bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimized construction of microsatellite‐enriched libraries
Authors: TECHEN NATASCHA , ARIAS RENÉE S. , GLYNN NEIL C. , PAN ZHIQIANG , KHAN IKHLAS A. , SCHEFFLER BRIAN E.
Published: December 2009

The construction of microsatellite‐enriched libraries is an indispensable tool to search for molecular markers as complete genome sequences are still not available for the majority of species of interest. Numerous protocols are available in the literature for the construction of these libraries; however, sometimes their low efficiency or lack of optimization in the protocols can restrict their efficacy. We have designed and tested various adapters and ligation methods; we also tested oligo‐repeat combinations and hybridization temperatures, and created libraries with this new protocol for four organisms: Ipomoea batatas (L.) Lam, Chionanthus retusus Lindley & Paxton, Rotylenchulus reniformis Linford & Olivera and Puccinia kuehnii W. Krüger. The number of microsatellites detected for these species ranged from 2494 to 3919 per Mb of nonredundant sequence, that was 0.86 and 1.53 microsatellites per contig, with 37–66% of di‐nucleotide motifs and 21–49% of tri‐ to octa‐nucleotide repeats combined. A simplified protocol is provided for the successful generation of SSR‐enriched libraries.
Combined effects of two stressors on Kenyan coral reefs are additive or antagonistic, not synergistic
Authors: Darling Emily S. , McClanahan Timothy R. , Côté Isabelle M.
Published: December 2009

A challenge for conservation science is predicting the impacts of co‐occurring human activities on ecological systems. Multiple anthropogenic and natural stressors impact ecosystems globally and are expected to jeopardize their ecological functions and the success of conservation and management initiatives. The possibility that two or more stressors interact synergistically is of particular concern, but such nonadditive effects remain largely unidentified in nature. A long‐term data set of hard coral cover from Kenyan reefs was used to examine the independent and interactive effects of two stressors: fishing and a temperature anomaly in 1998 that caused mass coral bleaching and mortality. While both stressors decreased coral cover, fishing by 51% and bleaching by 74%, they did not interact synergistically. Instead, their combined effect was antagonistic or weakly additive. The observed nonsynergistic response may be caused by the presence of one dominant stressor, bleaching, and cotolerance of coral taxa to both bleaching and fishing stressors. Consequently, coral bleaching has been the dominant driver of coral loss on Kenyan reefs and while marine reserves offer many benefits to reef ecosystems, they may not provide corals with a refuge from climate change.
Rapid in situ detection of virulent Vibrio vulnificus strains in raw oyster matrices using real‐time PCR
Authors: Baker‐Austin Craig , Gore Anthony , Oliver James D. , Rangdale Rachel , McArthur J Vaun , Lees David N.
Published: December 2009

Vibrio vulnificus is a Gram‐negative bacterial pathogen responsible for the vast majority of bacterially mediated fatalities from the consumption of raw or undercooked seafood in the USA. Vibrio vulnificus‐associated septicaemia can occur rapidly (< 24 h); however, methods for the isolation and confirmation of V. vulnificus from seafood samples typically require several days. A real‐time PCR assay was developed for V. vulnificus biotype 1 that provides a rapid means of identifying a gene fragment (vcgC) previously indicated as a strong predictor of potential virulence. PCR probe specificity was confirmed by amplification of 17 clinical V. vulnificus strains and by the lack of amplification with seven non‐pathogenic V. vulnificus isolates and a wide range of closely related bacteria. Oyster and seawater samples were amended with a range of environmentally realistic concentrations of C‐genotype V. vulnificus cells, which were quantitatively and unambiguously identified according to biotype. Of some significance, we utilized a sample processing and nucleic acid extraction procedure that allowed identification of pathogenic strains of V. vulnificus from oyster matrices without prior enrichment or culturing of strains. This outlined approach allowed the detection of as little as 50 cfu of V. vulnificus in less than 5 h, which compares favourably with culture‐based approaches. The results indicate the applicability of this approach for monitoring purposes or as a potential diagnostic tool in clinical settings.
The Influence of Grooves on the Fully Wetted and Aerated Flow Between Open Clutch Plates
Authors: Aphale Chinar R. , Schultz William W. , Ceccio Steven L.
Published: December 2009

