bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular pathogenicity of Streptococcus anginosus
Authors: Asam D. , Spellerberg B.
Published: May 2014

Streptococcus anginosus and the closely related species Streptococcus constellatus and Streptococcus intermedius, are primarily commensals of the mucosa. The true pathogenic potential of this group has been under‐recognized for a long time because of difficulties in correct species identification as well as the commensal nature of these species. In recent years, streptococci of the S. anginosus group have been increasingly found as relevant microbial pathogens in abscesses and blood cultures and they play a pathogenic role in cystic fibrosis. Several international studies have shown a surprisingly high frequency of infections caused by the S. anginosus group. Recent studies and a genome‐wide comparative analysis suggested the presence of multiple putative virulence factors that are well‐known from other streptococcal species. However, very little is known about the molecular basis of pathogenicity in these bacteria. This review summarizes our current knowledge of pathogenicity factors and their regulation in S. anginosus.
Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries‐free and caries‐positive human subjects
Authors: Hong S.W. , Seo D.‐G. , Baik J.E. , Cho K. , Yun C.‐H. , Han S.H.
Published: May 2014

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell‐wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA‐binding proteins (Sm.LTA‐BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA‐BPs in the saliva of caries‐free and caries‐positive human subjects using Sm.LTA‐conjugated beads and LTQ‐Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N‐hydroxysuccinimidyl‐Sepharose® 4 Fast Flow beads (Sm.LTA‐beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon‐γ‐inducible protein 10 in a murine macrophage cell line and activation of Toll‐like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA‐BPs were isolated from pooled saliva prepared from 10 caries‐free or caries‐positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high‐resolution mass spectrometry. A total of 8 and 12 Sm.LTA‐BPs were identified with statistical significance in the pooled saliva from the caries‐free and caries‐positive human subjects, respectively. Unique Sm.LTA‐BPs found in caries‐free saliva included histone H4, profilin‐1 and neutrophil defensin‐1, and those in caries‐positive saliva included cystatin‐C, cystatin‐SN, cystatin‐S, cystatin‐D, lysozyme C, calmodulin‐like protein 3 and β‐actin. The Sm.LTA‐BPs found in both groups were hemoglobin subunits α and β, prolactin‐inducible protein, protein S100‐A9, and SPLUNC2. Collectively, we identified Sm.LTA‐BPs in the saliva of caries‐free and caries‐positive subjects, which exhibit differential protein profiles.
Intraoperative neurophysiological monitoring in pediatric neurosurgery
Authors: Busso Veronica O. , McAuliffe John J.
Published: May 2014

The use of intraoperative neurophysiological monitoring (IONM) in pediatric neurosurgery is not new; however, its application to a wider range of procedures is a relatively new development. The purpose of this article is to review the physiology underlying the commonly employed IONM modalities and to describe their application to a subset of pediatric neurosurgical procedures.
Ultrasound‐guided rectus sheath block or wound infiltration in children: a randomized blinded study of analgesia and bupivacaine absorption
Authors: Flack Sean H. , Martin Lizabeth D. , Walker Benjamin J. , Bosenberg Adrian T. , Helmers Laurilyn D. , Goldin Adam B. , Haberkern Charles M.
Published: May 2014

Background

Rectus sheath block can provide analgesia following umbilical hernia repair. However, conflicting reports on its analgesic effectiveness exist. No study has investigated plasma local anesthetic concentration following ultrasound‐guided rectus sheath block (USGRSB) in children.

Objectives

Compare the effectiveness and bupivacaine absorption following USGRSB or wound infiltration (WI) for umbilical hernia repair in children.

Methods

A randomized blinded study comparing WI with USGRSB in 40 children undergoing umbilical hernia repair was performed. Group WI (n = 20) received wound infiltration 1 mg·kg−1 0.25% bupivacaine. Group RS (n = 20) received USGRSB 0.5 mg·kg−1 0.25% bupivacaine per side in the posterior rectus sheath compartment. Pain scores and rescue analgesia were recorded. Blood samples were drawn at 0, 10, 20, 30, 45, and 60 min.

