bluuebunny/crossref_metadata_2025 huggingface dataset:
Major breed distribution of canine patients enucleated or eviscerated due to glaucoma following routine cataract surgery as well as common histopathologic findings within enucleated globes
Authors: Scott Erin M. , Esson Douglas W. , Fritz Kevin J. , Dubielzig Richard R.
Published: February 2013

Purpose

To identify the most significantly represented breeds that were enucleated or eviscerated due to intractable glaucoma following elective cataract surgery and to describe the major histopathologic features of enucleated globes.

Methods

A retrospective review of theComparativeOcularPathology Laboratory ofWisconsin database revealed 153 canine evisceration or enucleation submissions that met inclusion criteria. The most commonly represented breeds were identified, and enucleated globes of these breeds were further evaluated histologically (n = 69). Following standard hematoxylin & eosin staining, globes were evaluated for the presence of; corneal pathology, goniodysgenesis, pigment dispersion, pre‐iridal fibrovascular membranes (PIFVMs), peripheral anterior, and/or posterior synechiation, inflammatory infiltrates, lens pathology, retinal pathology, and/or endophthalmitis.

Results

The most significantly represented breeds were the Cocker Spaniel (11.7%),BostonTerrier (10.5%),LabradorRetriever (6.5%),BichonFrise (6.5%),ShihTzu (5.8%), andJackRussellTerrier (5.8%). Common histopathologic findings included inflammatory keratitis, the presence ofPIFVMs, and lymphoplasmacytic uveal infiltrates.

Conclusions

TheCockerSpaniel,BostonTerrier,LabradorRetriever,BichonFrise,ShihTzu, andJackRussellTerrier may be at increased risk of the development of glaucoma following elective cataract surgery. These findings have potential clinical implications.
Size‐related assessment on viability and insulin secretion of caprine islets in vitro
Authors: Vakhshiteh Faezeh , Allaudin Zeenathul Nazariah , Mohd Lila Mohd Azmi B. , Hani Homayoun
Published: February 2013

Background

The successful isolation, purification, and culture of caprine islets has recently been reported. The present study shows arange of size distribution in caprine islet diameter from 50 to 250 μm, in which 80% of the total islet yield was comprised of small islets.

Methods

Caprine islets were isolated and purified. Islets were handpicked and the diameter of the islets was recorded using light microscopy. Viablility of the islets was analyzed by confocal microscopy. Insulin secretion assay was carried out and analyzed by ELISA.

Results

When tested at 48 h after isolation, these small islets were 29.3% more viable compared to the large‐sized islets. Large islets showed a high ratio (P < 0.01) of central core necrosis (29.5% ± 1.92) whilst no significant core death was observed in small islets (2.33% ± 0.59). The annexin assay demonstrated 5.21% ± 0.97 and 7.34% ± 0.78 apoptotic death for small and large islets, respectively. During static incubation, small islets released 2.89‐fold (1.39 ± 0.2 ng/IE) higher insulin level under low glucose induction (3.3 mm) and simultaneously 2.92‐fold (2.95 ± 0.33 ng/IE) more insulin under high glucose condition (16.7 mm) in comparison to large islets at the same islet equivalents (P < 0.05).

Conclusion

The present findings evidenced the superior quality of smaller caprine islets compared to larger ones under an optimized basal maintenance condition. As it is equally important to preserve the quality of larger caprine islets, this work warrants further investigation on special culture conditions to support these islets.
Risk of Infectious Gastroenteritis in Young Children Living in Québec Rural Areas with Intensive Animal Farming: Results of a Case–Control Study (2004–2007)
Authors: Levallois P. , Chevalier P. , Gingras S. , Déry P. , Payment P. , Michel P. , Rodriguez M.
Published: February 2013

This study was designed to evaluate the epidemiology of severe gastroenteritis in children living in Québec rural areas with intensive livestock activities. From September 2005 through June 2007, 165 cases of gastroenteritis in children aged from 6 months to 5 years, hospitalized or notified to the public health department were enrolled, and 326 eligible controls participated. The parents of cases and controls were asked questions about different gastroenteritis risk factors. The quality of the drinking water used by the participants was investigated for microbial indicators as well as for four zoonotic bacterial pathogens (Campylobacter spp, Escherichia coli, Salmonella spp and Yersinia spp) and two enteric parasites (Cryptosporidium spp and Giardia spp). From 134 stool specimen analysed, viruses were detected in 82 cases (61%), while 28 (21%) were found with at least one of the bacteria investigated, and five cases were infected by parasites. Campylobacteriosis was the main bacterial infection (n = 15), followed by Salmonella sp (n = 7) and E. coli O157:H7 (n = 5) among cases with bacterial gastroenteritis. No significant difference was found between cases and controls regarding the quality of water consumed; the frequency of faecal contamination of private wells was also similar between cases and controls. Considering the total cases (including those with a virus), no link was found between severe gastroenteritis and either being in contact with animals or living in a municipality with the highest animal density (4th quartile). However, when considering only cases with a bacterial or parasite infection (n = 32), there was a weak association with pig density that was not statistically significant after adjusting for potential confounders. Contact with domestic, zoo or farm animals were the only environmental factor associated with the disease.
The André–Oort conjecture for Drinfeld modular varieties
Authors: Hubschmid Patrik
Published: February 2013