In a disengaged or open clutch mode, one plate rotates while the other is stationary. This speed difference between the two plates (on the order of 1000 rpm) and the small clearance between them (on the order of 100 μm) results in large velocity gradients. Transmission fluid is passed between clutches since during reengagement; this oil provides lubrication and carries heat away. However, during the disengaged mode the shearing of this oil as it passes between the plates results in viscous drag that wastes power. Introduction of air between the two plates during the disengaged mode, referred to as aeration, is the most significant way of reducing this friction drag due to low viscosity of air compared with oil. Open clutch drag reduction is enhanced by providing grooves on one of the plates since they are known to promote aeration. Yet, a continuous supply of lubrication oil is necessary, even during disengagement. This study examines the underlying processes responsible for the oil flow between grooved disks and possible aeration through a combination of experiments and numerical computations. A two-dimensional model of the three-dimensional, single-phase flow between a stationary and a rotating clutch plate is presented, which is capable of describing pressures and shear stress distribution for plates with radial grooved geometries. The computational fluid dynamics code FLUENT® is used to examine the single-phase and aerated flows between the plates. These results are compared with accompanying experimental observations. We also examine new groove designs to study their efficiency in promoting aeration. Finally, we propose reasons for grooves promoting aeration in clutches.
A Transient Mixed Elastohydrodynamic Lubrication Model for Spur Gear Pairs
Authors: Li S. , Kahraman A.
Published: December 2009

In this study, a transient, non-Newtonian, mixed elastohydrodynamic lubrication (EHL) model of involute spur gear tooth contacts is proposed. Unlike the contact between two cylindrical rollers, spur gear contacts experience a number of time-varying contact parameters including the normal load, radii of curvature, surface velocities, and slide-to-roll ratio. The proposed EHL model is designed to continuously follow the contact of a tooth pair from the root to the tip to capture the transient characteristics of lubricated spur gear contacts due to these parameter variations, instead of analyzing the contact at discrete positions assuming time-invariant parameters. The normal tooth force along the line of action is predicted by using a gear load distribution formulation and the contact radii and tangential surface velocities are computed from the kinematics and geometry of involute profiles. A unified numerical approach is adapted for handling asperity interaction in mixed EHL conditions. The differences between the transient and discrete EHL analyses are shown for a spur gear pair having smooth surfaces and different tooth profile modifications. The transient behavior predicted by the proposed model is found to be mainly due to the squeezing and pumping effects caused by sudden load changes. The lubrication behavior under rough conditions is also investigated at different operating conditions.
Vibration and Acoustic Emission of Linear-Guideway Type Recirculating Ball Bearings With a Millimeter-Sized Artificial Defect in the Carriage
Authors: Ohta Hiroyuki , Matsuura Kazuya , Kato Soichiro , Igarashi Yutaka
Published: December 2009

This paper deals with vibrations and acoustic emissions (AEs) of linear-guideway type recirculating ball bearings with a millimeter-sized artificial defect in the carriage. The vibration and AE of one normal bearing without a defect (Type N), and six defective bearings (Types D1–D6) were measured using a linear velocity of 1 m/s. The defects in the bearings range from 1.87 mm to 6.77 mm in length, 2.45 mm to 3.80 mm in width, and 23.3 μm to 68.0 μm in depth. The experimental results show that the pulse amplitudes of the vibrations and AE (both the peak-to-peak and RMS values) of the defective bearings have a tendency to be greater than those of the normal bearing. Both the measured vibration and AE components over 30 kHz increased in the carriages with defects. To explain these increases in the defective bearings, a collision model with balls and one defect in the carriage is presented. That collision model shows that the increases in both the vibrations and AE of the defective bearings are caused by increases in the defect angle. A reasonable correlation exists between the presented collision model and the measured vibrations or AE of the defective bearings.
On the Modeling of Quasi-Steady and Unsteady Dynamic Friction in Sliding Lubricated Line Contact
Authors: Sojoudi H. , Khonsari M. M.
Published: December 2009

A simple but realistic dynamic friction model for the lubricated sliding contact is developed based on decoupling the steady and unsteady terms in Reynolds equation. The model realistically captures the physics of friction behavior both when speed is increased unidirectionally or when operating under oscillating condition. The model can simulate the transition from boundary to mixed to full film regimes as the speed is increased. Two different classes of simulations are performed to show the utility of the model: the so-called quasisteady, where the sliding velocity is varied very slowly, and the oscillating sliding velocity, where the friction coefficient exhibits a hysteresis type behavior. Both categories of simulation are verified by comparing the results with published experimental data.
The Effect of Surface Conditions on Friction by Tip Test
Authors: Jung Ki-Ho , Lee Hyun-Chul , Ajiboye Joseph S. , Kang Seong-Hoon , Im Yong-Taek
Published: December 2009

In the present investigation, a tip test based on upsetting and backward extrusion was utilized to characterize the effect of surface roughness of the billet and forming tools, and the type of lubricants on friction. For the test, cylindrical specimens made of aluminum alloys of 6061-O and 2024-O, and single punch and two die sets with different surface topologies, were used with four lubricants such as VG32, VG100, corn oil, and grease. The load levels and tip distances were measured for both materials, and compared with each other to determine shear friction factors at the punch and counter punch interfaces separately, depending on the variation in surface topologies and lubrications using finite element simulations. As a result, a linear relationship among the dimensionless load, tip distance, and shear friction factors at the punch and counter punch interfaces was derived for the experimental conditions investigated. The slope change of this linear relationship from negative to positive clearly depends on the variation in surface conditions at the billet/punch and billet/counter punch interfaces. Also, it was demonstrated that the dimensionless tip distance for the frictionless case can be extrapolated from the experimental data. This value can be used for characterizing the relative effect on friction due to surface conditions at the punch and counter punch, and lubrication quality of the lubricant for the given processing conditions.
On the Behavior of Misaligned Journal Bearings Based on Mass-Conservative Thermohydrodynamic Analysis
Authors: Jang J. Y. , Khonsari M. M.
Published: December 2009