Results

Patients in the WI group had a twofold increased risk of requiring morphine (hazard ratio 2.06, 95% CI 1.01, 4.20, P = 0.05). When required, median time to first morphine dose was longer in the USGRSB group (65.5 min vs 47.5 min, P = 0.049). Peak plasma bupivacaine concentration was higher following USGRSB than WI (median: 631.9 ng·ml−1 IQR: 553.9–784.1 vs 389.7 ng·ml−1 IQR: 250.5–502.7, P = 0.002). Tmax was longer in the USGRSB group (median 45 min IQR: 30–60 vs 20 min IQR: 20–45, P = 0.006).

Conclusions

USGRSB provides more effective analgesia than WI for umbilical hernia repair. USGRSB with 1 mg·kg−1 0.25% bupivacaine is associated with safe plasma bupivacaine concentration that peaks higher and later than WI. Caution against using larger volumes of higher concentration local anesthetic for USGRSB is advised.
Optical properties of developing pip and stone fruit reveal underlying structural changes
Authors: Seifert Birgit , Zude Manuela , Spinelli Lorenzo , Torricelli Alessandro
Published: May 2014

Analyzing the optical properties of fruits represents a powerful approach for non‐destructive observations of fruit development. With classical spectroscopy in the visible and near‐infrared wavelength ranges, the apparent attenuation of light results from its absorption or scattering. In horticultural applications, frequently, the normalized difference vegetation index (NDVI) is employed to reduce the effects of varying scattering properties on the apparent signal. However, this simple approach appears to be limited. In the laboratory, with time‐resolved reflectance spectroscopy, the absorption coefficient, μa, and the reduced scattering coefficient, μs′, can be analyzed separately. In this study, these differentiated optical properties were recorded (540–940 nm), probing fruit tissue from the skin up to 2 cm depth in apple (Malus × domestica ‘Elstar’) and plum (Prunus domestica ‘Tophit plus’) harvested four times (65–145 days after full bloom). The μa spectra showed typical peak at 670 nm of the chlorophyll absorption. The μs′ at 670 nm in apple changed by 14.7% (18.2–15.5 cm−1), while in plum differences of 41.5% (8.5–5.0 cm−1) were found. The scattering power, the relative change of μs′, was zero in apple, but enhanced in plum over the fruit development period. This mirrors more isotropic and constant structures in apple compared with plum. For horticultural applications, the larger variability in scattering properties of plum explains the discrepancy between commercially assessed NDVI values or similar indices and the absolute μa values in plum (R < 0.05), while the NDVI approach appeared reasonable in apple (R ≥ 0.80).
The barley anion channel, HvALMT1, has multiple roles in guard cell physiology and grain metabolism
Authors: Xu Muyun , Gruber Benjamin D. , Delhaize Emmanuel , White Rosemary G. , James Richard A. , You Jiangfeng , Yang Zhenming , Ryan Peter R.
Published: May 2014

The barley (Hordeum vulgare) gene HvALMT1 encodes an anion channel in guard cells and in certain root tissues indicating that it may perform multiple roles. The protein localizes to the plasma membrane and facilitates malate efflux from cells when constitutively expressed in barley plants and Xenopus oocytes. This study investigated the function of HvALMT1 further by identifying its tissue‐specific expression and by generating and characterizing RNAi lines with reduced HvALMT1 expression. We show that transgenic plants with 18–30% of wild‐type HvALMT1 expression had impaired guard cell function. They maintained higher stomatal conductance in low light intensity and lost water more rapidly from excised leaves than the null segregant control plants. Tissue‐specific expression of HvALMT1 was investigated in developing grain and during germination using transgenic barley lines expressing the green fluorescent protein (GFP) with the HvALMT1 promoter. We found that HvALMT1 is expressed in the nucellar projection, the aleurone layer and the scutellum of developing barley grain. Malate release measured from isolated aleurone layers prepared from imbibed grain was significantly lower in the RNAi barley plants compared with control plants. These data provide molecular and physiological evidence that HvALMT1 functions in guard cells, in grain development and during germination. We propose that HvALMT1 releases malate and perhaps other anions from guard cells to promote stomatal closure. The likely roles of HvALMT1 during seed development and grain germination are also discussed.
Facial, verbal, and symbolic stimuli differently affect the right hemisphere preponderance of stimulus‐preceding negativity
Authors: Ohgami Yoshimi , Kotani Yasunori , Arai Jun‐Ichirou , Kiryu Shigeru , Inoue Yusuke
Published: May 2014