We consider the analogue of the André–Oort conjecture for Drinfeld modular varieties which was formulated by Breuer. We prove this analogue for special points with separable reflex field over the base field by adapting methods which were used by Klingler and Yafaev to prove the André–Oort conjecture under the generalized Riemann hypothesis in the classical case. Our result extends results of Breuer showing the correctness of the analogue for special points lying in a curve and for special points having a certain behaviour at a fixed set of primes.
Computing zeta functions of nondegenerate hypersurfaces with few monomials
Authors: Sperber Steven , Voight John
Published: February 2013

Using the cohomology theory of Dwork, as developed by Adolphson and Sperber, we exhibit a deterministic algorithm to compute the zeta function of a nondegenerate hypersurface defined over a finite field. This algorithm is particularly well suited to work with polynomials in small characteristic that have few monomials (relative to their dimension). Our method covers toric, affine, and projective hypersurfaces, and also can be used to compute the L-function of an exponential sum.
A comprehensive perturbation theorem for estimating magnitudes of roots of polynomials
Authors: Pakdemirli M. , Sarı G.
Published: February 2013

A comprehensive new perturbation theorem is posed and proven to estimate the magnitudes of roots of polynomials. The theorem successfully determines the magnitudes of roots for arbitrary degree of polynomial equations with no restrictions on the coefficients. In the previous papers ‘Pakdemirli and Elmas, Appl. Math. Comput. 216 (2010) 1645–1651’ and ‘Pakdemirli and Yurtsever, Appl. Math. Comput. 188 (2007) 2025–2028’, the given theorems were valid only for some restricted coefficients. The given theorem in this work is a generalization and unification of the past theorems and valid for arbitrary coefficients. Numerical applications of the theorem are presented as examples. It is shown that the theorem produces good estimates for the magnitudes of roots of polynomial equations of arbitrary order and unrestricted coefficients.
Full-Coverage Film Cooling: Film Effectiveness and Heat Transfer Coefficients for Dense Hole Arrays at Different Hole Angles, Contraction Ratios, and Blowing Ratios
Authors: Ligrani Phil , Goodro Matt , Fox Mike , Moon Hee-Koo
Published: February 2013

Experimental results are presented for a full-coverage film cooling arrangement which simulates a portion of a gas turbine engine, with appropriate streamwise static pressure gradient. The test surface utilizes varying blowing ratio (BR) along the length of the contraction passage which contains the cooling hole arrangement. For the different experimental conditions examined, film cooling holes are sharp-edged and streamwise inclined either at 20 deg or 30 deg with respect to the liner surface. The film cooling holes in adjacent streamwise rows are staggered with respect to each other. Data are provided for turbulent film cooling, contraction ratios of 1, 3, 4, and 5, blowing ratios (at the test section entrance) of 2.0, 5.0, and 10.0, coolant Reynolds numbers Refc of 10,000–12,000, freestream temperatures from 75 °C to 115 °C, a film hole diameter of 7 mm, and density ratios from 1.15 to 1.25. Nondimensional streamwise and spanwise film cooling hole spacings, X/D and Y/D, are 6, and 5, respectively. When the streamwise hole inclination angle is 20 deg spatially averaged and line-averaged adiabatic effectiveness values at each x/D location are about the same as the contraction ratio varies between 1, 3, and 4, with slightly higher values at each x/D location when the contraction ratio Cr is 5. For each contraction ratio, there is a slight increase in effectiveness when the blowing ratio is increased from 2.0 to 5.0 but there is no further substantial improvement when the blowing ratio is increased to 10.0. Overall, line-averaged and spatially averaged-adiabatic film effectiveness data, and spatially averaged heat transfer coefficient data are described as they are affected by contraction ratio, blowing ratio, hole angle α, and streamwise location x/D. For example, when α = 20 deg, the detrimental effects of mainstream acceleration are apparent since heat transfer coefficients for contraction ratios Cr of 3 and 5 are often higher than values for Cr = 1, especially for x/D > 100.
Application of Lamé's Super Ellipsoids to Model Initial Imperfections
Authors: Elishakoff Isaac , Bekel Yannis
Published: February 2013