Misalignment affects nearly all the bearing performance parameters including the cavitation, thermal field, leakage flow-rate, and moments. The present paper provides a comprehensive analysis of misaligned journal bearings based on a three-dimensional mass-conservative thermohydrodynamic model that appropriately takes into account the film rupture and reformation. An extensive set of numerical solutions are presented to closely examine the effects of misalignment in journal bearings.
A Contact Stiffness Model of Machined Plane Joint Based on Fractal Theory
Authors: Jiang Shuyun , Zheng Yunjian , Zhu Hua
Published: December 2009

A general contact stiffness model is proposed in this paper to study the contacts between rough surfaces of machined plane joints. The proposed model uses fractal geometry for surface topography description, elastic-plastic deformation of contacting asperities, and size-dependent contact stiffness of microcontacts, where the contact stiffness is derived from Hertz contact theory. Three cast iron specimens are produced from different machining methods (milling, grinding, and scraping), and their rough surface profiles are extracted. The structure function method was used to calculate each profile’s fractal dimension and scale coefficient. Both theoretical analysis and experimental results of contact stiffness are obtained for these specimens under different contact loads. The comparison between the theoretical contact stiffness and the experimental results at the interface indicates that the present fractal model for the contact stiffness is appropriate and the theoretical contact stiffness is consistent with the experimental data.
A New Analytic Approximation for the Hydrodynamic Forces in Finite-Length Journal Bearings
Authors: Bastani Yaser , de Queiroz Marcio
Published: December 2009

A new method for determining a closed-form expression for the hydrodynamic forces in finite-length plain journal bearings is introduced. The method is based on applying correction functions to the force models of the infinitely long (IL) or infinitely short (IS) bearing approximation. The correction functions are derived by modeling the ratio between the forces from the numerical integration of the two-dimensional Reynolds equation and the forces from either the IL or IS bearing approximation. Low-order polynomial models, dependent on the eccentricity ratio and aspect ratio, are used for the correction functions. A comparative computational study is presented for the steady-state behavior of the bearing system under static and unbalance loads. The results show the proposed models outperforming the standard limiting approximations as well as a model based on the finite-length impedance method.
An Analytical Solution to the Kinetics of Breakable Filament Assembly
Authors: Knowles Tuomas P. J. , Waudby Christopher A. , Devlin Glyn L. , Cohen Samuel I. A. , Aguzzi Adriano , Vendruscolo Michele , Terentjev Eugene M. , Welland Mark E. , Dobson Christopher M.
Published: December 2009

Dissecting Amyloid Formation

Amyloid fibrils are associated with clinical disorders ranging from Alzheimer's disease to type II diabetes. Their self-assembly can be described by a master equation that takes into account nucleation-dependent polymerization and fragmentation.

Knowles
et al.

(p.
1533
) now present an analytical solution to the master equation, which shows that amyloid growth kinetics is often limited by the fragmentation rate rather than by the rate of primary nucleation. In addition, the results reveal relationships between system properties (scaling laws) that provide mechanistic insight not only into amyloid growth, but also into related self-assembly processes.
Positively Selected
G6PD
-Mahidol Mutation Reduces
Plasmodium
vivax
Density in Southeast Asians
Authors: Louicharoen Chalisa , Patin Etienne , Paul Richard , Nuchprayoon Issarang , Witoonpanich Bhee , Peerapittayamongkol Chayanon , Casademont Isabelle , Sura Thanyachai , Laird Nan M. , Singhasivanon Pratap , Quintana-Murci Lluis , Sakuntabhai Anavaj
Published: December 2009

Ghosts of Selection

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency of humans, and it has been long suspected to exert an effect on
Plasmodium falciparum
malaria in Africa. Likewise, the increase in prevalence of the G6PD-Mahidol 487
A
allele among Karen people in Thailand, who only in the past few thousand years have migrated into malarious zones, may be the result of selection by
Plasmodium vivax
malaria.
P. vivax
has recently been implicated in more severe disease than previously suspected, providing both a direct selective effect through mortality and an indirect selective effect through morbidity and reproductive failure.

Louicharoen
et al.