The present study investigated whether the right hemisphere preponderance of stimulus‐preceding negativity (SPN) was affected by different categories of visual feedback stimulus. A time estimation task was performed with facial, verbal, symbolic, and no‐feedback conditions. A principal component analysis identified an early component ofSPNin addition to a late component that was morphologically similar to the originalSPN. Motivational scores in the verbal and facial conditions were higher than that in the symbolic condition. Significant right hemisphere preponderance of the lateSPNwas observed in the symbolic condition but not in the verbal condition, whereas right hemisphere preponderance of the earlySPNwas observed in the facial condition. The right hemisphere preponderance was influenced by the category of visual feedback stimulus through stimulus‐related activation and the effect of the motivational level.
Modulation of the initial light reflex during affective picture viewing
Authors: Henderson Robert R. , Bradley Margaret M. , Lang Peter J.
Published: May 2014

An initial reflexive constriction of the pupil to stimulation—the light reflex—is primarily modulated by brightness, but is attenuated when participants are under threat of shock (i.e., fear‐inhibited light reflex). The present study assessed whether the light reflex is similarly attenuated when viewing emotional pictures. Pupil diameter was recorded while participants viewed erotic, violent, and neutral scenes that were matched in brightness; scrambled versions identical in brightness were also presented as an additional control. Compared to viewing neutral scenes, the light reflex was reliably modulated by hedonic content, with significant attenuation both when viewing unpleasant as well as pleasant pictures. No differences in the light reflex were found among scrambled versions. Thus, emotional modulation of the initial light reflex is not confined to a context of fear and is not indicative of brightness differences when viewing pictures of natural scenes.
Aspen Restoration Using Beaver on the Northern Yellowstone Winter Range under Reduced Ungulate Herbivory
Authors: Runyon Molly J. , Tyers Daniel B. , Sowell Bok F. , Gower Claire N.
Published: May 2014

Ungulate browsing and lack of overstory disturbance have historically prevented aspen regeneration on the Northern Yellowstone Winter Range (NYWR). Aspen clones regenerate if sprouts are produced that grow into recruitment stems (>2 m tall) and replace the mature overstory. Beaver reintroduced in 1991 to Eagle Creek on the NYWR facilitated aspen restoration by removing overstory trees and increasing sprouting. However, intense ungulate browsing, primarily from the Northern Yellowstone elk herd, was preventing aspen recruitment in Eagle Creek as of 2005. Since 2005, wolf predation has contributed to a 56% decrease in this elk herd. We investigated the effects of beaver reintroduction, ungulate herbivory, and predator‐mediated declines in elk numbers on aspen regeneration in Eagle Creek from 1997 to 2012. Aerial photos of Eagle Creek in 2005 and 2011 showed that the aspen overstory has not been replaced 21 years after beaver reintroduction (p > 0.05). Sprouting and recruitment were investigated using 4‐m radius circular plots (n = 31) established throughout Eagle Creek in 1997 and monitored annually until 2012. Beaver activity stimulated sprouting in 71% of these plots. In 2012, 77% of the plots had ≥1 recruitment stem and 75% of the paired plots associated with exclosures (n = 16) had aspen stems with an average height ≥2 m. Recent increases in aspen recruitment in Eagle Creek indicate that aspen communities are regenerating. This has likely resulted from decreased ungulate browsing pressure on aspen saplings from 2005 to 2012. These findings are consistent with the predictions of a density‐mediated trophic cascade following wolf reintroduction.
Establishment of Restoration Trajectories for Upland Tundra Communities on Diamond Mine Wastes in the Canadian Arctic
Authors: Anne Naeth M. , Wilkinson Sarah R.
Published: May 2014