In this paper, we investigate initial imperfections of structures that are modeled as belonging to a super ellipse or super ellipsoid. This extends previous analysis of regular ellipsoidal sets. We determine minimum area or minimum volume to which available data may belong. The methodology is illustrated on the case of impact buckling of a column possessing initial imperfections.
Self-Regulation of Motor Force Through Chemomechanical Coupling in Skeletal Muscle Contraction
Authors: Chen Bin
Published: February 2013

It is intriguing how the mechanics of molecular motors is regulated to perform the mechanical work in living systems. In sharp contrast to the conventional wisdom, recent experiments indicated that motor force maintains ∼6 pN upon a wide range of filament loads during skeletal muscle contraction at the steady state. Here we find that this rather precise regulation which takes place in an essentially chaotic system, can be due to that a “working” motor is arrested in a transitional state when the motor force is ∼6 pN. Our analysis suggests that the motor force can be self-regulated through chemomechanical coupling, and motor force homeostasis is a built-in feature at the level of a single motor, which provides insights to understanding the coordinated function of multiple molecular motors existing in various physiological processes. With a coupled stochastic-elastic numerical framework, the kinetic model for a Actin-myosin-ATP cycle constructed in this work might pave the way to decently investigate the transient behaviors of the skeletal muscle or other actomyosin complex structures.
MBE grown GaAsBi/GaAs double quantum well separate confinement heterostructures
Authors: Fan Dongsheng , Grant Perry C. , Yu Shui-Qing , Dorogan Vitaliy G. , Hu Xian , Zeng Zhaoquan , Li Chen , Hawkridge Michael E. , Benamara Mourad , Mazur Yuriy I. , Salamo Gregory J. , Johnson Shane R. , Wang Zhiming M.
Published: February 2013

GaAsBi/GaAs double quantum wells and double quantum well separate confinement heterostructures are grown at low temperatures using molecular beam epitaxy. Methods of achieving identical quantum wells in double quantum well structures without growth interruption are proposed and implemented. Cross-sectional transmission electron microscopy and room temperature photoluminescence measurements indicate that the samples have excellent structural and optical properties. The high optical quality of the samples is attributed to the surfactant effect of Bi throughout the low temperature growth of GaAs and AlGaAs layers. The proposed approach can be extended to grow laser diode structures with multiple quantum well separate confinement heterostructures containing more identical quantum wells.
Tight‐frame based iterative image reconstruction for spectral breast CT
Authors: Zhao Bo , Gao Hao , Ding Huanjun , Molloi Sabee
Published: February 2013

Purpose:

To investigate tight‐frame based iterative reconstruction (TFIR) technique for spectral breast computed tomography (CT) using fewer projections while achieving greater image quality.

Methods:

The experimental data were acquired with a fan‐beam breast CT system based on a cadmium zinc telluride photon‐counting detector. The images were reconstructed with a varying number of projections using the TFIR and filtered backprojection (FBP) techniques. The image quality between these two techniques was evaluated. The image's spatial resolution was evaluated using a high‐resolution phantom, and the contrast to noise ratio (CNR) was evaluated using a postmortem breast sample. The postmortem breast samples were decomposed into water, lipid, and protein contents based on images reconstructed from TFIR with 204 projections and FBP with 614 projections. The volumetric fractions of water, lipid, and protein from the image‐based measurements in both TFIR and FBP were compared to the chemical analysis.

Results:

The spatial resolution and CNR were comparable for the images reconstructed by TFIR with 204 projections and FBP with 614 projections. Both reconstruction techniques provided accurate quantification of water, lipid, and protein composition of the breast tissue when compared with data from the reference standard chemical analysis.

Conclusions:

Accurate breast tissue decomposition can be done with three fold fewer projection images by the TFIR technique without any reduction in image spatial resolution and CNR. This can result in a two‐third reduction of the patient dose in a multislit and multislice spiral CT system in addition to the reduced scanning time in this system.
Structural Biological Materials: Critical Mechanics-Materials Connections
Authors: Meyers Marc André , McKittrick Joanna , Chen Po-Yu
Published: February 2013

The Building Blocks of Life

Biological organisms are often limited in the resources that they can use to make structural materials. Primary building blocks may be weak or brittle materials, such as minerals and biopolymers, and processing conditions by default have to be mild. Despite this, a wide range of strong and tough structures exist, including shells, bones, quills, and fibers.

Meyers
et al.