(p.
1546
) link population-genetic evidence for positive selection in an 8-year family-based study of 3000 Karen individuals and reveal that there is an association between the presence of the G6PD-Mahidol 487
A
allele and a reduction in the density of
P. vivax
parasites circulating in the bloodstreams of infected individuals. The mutation appears to exert its effect on the physiology of immature red blood cells, which are the preferred niche for
P. vivax
but not of
P. falciparum
.
Meteorite Kr in Earth’s Mantle Suggests a Late Accretionary Source for the Atmosphere
Authors: Holland Greg , Cassidy Martin , Ballentine Chris J.
Published: December 2009

Gas Leak Inspection

The solid portion of Earth was formed from accretion of material and debris formed in the primitive Solar System. Earth's early evolution included the differentiation of its interior and the development of a primordial atmosphere. Heavy noble gases in the atmosphere could have been acquired during the initial accretion process or may have accumulated later through gravitational volatile capture.

Holland
et al.

(p.
1522
) show that Kr and Xe trapped in the upper mantle have isotopic signatures characteristic of early Solar System material similar to meteorites rather than those of the modern atmosphere and oceans. Thus, noble gases trapped within the young Earth did not contribute to Earth's later atmospheric composition.
A Reinnervating MicroRNA
Authors: Brown Robert H.
Published: December 2009

A microRNA expressed in skeletal muscle promotes the regeneration of neuromuscular synapses after injury and in a neurodegenerative disease model.
Biomass Fuel Starts to See the Light
Authors: Service Robert F.
Published: December 2009

Even with a major push, commercial plants capable of turning CO
2
or water into liquid fuels are still likely to be 2 decades away. A simpler version of the technology, however, already appears headed to market.
HIV Natural Resistance Field Finally Overcomes Resistance
Authors: Cohen Jon
Published: December 2009

People who fend off HIV despite repeated exposures may have genetic or immunologic factors working for them that can help guide AIDS vaccine research.
Evaluation of Three Multiplex Flow Immunoassays Compared to an Enzyme Immunoassay for the Detection and Differentiation of IgG Class Antibodies to Herpes Simplex Virus Types 1 and 2
Authors: Binnicker M. J. , Jespersen D. J. , Harring J. A.
Published: December 2009

The diagnosis of herpes simplex virus (HSV) infections is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture. However, in instances of subclinical or unrecognized HSV infection, serologic testing for IgG class antibodies to type-specific HSV glycoprotein G (gG) may be useful. This study evaluated and compared the performances of three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) for the simultaneous detection of gG type-specific IgG antibodies to HSV types 1 and 2 (HSV-1 and HSV-2). Serum specimens (
n
= 505) submitted for routine gG type-specific HSV IgG testing by enzyme immunoassay (EIA) (HerpeSelect; Focus Diagnostics) were also tested by the three multiplex flow immunoassays. Specimens showing discordant results were tested by HSV type-specific Western blotting (WB). For HSV-1 IgG, the AtheNA, BioPlex, and Plexus assays demonstrated agreements of 94.9% (479/505 specimens), 97.8% (494/505 specimens), and 97.4% (492/505 specimens), respectively, with the results of EIA. For HSV-2 IgG, the AtheNA, BioPlex, and Plexus assays showed agreements of 87.9% (444/505 specimens), 97.2% (491/505 specimens), and 96.8% (489/505 specimens), respectively, with EIA results. Timing studies showed that the AtheNA, BioPlex, and Plexus assays could provide complete analysis of 90 serum specimens in 3.1, 1.5, and 2.9 h, respectively, versus 3.1 h by EIA. These findings suggest that the gG type-specific HSV IgG multiplex immunoassays may be beneficial to high-volume clinical laboratories experiencing significant increases in the number of specimens submitted for HSV serologic testing. The evaluated systems provide comparable results to those of EIA, while reducing hands-on time and eliminating the necessity to aliquot specimens prior to testing.
A Live Oral Recombinant
Salmonella enterica
Serovar Typhimurium Vaccine Expressing
Clostridium perfringens
Antigens Confers Protection against Necrotic Enteritis in Broiler Chickens
Authors: Kulkarni R. R. , Parreira V. R. , Jiang Y.-F. , Prescott J. F.
Published: December 2009

Necrotic enteritis (NE) in broiler chickens is caused by
Clostridium perfringens
, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for
Salmonella
and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated
Salmonella enterica
serovar Typhimurium χ9352 vaccine vehicle. The expression of these
C
lostridium perfringens
proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with
Salmonella
expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against
Clostridium
and
Salmonella
antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a
Salmonella
vaccine vehicle.
Optimal Threshold for a Positive Hybrid Capture 2 Test for Detection of Human Papillomavirus: Data from the ARTISTIC Trial
Authors: Sargent A. , Bailey A. , Turner A. , Almonte M. , Gilham C. , Baysson H. , Peto J. , Roberts C. , Thomson C. , Desai M. , Mather J. , Kitchener H.
Published: December 2009