Mining in the arctic amplifies restoration challenges due to inherent environmental conditions by removing soil, vegetation, and the propagule bank, adding coarse textured wastes with low water holding capacity and nutrients, and introducing salt and metal contamination. Short‐term reclamation focuses on rebuilding soil and providing rapid native plant cover for erosion control, supporting longer term reestablishment of ecological processes for sustainable tundra communities that provide essential wildlife habitat. This study evaluated methods to restore soil and plant communities 5 years after implementation of treatments at a diamond mine in the Canadian arctic. Five substrates including mine waste materials (processed kimberlite, glacial till, gravel, and mixes), four amendments (inorganic fertilizer, salvaged soil, sewage sludge, and water treatment sludge), five native species seed mixes and natural recovery were investigated. Soil and plant response were assessed annually. Soil chemistry was ameliorated with time. Chromium, cobalt, and nickel concentrations in processed kimberlite remained high and potentially toxic to plants. Adding fine textured materials such as glacial till to mine wastes improved nutrient and water retention, which in turn enhanced revegetation. Sewage and inorganic fertilizer increased available nitrogen and phosphorus, plant density and cover. Soil amendment increased species richness. Seeding was essential to establish a vegetation cover. After 5 years, seed mix composition and diversity had no effect on plant community development; soil and plant community properties among treatments changed considerably, providing evidence that restoration in the arctic is dynamic yet slow and success cannot be determined in the short term.
The Relationship between ‘Non‐successor’ Farm Offspring and the Continuity of the Irish Family Farm
Authors: Cassidy Anne , McGrath Brian
Published: May 2014

Studies of farm families have largely neglected the position of farm offspring who, through necessity or choice, live their lives away from the farm. This article explores how Irish farming offspring who will not or are highly unlikely to be farm successors frame their relationship with the farm, as well as their attitude to and role in the succession process and the continuity of the farm within the family. Particularly, the concern is to know how attachment to/detachment from the farm and home life are shaped and the implications for how they construct their identities. The article is based on a qualitative narrative study of 30 young adults from farm backgrounds attending university. It is argued that the ‘non‐successors’ in this cohort have a deep attachment to the farm as an enduring place in their lives. This has key implications for the desire to retain the farm within the family. The article demonstrates that while there is acceptance of enduring gendered cultural scripts surrounding succession, non‐successors demonstrate their attachments in key terms, namely through a collective and secure sense of ownership; a sense of responsibility in maintaining the intergenerational legacy and continuity; and the articulation of the farm as a repository of memories.
Characterization of the novel KIR2DL3*029 allele identified in a southern Chinese Han individual
Authors: Deng Z.‐H. , Zhen J.‐X.
Published: May 2014

KIR2DL3*029 differs from the closest allele KIR2DL3*00101 by a single missense mutation at CDS nt505 C > A.
Robust Nonfragile Stabilizing Controller for an Uncertain Stochastic Nonlinear System With Interval Time-Varying State-Delays: Prescribed H∞ Performance Level
Authors: Ramakrishnan K. , Ray G.
Published: May 2014

In this technical brief, delay-dependent nonfragile H∞ control problem of a class of stochastic nonlinear systems with interval time-varying state-delays has been considered using Lyapunov–Krasovskii (LK) functional approach. By exploiting a candidate LK functional and using free-weighting matrix technique, a less conservative delay-dependent stabilization criterion is presented for the existence of a nonfragile memoryless state-feedback controller, which ensures stochastic stability as well as a prescribed H∞ performance level of the closed-loop system in the presence admissible parametric uncertainties in the system as well as in the controller gains and exogenous input signal. Since the resulting stabilization criterion is in terms of nonlinear matrix inequalities (NLMIs), it is solved using cone complementarity algorithm (CCA) to obtain a stabilizing controller. A numerical example is presented to illustrate the effectiveness of the proposed result.
Correlation between microstructure and thermionic electron emission from Os-Ru thin films on dispenser cathodes
Authors: Swartzentruber Phillip D. , John Balk Thomas , Effgen Michael P.
Published: May 2014