(p.
773
) review a wide range of materials and architectures used in nature to make strong and tough materials and show how many of the design principles have also been used or are being considered for manmade materials and structures.
Comment on "Evolutionary Trade-Offs, Pareto Optimality, and the Geometry of Phenotype Space"
Authors: Edelaar Pim
Published: February 2013

Shoval
et al
. (Reports, 1 June 2012, p. 1157) showed how configurations of phenotypes may identify tasks that trade off with each other, using randomizations assuming independence of data points. I argue that this assumption may not be correct for most and possibly all examples and led to pseudoreplication and inflated significance levels. Improved statistical testing is necessary to assess how the theory applies to empirical data.
Prediction Error Governs Pharmacologically Induced Amnesia for Learned Fear
Authors: Sevenster Dieuwke , Beckers Tom , Kindt Merel
Published: February 2013

Overwriting Human Memory

A consolidated fear memory can enter a transient labile phase upon its reactivation. Pharmacological blockade of the subsequent protein synthesis-dependent restabilization produces a memory deficit.

Sevenster
et al.

(p.
830
) found that prediction error (a discrepancy between actual and expected events) is a necessary condition for reconsolidation of a fear memory in human subjects. Retrieval cued by a negative prediction error, for example, like a conditioned stimulus presented in the absence of the unconditioned stimulus, resulted in labilization and subsequent reconsolidation was blocked by the drug propranolol. However, if there was no prediction error because the conditioned stimulus and the aversive unconditioned stimulus were presented simultaneously, the memory did not enter a labile state and its reconsolidation could not be blocked.
A Unique Piece of Mars
Authors: Humayun Munir
Published: February 2013

A meteorite with a chemical makeup resembling that of rocks from Mars' Gusev Crater has been identified.

[Also see Research Article by

Agee
et al.

]
Sensing the Dark Side of DNA
Authors: O'Neill Luke A. J.
Published: February 2013

An enzyme that senses foreign and mislocated DNA in the cell cytoplasm acts as an alarm that triggers host defense responses.

[Also see Research Article by

Sun
et al.

and Report by

Wu
et al.

]
New Tool for Genome Surgery
Authors: Oost John van der
Published: February 2013

A bacterial system that uses RNA to edit DNA is harnessed for engineering mammalian genomes.

[Also see Reports by

Cong
et al.

and

Mali
et al.

]
Novel Aspects of the Z and R3 Antigens of Streptococcus agalactiae Revealed by Immunological Testing
Authors: Maeland Johan A. , Radtke Andreas , Lyng Randi V. , Mavenyengwa Rooyen T.
Published: February 2013

Group B streptococci (GBS) are important human and bovine pathogens which can be classified by a variety of phenotype- and gene-based techniques. The capsular polysaccharide and strain-variable, surface-anchored proteins are particularly important phenotypic markers. In an earlier study, a previously unrecognized protein antigen called Z was described. It was expressed by 27.2% of GBS strains from Zimbabwe, usually in combination with R3 protein expression. In this study, a putative Z-specific antiserum actually contained antibodies against two different antigens named Z1 and Z2; Z1 was >250 kDa in molecular mass. Z1, Z2, and R3 generated multiple stained bands on Western blots and showed similar chromatographic characteristics with respect to molecular mass, aggregate formation, and charge. Of 28 reference and prototype GBS strains examined, 8/28 (28.5%) isolates expressed one, two, or all three of the Z1, Z2, and R3 antigens; 4/28 expressed all three antigens; 2/28 expressed Z2 and R3; 1/28 expressed Z1 only; and 1/28 expressed R3 only. Twenty (71.5%) of the 28 isolates expressed none of the three antigens. Expression of one or more of these antigens was shown by isolates of the capsular polysaccharide types Ia, Ib, V, and IX and NT strains and occurred in combination with expression of various other strain-variable and surface-localized protein antigens. When used as serosubtype markers, Z1, Z2, and R3 affected existing GBS serotype designations for some of the isolates. For instance, the R3 reference strain Prague 10/84 (ATCC 49447) changed serotype markers from V/R3 to V/R3, Z1, and Z2. Other isolates may change correspondingly, implying consequences for GBS serotyping and research.
Adjuvanticity of a Recombinant Calreticulin Fragment in Assisting Anti-β-Glucan IgG Responses in T Cell-Deficient Mice
Authors: Li Wei-Ji , Long Kai , Dong Hong-Liang , Gao Xiao-Ming
Published: February 2013