We present data on the use of the Hybrid Capture 2 (HC2) test for the detection of high-risk human papillomavirus (HR HPV) with different thresholds for positivity within a primary screening setting and as a method of triage for low-grade cytology. In the ARTISTIC population-based trial, 18,386 women were screened by cytology and for HPV. Cervical intraepithelial neoplasia lesions of grade two and higher (CIN2+ lesions) were identified for 453 women within 30 months of an abnormal baseline sample. When a relative light unit/cutoff (RLU/Co) ratio of ≥1 was used as the threshold for considering an HC2 result positive, 15.6% of results were positive, and the proportion of CIN2+ lesions in this group was 14.7%. The relative sensitivity for CIN2+ lesion detection was 93.4%. When an RLU/Co ratio of ≥2 was used as the threshold, there was a 2.5% reduction in positivity, with an increase in the proportion of CIN2+ lesions detected. The relative sensitivity decreased slightly, to 90.3%. Among women with low-grade cytology, HPV prevalences were 43.7% and 40.3% at RLU/Co ratios of ≥1 and ≥2, respectively. The proportions of CIN2+ lesions detected were 17.3% and 18.0%, with relative sensitivities of 87.7% at an RLU/Co ratio of ≥1 and 84.2% at an RLU/Co ratio of ≥2. At an RLU/Co ratio of ≥1, 68.3% of HC2-positive results were confirmed by the Roche line blot assay, compared to 77.2% of those at an RLU/Co ratio of ≥2. Fewer HC2-positive results were confirmed for 35- to 64-year-olds (50.3% at an RLU/Co ratio of ≥1 and 63.2% at an RLU/Co ratio of >2) than for 20- to 34-year-olds (78.7% at an RLU/Co ratio of ≥1 and 83.7% at an RLU/Co ratio of >2). If the HC2 test is used for routine screening as an initial test or as a method of triage for low-grade cytology, we would suggest increasing the threshold for positivity from the RLU/Co ratio of ≥1, recommended by the manufacturer, to an RLU/Co ratio of ≥2, since this study has shown that a beneficial balance between relative sensitivity and the proportion of CIN2+ lesions detected is achieved at this threshold.
Wound Botulism Complicating Internal Fixation of a Complex Radial Fracture
Authors: Taylor Steve M. , Wolfe Cameron R. , Dixon Terry C. , Ruch David S. , Cox Gary M.
Published: December 2009

Botulism developed in a patient following surgical repair of an open radial fracture. Symptoms resolved after treatment with antitoxin and antibiotics, and hardware excision was deferred. Subsequent osteomyelitis necessitated hardware exchange, and wound cultures grew
Clostridium argentinense
. This case highlights the management of botulism associated with orthopedic hardware.
Serotyping Pneumococcal Meningitis Cases in the African Meningitis Belt by Use of Multiplex PCR with Cerebrospinal Fluid
Authors: Njanpop Lafourcade Berthe-Marie , Sanou Oumarou , van der Linden Mark , Levina Natalia , Karanfil Meryem , Yaro Seydou , Tamekloe Tsidi A. , Mueller Judith E.
Published: December 2009

We reformulated a multiplex PCR algorithm for serotyping of pneumococcal meningitis directly on cerebrospinal fluid (CSF). Compared to established methods on isolates, CSF-based PCR had at least 80% sensitivity and 100% specificity. In regional meningitis surveillance, CSF-based PCR increased the serotype information yield from 40% of cases (isolate testing) to 90%.
Comparison of the Abbott RealTi
m
e CT New Formulation Assay with Two Other Commercial Assays for Detection of Wild-Type and New Variant Strains of
Chlamydia trachomatis
Authors: Møller Jens Kjølseth , Pedersen Lisbeth Nørum , Persson Kenneth
Published: December 2009

In an analytical-method comparison study of clinical samples, the Abbott RealTi
m
e CT new formulation assay (
m
2000 real-time PCR), consisting of a duplex PCR targeting different parts of the cryptic plasmid in
Chlamydia trachomatis
, was compared both with version 2 of the Roche Cobas TaqMan CT assay, comprising a duplex PCR for a target in the cryptic plasmid and the
omp1
gene, and with the Gen-Probe Aptima Combo 2 assay (AC2) targeting the
C. trachomatis
23S rRNA molecule. First-catch urine samples from Sweden were tested in Malmö, Sweden, for
C. trachomatis
with the
m
2000 real-time PCR assay and with an in-house PCR for the new variant
C. trachomatis
strain with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark, where they were further examined with the TaqMan CT and AC2 assays. A positive prevalence of 9.1% (148/1,632 urine samples examined) was detected according to the combined reference standard. The sensitivities and specificities of the three assays were as follows: for the Abbott
m
2000 assay, 95.3% (141/148) and 99.9% (1,483/1,485), respectively; for the Roche TaqMan assay, 82.4% (122/148) and 100.0% (1,485/1,485); and for the Gen-Probe AC2 assay, 99.3% (147/148) and 99.9% (1,484/1,485). The plasmid mutant strain was detected in 24% (36/148) of the
C. trachomatis
-positive samples. There is a difference in sensitivity between the new formulations of the Abbott and the Roche assays, but both assays detected the wild-type and new variant
C. trachomatis
strains equally well.
Correlation of Genotype and
In Vitro
Susceptibilities of
Cryptococcus gattii
Strains from the Pacific Northwest of the United States
Authors: Iqbal Naureen , DeBess Emilio E. , Wohrle Ron , Sun Ben , Nett Randall J. , Ahlquist Angela M. , Chiller Tom , Lockhart Shawn R.
Published: December 2009