Osmium-ruthenium films with different microstructures were deposited onto dispenser cathodes and subjected to 1000 h of close-spaced diode testing. Tailored microstructures were achieved by applying substrate biasing during deposition, and these were evaluated with scanning electron microscopy, x-ray diffraction, and energy dispersive x-ray spectroscopy before and after close-spaced diode testing. Knee temperatures determined from the close-spaced diode test data were used to evaluate cathode performance. Cathodes with a large {10-11} Os-Ru film texture possessed comparatively low knee temperatures. Furthermore, a low knee temperature correlated with a low effective work function as calculated from the close-spaced diode data. It is proposed that the formation of strong {10-11} texture is responsible for the superior performance of the cathode with a multilayered Os-Ru coating.
New strategies for developing cardiovascular stent surfaces with novel functions (Review)
Authors: Qi Pengkai , Chen Si , Liu Tao , Chen Jialong , Yang Zhilu , Weng Yajun , Chen Junying , Wang Jin , Maitz Manfred F. , Huang Nan
Published: May 2014

In this review, the authors summarize the developments in surface modification of cardiovascular materials especially in author's laboratory. The authors focus on three different strategies to construct multifunctional surfaces including coimmobilization of various biomolecules on stent surfaces, stem cell based therapy systems, and a single-molecule multipurpose modification strategy in vascular interventional therapy. The roles of various molecules like heparin, gallic acid, various aptamers, and nitric oxide are highlighted in the new strategies for developing cardiovascular stent surfaces with novel functions including excellent hemocompatibility, inhibiting smooth muscle cells proliferation, and native endothelium regeneration. The success of these multifunctional surfaces provides the tremendous potential in designing the next generation of vascular stents.
Contributions to Protection from Streptococcus pneumoniae Infection Using the Monovalent Recombinant Protein Vaccine Candidates PcpA, PhtD, and PlyD1 in an Infant Murine Model during Challenge
Authors: Verhoeven David , Perry Sheldon , Pichichero Michael E.
Published: May 2014

A vaccine consisting of several conserved proteins with different functions directing the pathogenesis of pneumonia and sepsis would be preferred for protection against infection byStreptococcus pneumoniae. Infants will be the major population targeted for next-generation pneumococcal vaccines. Here, we investigated the potential efficacy provided by three recombinant pneumococcal vaccine candidate proteins—pneumococcal histidine triad D (PhtD), detoxified pneumolysin derivative (PlyD1), and pneumococcal choline-binding protein A (PcpA)—for reducing pneumonia and sepsis in an infant mouse vaccine model. We found vaccination with PhtD and PcpA provided high IgG antibody titers after vaccination in infant mice, similar to adult mice comparators. PlyD1-specific total IgG was significantly lower in infant mice, with minimal boosting with the second and third vaccinations. Similar isotypes of IgG for PhtD and PlyD1 were generated in infant compared to adult mice. Although lower total specific IgG to all three proteins was elicited in infant than in adult mice, the infant mice were protected from bacteremic pneumonia and sepsis mortality (PlyD1) and had lower lung bacterial burdens (PcpA and PhtD) after challenge. The observed immune responses coupled with bacterial reductions elicited by each of the monovalent proteins support further testing in human infant clinical trials.
Use of the Bruker MALDI Biotyper for Identification of Molds in the Clinical Mycology Laboratory
Authors: Schulthess Bettina , Ledermann Raphael , Mouttet Forouhar , Zbinden Andrea , Bloemberg Guido V. , Böttger Erik C. , Hombach Michael
Published: May 2014