Polysaccharide-encapsulated fungi are the chief source of diseases in immunocompromised hosts such as those infected with human immunodeficiency virus or neutropenia patients. Currently available polysaccharide-protein conjugate vaccines are mainly T cell dependent and are usually ineffective in weakened immune systems. In this study, laminarin, a well-characterized β-1,3-glucan, was conjugated with a prokaryotically expressed recombinant fragment (amino acids [aa] 39 to 272) of calreticulin (rCRT/39–272), which exhibits extraordinarily potent immunogenicity and adjuvanticity in experimental animals. The resultant conjugate reserves the immunostimulatory effect of rCRT/39–272 on naïve murine B cells and is capable of eliciting anti-β-glucan IgG (mostly IgG1) responses in not only BALB/c mice but also athymic nude mice. Laminarin-CRT-induced mouse antibodies (Abs) are able to bind with
Candida albicans
and inhibit its growth
in vitro
. In addition, vaccination with laminarin-CRT partially protects mice from lethal
C. albicans
challenge. These results imply that rCRT/39–272 could be used as an ideal carrier or adjuvant for carbohydrate vaccines aimed at inducing or boosting IgG responses to fungal infections in immunodeficient hosts.
Hemin-Binding Proteins as Potent Markers for Serological Diagnosis of Infections with Bartonella quintana
Authors: Matsuoka Mayumi , Sasaki Toshinori , Seki Naomi , Kobayashi Mutsuo , Sawabe Kyoko , Sasaki Yuko , Shibayama Keigo , Sasaki Tsuguo , Arakawa Yoshichika
Published: February 2013

It is difficult to distinguish infections with differentBartonellaspecies using commercially available immunofluorescence (indirect immunofluorescent antibody [IFA]) assay kits. To identify appropriate proteins for serodiagnosis ofBartonella quintanainfections, we investigated the antigenicity ofB. quintanaproteins using sera from homeless people with highB. quintanaIgG titers in IFA assay. These sera reacted strongly to an outer membrane protein, hemin-binding protein D (HbpD). Further, serum from an endocarditis patient infected withB. quintanareacted to HbpB and HbpD. To locate the antigenic sites within the proteins, we generated deletion mutants of HbpB and HbpD. Amino acid residues 89 to 220 of HbpB and 151 to 200 of HbpD were identified as the minimum regions required for recognition by these sera. Several oligopeptides comprising parts of the minimum regions of HbpB and HbpD were synthesized, and their immunoreactivity with the above-mentioned sera was tested by enzyme-linked immunosorbent assay (ELISA). Serum from the endocarditis patient reacted similarly to synthetic peptides HbpB2 (amino acid residues 144 to 173 of HbpB) and HbpD3 (151 to 200 residues of HbpD), while sera from the other subjects reacted to HbpD3. These results indicate that synthetic peptides HbpB2 and HbpD3 might be suitable for developing serological tools for differential diagnosis ofB. quintanainfections from otherBartonellainfections.
Molecular Characterization of Streptococcus pneumoniae Serotype 12F Isolates Associated with Rural Community Outbreaks in Alaska
Authors: Zulz Tammy , Wenger Jay D. , Rudolph Karen , Robinson D. Ashley , Rakov Alexey V. , Bruden Dana , Singleton Rosalyn J. , Bruce Michael G. , Hennessy Thomas W.
Published: February 2013

Outbreaks of invasive pneumococcal disease (IPD) caused by
Streptococcus pneumoniae
serotype 12F were observed in two neighboring regions of rural Alaska in 2003 to 2006 and 2006 to 2008. IPD surveillance data from 1986 to 2009 and carriage survey data from 1998 to 2004 and 2008 to 2009 were reviewed to identify patterns of serotype 12F transmission. Pulsed-field gel electrophoresis was performed on all available isolates, and selected isolates were characterized by additional genetic subtyping methods. Serotype 12F IPD occurred in two waves in Alaska between 1986 and 2008. While cases of disease occurred nearly every year in Anchorage, in rural regions, 12F IPD occurred with rates 10- to 20-fold higher than those in Anchorage, often with many years between disease peaks and generally caused by a single predominant genetic clone. Carriage occurred predominantly in adults, except early in the rural outbreaks, when most carriage was in persons <18 years old. In rural regions, carriage of 12F disappeared completely after outbreaks. Different 12F clones appear to have been introduced episodically into rural populations, spread widely in young, immunologically naïve populations (leading to outbreaks of IPD lasting 1 to 3 years), and then disappeared rapidly from the population. Larger population centers might have been the reservoir for these clones. This epidemiologic pattern is consistent with a highly virulent, but immunogenic, form of pneumococcus.
“Candidatus Mycoplasma haemomacaque” and Bartonella quintana Bacteremia in Cynomolgus Monkeys
Authors: Maggi Ricardo G. , Mascarelli Patricia E. , Balakrishnan Nandhakumar , Rohde Cynthia M. , Kelly Catherine M. , Ramaiah Lila , Leach Michael W. , Breitschwerdt Edward B.
Published: February 2013

