCl). We have also shown that the extreme C terminus of SeV N
TAIL
does not interact with PX, which is in contrast to MV, where a second binding site was identified. In addition, the interaction surfaces of the MV proteins are hydrophobic and a stronger binding constant was found. This gives a good illustration of how selection pressure allowed the C-terminal domains of N and P proteins to evolve concomitantly within this family of viruses in order to lead to protein complexes having the same three-dimensional fold, and thus the same function, but with completely different binding interfaces.
Predictable αβ T-Cell Receptor Selection toward an HLA-B*3501-Restricted Human Cytomegalovirus Epitope
Authors: Brennan Rebekah M. , Miles John J. , Silins Sharon L. , Bell Melissa J. , Burrows Jacqueline M. , Burrows Scott R.
Published: April 2007

Human cytomegalovirus (HCMV) elicits a very large burden on the immune system, with approximately one in ten T cells being reserved solely to manage this infection. However, information on the clonotypic composition of these vast T-cell populations is limited. In this study, we sequenced 116 T-cell receptor (TcR) α/β-chains specific for the highly immunogenic HLA-B*3501-resticted epitope IPSINVHHY from the pp65 antigen. Interestingly, T cells recovered from all donors bore an identical or near-identical TRBV28/TRBJ1-4/TRAV17/TRAJ33 TcR. The ability to predict the responding αβ TcR repertoire before viral infection should prove a powerful tool for basic and clinical immunology.
DNA Immunization with Plasmids Encoding Fusion and Nucleocapsid Proteins of Bovine Respiratory Syncytial Virus Induces a Strong Cell-Mediated Immunity and Protects Calves against Challenge
Authors: Boxus Mathieu , Tignon Marylène , Roels Stefan , Toussaint Jean-François , Walravens Karl , Benoit Marie-Ange , Coppe Philippe , Letesson Jean-Jacques , Letellier Carine , Kerkhofs Pierre
Published: April 2007

Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed two codon-optimized plasmids encoding the bovine RSV fusion (F) and nucleocapsid (N) proteins and assessed their immunogenicity in young calves. Two administrations of both plasmids elicited low antibody levels but primed a strong cell-mediated immunity characterized by lymphoproliferative response and gamma interferon production in vitro and in vivo. Interestingly, this strong cellular response drastically reduced viral replication, clinical signs, and pulmonary lesions after a highly virulent challenge. Moreover, calves that were further vaccinated with a killed-virus vaccine developed high levels of neutralizing antibody and were fully protected following challenge. These results indicate that DNA vaccination could be a promising alternative to the classical vaccines against RSV in cattle and could therefore open perspectives for vaccinating young infants.