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is increasingly used for the identification of bacteria and fungi in the diagnostic laboratory. We evaluated the mold database of Bruker Daltonik (Bremen, Germany), the Filamentous Fungi Library 1.0. First, we studied 83 phenotypically and molecularly well-characterized, nondermatophyte, nondematiaceous molds from a clinical strain collection. Using the manufacturer-recommended interpretation criteria, genus and species identification rates were 78.3% and 54.2%, respectively. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification to 71.1% without increasing misidentifications. In a subsequent prospective study, 200 consecutive clinical mold isolates were identified by the MALDI Biotyper and our conventional identification algorithm. Discrepancies were resolved by ribosomal DNA (rDNA) internal transcribed spacer region sequence analysis. For the MALDI Biotyper, genus and species identification rates were 83.5% and 79.0%, respectively, when using a species cutoff of 1.7. Not identified were 16.5% of the isolates. Concordant genus and species assignments of MALDI-TOF MS and the conventional identification algorithm were observed for 98.2% and 64.2% of the isolates, respectively. Four erroneous species assignments were observed using the MALDI Biotyper. The MALDI Biotyper seems highly reliable for the identification of molds when using the Filamentous Fungi Library 1.0 and a species cutoff of 1.7. However, expansion of the database is required to reduce the number of nonidentified isolates.
Azithromycin-Resistant Syphilis-Causing Strains in Sydney, Australia: Prevalence and Risk Factors
Authors: Read Phillip , Jeoffreys Neisha , Tagg Kaitlin , Guy Rebecca J. , Gilbert Gwendolyn L. , Donovan Basil
Published: May 2014

Azithromycin has shown high efficacy in randomized trials when used for treating infectious syphilis in Africa. However, its use in clinical practice has been limited by the development of antimicrobial drug resistance. Resistance has not previously been reported from Australasia. The aim of this study was to determine the prevalence of and risk factors for azithromycin-resistant syphilis-causing strains in Sydney, Australia. We evaluated 409 samples that were PCR positive for
Treponema pallidum
DNA collected between 2004 and 2011 for the presence of the A2058G mutation, which confers resistance to macrolide antibiotics such as azithromycin. Overall, 84% of samples harbored the mutation. The prevalence of the mutation increased during the study period (
P
trend, 0.003). We also collected clinical and demographic data on 220 patients from whom these samples had been collected to determine factors associated with the A2058G mutation; 97% were from men who have sex with men. Reporting sex in countries other than Australia was associated with less macrolide resistance (adjusted odds ratio, 0.25; 95% confidence interval, 0.09 to 0.66;
P
= 0.005), with other study factors showing no association (age, HIV status, recent macrolide use, stage of syphilis, or history of prior syphilis). Azithromycin cannot be recommended as an alternative treatment for syphilis in Sydney.
Infective Meningitis Caused by Phialemonium curvatum
Authors: Zou Yueli , Bi Yuhai , Bu Hui , He Yunying , Guo Li , Shi Dongyan
Published: May 2014

Infections caused by rarely encountered fungal pathogens have increased in recent decades.
Phialemonium
species are widely distributed in the environment and are also involved in human infections, affecting both immunocompromised and immunocompetent patients. The present study describes a case of meningitis caused by
Phialemonium curvatum
.
Development of Oxacillin Resistance in a Patient with Recurrent Staphylococcus aureus Bacteremia
Authors: Burd Eileen M. , Alam Mohammad Tauqeer , Passalacqua Karla D. , Kalokhe Ameeta S. , Eaton Molly E. , Satola Sarah W. , Kraft Colleen S. , Read Timothy D.
Published: May 2014

Whole-genome sequencing was used to compare longitudinal isolates of
Staphylococcus aureus
that developed resistance to oxacillin (MIC up to 16 μg/ml). The
mecA
gene was absent. A novel 5-bp TATCC frameshift insertion in a gene encoding an ABC transporter similar to that of the teichoic acid translocation ATP-binding protein TagH and a 3-bp GCT nonframeshift insertion in the
pdhA
pyruvate dehydrogenase gene were detected in the oxacillin-resistant isolates.
A Rapid Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant Enterobacteriaceae
Authors: Lau Anna F. , Wang Honghui , Weingarten Rebecca A. , Drake Steven K. , Suffredini Anthony F. , Garfield Mark K. , Chen Yong , Gucek Marjan , Youn Jung-Ho , Stock Frida , Tso Hanna , DeLeo Jim , Cimino James J. , Frank Karen M. , Dekker John P.
Published: May 2014