Here, we report latent infections with
Bartonella quintana
and a hemotropic
Mycoplasma
sp. in a research colony of cynomolgus monkeys (
Macaca fascicularis
). Sequence alignments, evolutionary analysis, and signature nucleotide sequence motifs of the hemotropic
Mycoplasma
16S rRNA and RNase P genes indicate the presence of a novel organism.
Development of a Pseudotyped-Lentiviral-Vector-Based Neutralization Assay for Chikungunya Virus Infection
Authors: Kishishita Natsuko , Takeda Naokazu , Anuegoonpipat Atchareeya , Anantapreecha Surapee
Published: February 2013

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever in Africa, South Asia, and Southeast Asia. Because the mosquito vectorAedes albopictusis present in habitats across Europe, North America, and East Asia, CHIKV has become a serious worldwide public health concern. Infection with CHIKV typically causes fever, rash, myalgia, and arthralgia. One of the important questions yet to be answered is how the host immune system is involved in the development of this disease. In this study, we prepared a CHIKV-pseudotyped lentiviral vector for use in a safe and convenient neutralization (NT) assay and analyzed its efficacy. The CHIKV-pseudotyped lentiviral vector was prepared by cotransfection with plasmids encoding the CHIKV glycoproteins E3, E2, 6k, and E1, packaging elements, and a luciferase reporter. This alternative to native CHIKV can be safely handled in a biosafety level 2 facility. The NT assay was optimized using sera from CHIKV-immunized mice and then applied to human patient sera. The majority of the serum samples from patients with chikungunya in Thailand showed robust neutralization activities, with titers that were tightly correlated with those determined by a conventional NT assay. Moreover, there was a strong correlation with the CHIKV antibody titers as determined by enzyme-linked immunosorbent assay. Thus, the CHIKV-pseudotyped-lentiviral-vector-based NT assay system is a powerful tool for examining the neutralization activity of patient sera, which will lead to a better understanding of the immune responses involved in CHIKV infection.
Outbreak Investigation Using High-Throughput Genome Sequencing within a Diagnostic Microbiology Laboratory
Authors: Sherry Norelle L. , Porter Jessica L. , Seemann Torsten , Watkins Andrew , Stinear Timothy P. , Howden Benjamin P.
Published: February 2013

Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant
Escherichia coli
outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the
bla
CTX-M-15
extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete
E. coli
genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications.
An RNA Element in Human Interleukin 6 Confers Escape from Degradation by the Gammaherpesvirus SOX Protein
Authors: Hutin Stephanie , Lee Yeon , Glaunsinger Britt A.
Published: February 2013

Several viruses express factors to silence host gene expression via widespread mRNA degradation. This phenotype is the result of the coordinated activity of the viral endonuclease SOX and the cellular RNA degradation enzyme Xrn1 during lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection. While most cellular transcripts are highly downregulated, a subset of host mRNA escapes turnover via unknown mechanisms. One of the most prominent escapees is the interleukin 6 (IL-6) mRNA, which accumulates robustly during KSHV lytic infection and is not subjected to SOX-induced degradation. Here we reveal that the IL-6 mRNA contains a dominant,
cis
-acting ∼100-nucleotide element within its 3′ untranslated region (UTR) that renders it directly refractory to cleavage by SOX. This element specifically interacts with a cellular protein complex both in SOX-transfected cells and in KSHV-infected B cells. Using a directed RNA pulldown approach, we identified two components of this complex to be the AU-rich element (ARE) binding proteins AUF1 and HuR. Depletion of these proteins significantly reduced the protective capacity of the IL-6 RNA element in SOX-expressing cells. These findings suggest that SOX activity may be directly counteracted by select RNA regulatory complexes and reveal a novel mechanism contributing to the robust expression of IL-6 during KSHV replication.
Antibody-Dependent, FcγRI-Mediated Neutralization of HIV-1 in TZM-bl Cells Occurs Independently of Phagocytosis
Authors: Perez Lautaro G. , Zolla-Pazner Susan , Montefiori David C.
Published: February 2013