Cryptococcus gattii
emerged in North America in 1999 as a human and veterinary pathogen on Vancouver Island, British Columbia. The emergent subtype, VGIIa, and the closely related subtype VGIIb can now be found in the United States in Washington, Oregon, and California. We performed multilocus sequence typing and antifungal susceptibility testing on 43 isolates of
C. gattii
from human patients in Oregon, Washington, California, and Idaho. In contrast to Vancouver Island, VGIIa was the most frequent but not the predominant subtype in the northwest United States. Antifungal susceptibility testing showed statistically significant differences in MICs between the subtypes. This is the first study to apply antifungal susceptibility testing to
C. gattii
isolates from the Pacific Northwest and the first to make direct comparisons between subtypes.
PCR Using Blood for Diagnosis of Invasive Pneumococcal Disease: Systematic Review and Meta-Analysis
Authors: Avni Tomer , Mansur Nariman , Leibovici Leonard , Paul Mical
Published: December 2009

The use of molecular-based methods for the diagnosis of bacterial infections in blood is appealing, but they have not yet passed the threshold for clinical practice. A systematic review of prospective and case-control studies assessing the diagnostic utility of PCR directly with blood samples for the diagnosis of invasive pneumococcal disease (IPD) was performed. A broad search was conducted to identify published and unpublished studies. Two reviewers independently extracted the data. Summary estimates for sensitivity and specificity with 95% confidence intervals (CIs) were calculated by using the hierarchical summary receiver operating characteristic method. The effects of sample processing, PCR type, the gene-specific primer, study design, the participants' age, and the source of infection on the diagnostic odds ratios were assessed through meta-regression. Twenty-nine studies published between 1993 and 2009 were included. By using pneumococcal bacteremia for case definition and healthy people or patients with bacteremia caused by other bacteria as controls (22 studies), the summary estimates for sensitivity and specificity were 57.1% (95% CI, 45.7 to 67.8%) and 98.6% (95% CI, 96.4 to 99.5%), respectively. When the controls were patients suspected of having IPD without pneumococcal bacteremia (26 studies), the respective values were 66.4% (95% CI, 55.9 to 75.6%) and 87.8% (95% CI, 79.5 to 93.1%). With lower degrees of proof for IPD (any culture or serology result and the clinical impression), the sensitivity of PCR decreased and the specificity increased. All analyses were highly heterogeneous. The use of nested PCR and being a child were associated with low specificity, while the use of a cohort study design was associated with a low sensitivity. The lack of an appropriate reference standard might have caused underestimation of the performance of the PCR. Currently available methods for PCR with blood samples for the diagnosis of IPD lack the sensitivity and specificity necessary for clinical practice.
Direct Serogrouping of
Streptococcus pneumoniae
Strains in Clinical Samples by Use of a Latex Agglutination Test
Authors: Sanz Juan Carlos , Culebras Esther , Ríos Esther , Rodríguez-Avial Iciar , Wilhelmi Isabel , Ramos Belén , Ordobás María , Picazo Juan José
Published: December 2009

Pneumotest-Latex (Statens Seruminstitut) was evaluated for direct serogrouping of
Streptococcus pneumoniae
strains in clinical samples from patients with invasive disease. The technique was accurate to its level of discrimination for 62 of 67 clinical samples (92.5%). Pneumotest-Latex would be a useful alternative for direct serogrouping of pneumococci in clinical samples.
K2 Serotype
Klebsiella pneumoniae
Causing a Liver Abscess Associated with Infective Endocarditis
Authors: Rivero Andres , Gomez Eric , Alland David , Huang David B. , Chiang Tom
Published: December 2009

Klebsiella pneumoniae
primary liver abscess (KPLA) is an emerging disease that is associated with distant septic complications. We report the first case of KPLA associated with infective endocarditis. The
K. pneumoniae
strain was a hypermucoid K2 serotype carrying the
rmpA
virulence-associated gene.
First Autochthonous Case of
Rhinocladiella mackenziei
Cerebral Abscess Outside the Middle East
Authors: Badali Hamid , Chander Jagdish , Bansal Shaifali , Aher Atul , Borkar Surendra S. , Meis Jacques F. , De Hoog G. Sybren
Published: December 2009