Carbapenem-resistant
Enterobacteriaceae
(CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the

bla
KPC

carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼11,109-Da MS peak corresponding to a gene product of the

bla
KPC

pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed
K. pneumoniae
outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of

bla
KPC

-negative clinical
Enterobacteriaceae
isolates. Notably, the gene characterized here is located adjacent to the

bla
KPC

Tn
4401
transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other

bla
KPC

Tn
4401
-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.
Impact of Isolated Hepatitis C Virus (HCV) Core-Specific Antibody Detection and Viral RNA Amplification among HCV-Seronegative Dialysis Patients at Risk for Infection
Authors: Barril Guillermina , Quiroga Juan A. , Arenas María Dolores , Espinosa Mario , García-Fernández Nuria , Cigarrán Secundino , Herrero José A. , del Peso Gloria , Caro Pilar , García-Agudo Rebeca , Amézquita Yésica , Blanco Ana , Martínez-Rubio Pilar , Alcázar José M. , González-Parra Emilio , Martín-Gómez Adoración , Castillo Inmaculada , Bartolomé Javier , Carreño Vicente
Published: May 2014

Amplification of hepatitis C virus (HCV) RNA from blood detected occult HCV infections in 30.9% of 210 HCV-seronegative dialysis patients with abnormal liver enzyme levels that had evaded standard HCV testing practices. Isolated HCV core-specific antibody detection identified three additional anti-HCV screening-negative patients lacking HCV RNA amplification in blood who were considered potentially infectious. Together, these findings may affect management of the dialysis setting.
Axonal and Transynaptic Spread of Prions
Authors: Shearin Harold , Bessen Richard A.
Published: May 2014

Natural transmission of prion diseases depends upon the spread of prions from the nervous system to excretory or secretory tissues, but the mechanism of prion transport in axons and into peripheral tissue is unresolved. Here, we examined the temporal and spatial movement of prions from the brain stem along cranial nerves into skeletal muscle as a model of axonal transport and transynaptic spread. The disease-specific isoform of the prion protein, PrP
Sc
, was observed in nerve fibers of the tongue approximately 2 weeks prior to PrP
Sc
deposition in skeletal muscle. Initially, PrP
Sc
deposits had a small punctate pattern on the edge of muscle cells that colocalized with synaptophysin, a marker for the neuromuscular junction (NMJ), in >50% of the cells. At later time points PrP
Sc
was widely distributed in muscle cells, but <10% of prion-infected cells exhibited PrP
Sc
deposition at the NMJ, suggesting additional prion replication and dissemination within muscle cells. In contrast to the NMJ, PrP
Sc
was not associated with synaptophysin in nerve fibers but was found to colocalize with LAMP-1 and cathepsin D during early stages of axonal spread. We propose that PrP
Sc
-bound endosomes can lead to membrane recycling in which PrP
Sc
is directed to the synapse, where it either moves across the NMJ into the postsynaptic muscle cell or induces PrP
Sc
formation on muscle cells across the NMJ.

IMPORTANCE
Prion diseases are transmissible and fatal neurodegenerative diseases in which prion dissemination to excretory or secretory tissues is necessary for natural disease transmission. Despite the importance of this pathway, the cellular mechanism of prion transport in axons and into peripheral tissue is unresolved. This study demonstrates anterograde spread of prions within nerve fibers prior to infection of peripheral synapses (i.e., neuromuscular junction) and infection of peripheral tissues (i.e., muscle cells). Within nerve fibers prions were associated with the endosomal-lysosomal pathway prior to entry into muscle cells. Since early prion spread is anterograde and endosome-lysosomal movement within axons is primarily retrograde, these findings suggest that endosome-bound prions may have an alternate fate that directs prions to the peripheral synapse.
Discovery and Evolution of Bunyavirids in Arctic Phantom Midges and Ancient Bunyavirid-Like Sequences in Insect Genomes
Authors: Ballinger Matthew J. , Bruenn Jeremy A. , Hay John , Czechowski Donna , Taylor Derek J.
Published: May 2014