We previously showed that expression of human FcγRI on TZM-bl cells potentiates neutralization by gp41 membrane-proximal external region (MPER)-specific antibodies. Here we show that lysosomotropic reagents known to block phagocytosis do not diminish this effect. We also show that FcγRI occasionally potentiates neutralization by antibodies against the V3 loop of gp120 and cluster I of gp41. We conclude that FcγRI provides a kinetic advantage for neutralizing antibodies against partially cryptic epitopes independent of phagocytosis.
RNA Helicase Signaling Is Critical for Type I Interferon Production and Protection against Rift Valley Fever Virus during Mucosal Challenge
Authors: Ermler Megan E. , Yerukhim Ekaterina , Schriewer Jill , Schattgen Stefan , Traylor Zachary , Wespiser Adam R. , Caffrey Daniel R. , Chen Zhijian J. , King Charles H. , Gale Michael , Colonna Marco , Fitzgerald Katherine A. , Buller R. Mark L. , Hise Amy G.
Published: February 2013

Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both
in vitro
assays and
in vivo
mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
Membrane Topology and Function of Dengue Virus NS2A Protein
Authors: Xie Xuping , Gayen Shovanlal , Kang CongBao , Yuan Zhiming , Shi Pei-Yong
Published: February 2013

Flavivirus nonstructural protein 2A (NS2A) is a component of the viral replication complex that functions in virion assembly and antagonizes the host immune response. Although flavivirus NS2A is known to associate with the endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on the ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are located in the ER lumen, with one segment (amino acids 30 to 52) that interacts with ER membrane without traversing the lipid bilayer; (ii) amino acids 69 to 209 form five transmembrane segments, each of which integrally spans the ER membrane; and (iii) the C-terminal tail (amino acids 210 to 218) is located in the cytosol. Nuclear magnetic resonance (NMR) structural analysis showed that the first membrane-spanning segment (amino acids 69 to 93) consists of two helices separated by a “helix breaker.” The helix breaker is formed by amino acid P85 and one positively charged residue, R84. Functional analysis using replicon and genome-length RNAs of DENV-2 indicates that P85 is not important for viral replication. However, when R84 was replaced with E, the mutation attenuated both viral RNA synthesis and virus production. Remarkably, an R84A mutation did not affect viral RNA synthesis but blocked intracellular formation of infectious virions. Collectively, the mutagenesis results demonstrate that NS2A functions in both DENV RNA synthesis and virion assembly/maturation. The topology model of DENV NS2A provides a good starting point for studying how flavivirus NS2A modulates viral replication and evasion of host immune response.
A Whole-Genome RNA Interference Screen for Human Cell Factors Affecting Myxoma Virus Replication
Authors: Teferi Wondimagegnehu M. , Dodd Kristopher , Maranchuk Rob , Favis Nicole , Evans David H.
Published: February 2013

Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes (“hits”) and nonsignificant genes (“nonhits”) of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G1, or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G1/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-d-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.
Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling
Authors: Kumar Anil , Bühler Sandra , Selisko Barbara , Davidson Andrew , Mulder Klaas , Canard Bruno , Miller Sven , Bartenschlager Ralf
Published: February 2013

Dengue virus (DENV) is an important human pathogen, especially in the tropical and subtropical parts of the world, causing considerable morbidity and mortality. DENV replication occurs in the cytoplasm; however, a high proportion of nonstructural protein 5 (NS5), containing methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities, accumulates in the nuclei of infected cells. The present study investigates the impact of nuclear localization of NS5 on its known functions, including viral RNA replication and subversion of the type I interferon response. By using a mutation analysis approach, we identified the most critical residues within the αβ nuclear localization signal (αβNLS), which are essential for the nuclear accumulation of this protein. Although we observed an overall correlation between reduced nuclear accumulation of NS5 and impaired RNA replication, we identified one mutant with drastically reduced amounts of nuclear NS5 and virtually unaffected RNA replication, arguing that nuclear localization of NS5 does not correlate strictly with DENV replication, at least in cell culture. Because NS5 plays an important role in blocking interferon signaling via STAT-2 (signal transducer and activator of transcription 2) degradation, the abilities of the NLS mutants to block this pathway were investigated. All mutants were able to degrade STAT-2, with accordingly similar type I interferon resistance phenotypes. Since the NLS is contained within the RdRp domain, the MTase and RdRp activities of the mutants were determined by using recombinant full-length NS5. We found that the C-terminal region of the αβNLS is a critical functional element of the RdRp domain required for polymerase activity. These results indicate that efficient DENV RNA replication requires only minimal, if any, nuclear NS5, and they identify the αβNLS as a structural element required for proper RdRp activity.
Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection
Authors: Amorim Maria Joao , Kao Richard Y. , Digard Paul
Published: February 2013

Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as “molecular staples” that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.
Genetic and Functional Characterization of the N-Terminal Region of the Hepatitis C Virus NS2 Protein
Authors: de la Fuente Cynthia , Goodman Zachary , Rice Charles M.
Published: February 2013