Cerebral phaeohyphomycosis due to
Rhinocladiella mackenziei
is a severe infection in the middle east, resulting in nearly 100% mortality despite the application of combined surgical and antifungal therapy and occurring occasionally in otherwise healthy patients. We report the first case of brain infection in a middle-aged male in India, where
R. mackenziei
is not endemic.
Crystal Structure of an Aquabirnavirus Particle: Insights into Antigenic Diversity and Virulence Determinism
Authors: Coulibaly Fasséli , Chevalier Christophe , Delmas Bernard , Rey Félix A.
Published: December 2009

Infectious pancreatic necrosis virus (IPNV), a pathogen of salmon and trout, imposes a severe toll on the aquaculture and sea farming industries. IPNV belongs to the
Aquabirnavirus
genus in the
Birnaviridae
family of bisegmented double-stranded RNA viruses. The virions are nonenveloped with a T=13
l
icosahedral capsid made by the coat protein VP2, the three-dimensional (3D) organization of which is known in detail for the family prototype, the infectious bursal disease virus (IBDV) of poultry. A salient feature of the birnavirus architecture is the presence of 260 trimeric spikes formed by VP2, projecting radially from the capsid. The spikes carry the principal antigenic sites as well as virulence and cell adaptation determinants. We report here the 3.4-Å resolution crystal structure of a subviral particle (SVP) of IPNV, containing 20 VP2 trimers organized with icosahedral symmetry. We show that, as expected, the SVPs have a very similar organization to the IBDV counterparts, with VP2 exhibiting the same overall 3D fold. However, the spikes are significantly different, displaying a more compact organization with tighter packing about the molecular 3-fold axis. Amino acids controlling virulence and cell culture adaptation cluster differently at the top of the spike, i.e., in a central bowl in IBDV and at the periphery in IPNV. In contrast, the spike base features an exposed groove, conserved across birnavirus genera, which contains an integrin-binding motif. Thus, in addition to revealing the viral antigenic determinants, the structure suggests that birnaviruses interact with different receptors for attachment and for cell internalization during entry.
An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome
Authors: Lelke Michaela , Brunotte Linda , Busch Carola , Günther Stephan
Published: December 2009

The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.
The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV
Authors: Ma Wenjun , Brenner Dominique , Wang Zhongfang , Dauber Bianca , Ehrhardt Christina , Högner Katrin , Herold Susanne , Ludwig Stephan , Wolff Thorsten , Yu Kangzhen , Richt Jürgen A. , Planz Oliver , Pleschka Stephan
Published: December 2009

A reassortant avian influenza virus (designated FPV NS GD), carrying the NS-segment of the highly pathogenic avian influenza virus (HPAIV) strain A/Goose/Guangdong/1/96 (GD; H5N1) in the genetic background of the HPAIV strain A/FPV/Rostock/34 (FPV; H7N1), was rescued by reverse genetics. Remarkably, in contrast to the recombinant wild-type FPV (rFPV), the reassortant virus was able to replicate more efficiently in different human cell lines and primary mouse epithelia cells without prior adaptation. Moreover, FPV NS GD caused disease and death in experimentally infected mice and was detected in mouse lungs; in contrast, rFPV was not able to replicate in mice effectively. These results indicated an altered host range and increased virulence. Furthermore FPV NS GD showed pronounced pathogenicity in chicken embryos. In an attempt to define the molecular basis for the apparent differences, we determined that NS1 proteins of the H5N1 and H7N1 strains bound the antiviral kinase PKR and the F2F3 domain of cleavage and polyadenylation specificity factor 30 (CPSF30) with comparable efficiencies
in vitro
. However, FPV NS GD infection resulted in (i) increased expression of NS1, (ii) faster and stronger PKR inhibition, and (iii) stronger beta interferon promoter inhibition than rFPV. Taken together, the results shed further light on the importance of the NS segment of an H5N1 strain for viral replication, molecular pathogenicity, and host range of HPAIVs and the possible consequences of a reassortment between naturally occurring H7 and H5 type HPAIVs.
The Murine Coronavirus Nucleocapsid Gene Is a Determinant of Virulence
Authors: Cowley Timothy J. , Long Simon Y. , Weiss Susan R.
Published: December 2009