The hepatitis C virus (HCV) NS2 protein has dual roles within the HCV life cycle. While well characterized as an autoprotease that cleaves the NS2/NS3 junction, NS2, primarily via its N-terminal region, is also involved in virion morphogenesis. In order to map the determinants necessary for infectious virus production and gain further insight into the multiple points at which NS2 may impact this process, a detailed mutational analysis of residues spanning amino acids (aa) 1 to 92 was performed. Initial block mutagenesis (5 or 7 amino acid residues) in both bicistronic and monocistronic HCV cell culture-based (HCVcc) genomes revealed that all but two blocks had various levels of impaired infectious virus production. None of these mutations affected RNA replication, indicating that the N-terminal region of NS2 is not required for NS2-3 processing and replicase assembly. Fine mapping identified 29 critical residues that, when mutated, yielded at least a 1 log decrease in infectious virus titers. These mutants were characterized further with respect to release of extracellular HCV RNA and core, intracellular infectivity, thermal stability of virus particles, and NS2 interactions. While the most severely debilitated mutants were impaired early in the assembly process, which is in agreement with previous reports, others targeted later steps of virus production, most notably egress. Thus, in addition to participating in early steps in virion assembly, this comprehensive mutagenesis study suggests yet another role for NS2 in later steps in virus production.
Analysis of the Early Steps of Herpes Simplex Virus 1 Capsid Tegumentation
Authors: Henaff Daniel , Rémillard-Labrosse Gaudeline , Loret Sandra , Lippé Roger
Published: February 2013

Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an
in vitro
nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay. As anticipated,
in vitro
cytoplasmic capsids did not harbor U
L
34, U
L
31, or viral glycoproteins but contained U
S
3. In agreement with previous findings, both nuclear and
in vitro
capsids were positive for ICP0 and ICP4. Unexpectedly, nuclear C capsids and cytoplasmic capsids produced
in vitro
without any cytosolic viral proteins also scored positive for U
L
36 and U
L
37. Immunoelectron microscopy confirmed that these tegument proteins were closely associated with nuclear capsids. When cytosolic viral proteins were present in the
in vitro
assay, no additional tegument proteins were detected on the capsids. As previously reported, the tegument was sensitive to high-salt extraction but, surprisingly, was stabilized by exogenous proteins. Finally, some tegument proteins seemed partially lost during egress, while others possibly were added at multiple steps or modified along the way. Overall, an emerging picture hints at the early coating of capsids with up to 5 tegument proteins at the nuclear stage, the shedding of some viral proteins during nuclear egress, and the acquisition of others tegument proteins during reenvelopment.
Multiple Pathways of Escape from HIV Broadly Cross-Neutralizing V2-Dependent Antibodies
Authors: Moore Penny L. , Sheward Daniel , Nonyane Molati , Ranchobe Nthabeleng , Hermanus Tandile , Gray Elin S. , Abdool Karim Salim S. , Williamson Carolyn , Morris Lynn
Published: February 2013

Broadly cross-neutralizing (BCN) antibodies are likely to be critical for an effective HIV vaccine. However, the ontogeny of such antibodies and their relationship with autologous viral evolution is unclear. Here, we characterized viral evolution in CAP256, a subtype C-infected individual who developed potent BCN antibodies targeting positions R166 and K169 in the V2 region. CAP256 was superinfected at 3 months postinfection with a virus that was highly sensitive to BCN V2-dependent monoclonal antibodies. The autologous neutralizing response in CAP256 was directed at V1V2, reaching extremely high titers (>1:40,000) against the superinfecting virus at 42 weeks, just 11 weeks prior to the development of the BCN response targeting the same region. Recombination between the primary and superinfecting viruses, especially in V2 and gp41, resulted in two distinct lineages by 4 years postinfection. Although neutralization of some CAP256 clones by plasma from as much as 2 years earlier suggested incomplete viral escape, nonetheless titers against later clones were reduced at least 40-fold to less than 1:1,000. Escape mutations were identified in each lineage, either at R166 or at K169, suggesting that strain-specific and BCN antibodies targeted overlapping epitopes. Furthermore, the early dependence of CAP256 neutralizing antibodies on the N160 glycan decreased with the onset of neutralization breadth, indicating a change in specificity. These data suggest rapid maturation, within 11 weeks, of CAP256 strain-specific antibodies to acquire breadth, with implications for the vaccine elicitation of BCN V2-dependent antibodies. Overall these studies demonstrate that ongoing viral escape is possible, even from BCN antibodies.