The murine coronavirus, mouse hepatitis virus (MHV) strain A59, causes acute encephalitis and chronic demyelinating disease as well as hepatitis in mice. The JHM strain (also called MHV-4 or JHM.SD) causes fatal encephalitis and only minimal hepatitis. Previous analysis of chimeric recombinant MHVs in which the spike gene, encoding the protein that mediates viral entry and cell-to-cell fusion, was exchanged between JHM and A59 showed that the spike plays a major role in determining organ tropism and neurovirulence but that other genes also play important roles in pathogenic outcome. Here, we have investigated the role of the nucleocapsid protein in MHV-induced disease. The multifunctional nucleocapsid protein is complexed with the genomic RNA, interacts with the viral membrane protein during virion assembly, and plays an import role in enhancing the efficiency of transcription. A pair of chimeric recombinant viruses in which the nucleocapsid gene was exchanged between JHM and A59 was selected and compared to wild-type parental strains in terms of virulence. Importantly, expression of the JHM nucleocapsid in the context of the A59 genome conferred increased mortality and spread of viral antigen in the mouse central nervous system compared to the parental A59 strain, while having little effect on the induction of hepatitis. While the JHM nucleocapsid did not appear to enhance neuron-to-neuron spread in primary neuronal cultures, the increased neurovirulence it conferred may be due in part to the induction of a less robust T-cell response than that induced by strain A59.
Importance of Hydrogen Bond Contacts between the N Protein and RNA Genome of Vesicular Stomatitis Virus in Encapsidation and RNA Synthesis
Authors: Rainsford Edward W. , Harouaka Djamila , Wertz Gail W.
Published: December 2009

Vesicular stomatitis virus (VSV) genomic RNA encapsidated by the nucleocapsid (N) protein is the template for transcription and replication by the viral polymerase. We analyzed the 2.9-Å structure of the VSV N protein bound to RNA (T. J. Green, X. Zhang, G. W. Wertz, and M. Luo, Science 313:357-360, 2006) and identified amino acid residues with the potential to interact with RNA via hydrogen bonds. The contributions of these interactions to N protein function were investigated by individually substituting the residues with alanine and assaying the effect of these mutations on N protein expression, on the ability of the N protein to interact with the phosphoprotein (P), and on its ability to encapsidate RNA and generate templates that can support transcription and RNA replication. These studies identified individual amino acids critical for N protein function. Nine nucleotides are associated with each N monomer and contorted into two quasi-helices within the N protein RNA binding cavity. We found that N protein residues that formed hydrogen bond contacts with the nucleotides in quasi-helix 2 were critical to the encapsidation of RNA and the production of templates that can support RNA synthesis. Individual hydrogen bond interactions between the N protein and the nucleotides of quasi-helix 1 were not essential for ribonucleoprotein (RNP) template function. Residue R143 forms a hydrogen bond with nucleotide 9, the nucleotide that extends between N monomers. R143A mutant N protein failed to encapsidate RNA and to support RNA synthesis and suppressed wild-type N protein function. These studies show a direct correlation between viral RNA synthesis and N protein residues structurally positioned to interact with RNA.
Fine Mapping of Pre-S Sequence Requirements for Hepatitis B Virus Large Envelope Protein-Mediated Receptor Interaction
Authors: Schulze Andreas , Schieck Alexa , Ni Yi , Mier Walter , Urban Stephan
Published: December 2009

Previous studies showed that the N-terminal 75 amino acids of the pre-S1 domain of the hepatitis B virus (HBV) L protein are essential for HBV and hepatitis delta virus (HDV) infectivity. Consistently, synthetic lipopeptides encompassing this sequence or only parts of it efficiently block HBV and HDV infection, presumably through specific interference with a cellular receptor. Crucial for both virus infectivity and the inhibitory activity of the peptides are N-terminal myristoylation and a highly conserved motif within the N-terminal 48 amino acids. To refine the sequence requirements, we synthesized a series of HBV pre-S1 peptides containing deletions, point mutations,
d
-amino acid exchanges, or genotype-specific sequence permutations. Using the HepaRG cell line and a genotype D-derived virus, we determined the specific inhibitory activities of the peptides and found that (i) lipopeptides with an artificial consensus sequence inhibit HBV genotype D infection more potently than the corresponding genotype D peptides; (ii) point mutations,
d
-amino acid exchanges, or deletions introduced into the highly conserved part of the pre-S1 domain result in an almost complete loss of activity; and (iii) the flanking sequences comprising amino acids 2 to 8, 16 to 20, and, to a less pronounced extent, 34 to 48 gradually increase the inhibitory activity, while amino acids 21 to 33 behave indifferently. Taken together, our data suggest that HBV pre-S1-mediated receptor interference and, thus, HBV receptor recognition form a highly specific process. It requires an N-terminal acyl moiety and a highly conserved sequence that is present in primate but not rodent or avian hepadnaviruses, indicating different entry pathways for the different family members.
During Lytic Infections, Herpes Simplex Virus Type 1 DNA Is in Complexes with the Properties of Unstable Nucleosomes
Authors: Lacasse Jonathan J. , Schang Luis M.
Published: December 2009

The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono- and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono- to polynucleosome sizes in complexes fractionating as cellular mono- to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono- to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.
Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic
Authors: Xu Rui , McBride Ryan , Paulson James C. , Basler Christopher F. , Wilson Ian A.
Published: December 2009

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 Å resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.