Bunyaviridae
is a large family of RNA viruses chiefly comprised of vertebrate and plant pathogens. We discovered novel bunyavirids that are approximately equally divergent from each of the five known genera. We characterized novel genome sequences for two bunyavirids, namely, Kigluaik phantom virus (KIGV), from tundra-native phantom midges (
Chaoborus
), and Nome phantom virus (NOMV), from tundra-invading phantom midges, and demonstrated that these bunyavirid-like sequences belong to an infectious virus by passaging KIGV in mosquito cell culture, although the infection does not seem to be well sustained beyond a few passages. Virus and host gene sequences from individuals collected on opposite ends of North America, a region spanning 4,000 km, support a long-term, vertically transmitted infection of KIGV in
Chaoborus trivittatus
. KIGV-like sequences ranging from single genes to full genomes are present in transcriptomes and genomes of insects belonging to six taxonomic orders, suggesting an ancient association of this clade with insect hosts. In
Drosophila
, endogenous virus genes have been coopted, forming an orthologous tandem gene family that has been maintained by selection during the radiation of the host genus. Our findings indicate that bunyavirid-host interactions in nonbloodsucking arthropods have been much more extensive than previously thought.

IMPORTANCE
Very little is known about the viral diversity in polar freshwater ponds, and perhaps less is known about the effects that climate-induced habitat changes in these regions will have on virus-host interactions in the coming years. Our results show that at the tundra-boreal boundary, a hidden viral landscape is being altered as infected boreal phantom midges colonize tundra ponds. Likewise, relatively little is known of the deeper evolutionary history of bunyavirids that has led to the stark lifestyle contrasts between some genera. The discovery of this novel bunyavirid group suggests that ancient and highly divergent bunyavirid lineages remain undetected in nature and may offer fresh insight into host reservoirs, potential sources of emerging disease, and major lifestyle shifts in the evolutionary history of viruses in the family
Bunyaviridae
.
Deep Sequencing of HIV-Infected Cells: Insights into Nascent Transcription and Host-Directed Therapy
Authors: Peng Xinxia , Sova Pavel , Green Richard R. , Thomas Matthew J. , Korth Marcus J. , Proll Sean , Xu Jiabao , Cheng Yanbing , Yi Kang , Chen Li , Peng Zhiyu , Wang Jun , Palermo Robert E. , Katze Michael G.
Published: May 2014

Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately sequenced total RNAs (Total RNAseq) and mRNAs (mRNAseq) from the same HIV-1-infected human CD4
+
T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more differentially expressed genes at 12 h postinfection by Total RNAseq than by mRNAseq. These expression changes were also evident by concurrent changes in introns and were recapitulated by later mRNA changes, revealing an unexpectedly significant delay between transcriptional initiation and mature mRNA production early after HIV-1 infection. We computationally derived and validated the underlying regulatory programs, and we predicted drugs capable of reversing these HIV-1-induced expression changes followed by experimental confirmation. Our results show that combined total and mRNA transcriptome analysis is essential for fully capturing the early host response to virus infection and provide a framework for identifying candidate drugs for host-directed therapy against HIV/AIDS.

IMPORTANCE
In this study, we used mass sequencing to identify genes differentially expressed in CD4
+
T cells during HIV-1 infection. To our surprise, we found many differentially expressed genes early after infection by analyzing both newly transcribed unprocessed pre-mRNAs and fully processed mRNAs, but not by analyzing mRNAs alone, indicating a significant delay between transcription initiation and mRNA production early after HIV-1 infection. These results also show that important findings could be missed by the standard practice of analyzing mRNAs alone. We then derived the regulatory mechanisms driving the observed expression changes using integrative computational analyses. Further, we predicted drugs that could reverse the observed expression changes induced by HIV-1 infection and showed that one of the predicted drugs indeed potently inhibited HIV-1 infection. This shows that it is possible to identify candidate drugs for host-directed therapy against HIV/AIDS using our genomics-based approach.
Coexistence of Hepatitis B Virus Quasispecies Enhances Viral Replication and the Ability To Induce Host Antibody and Cellular Immune Responses
Authors: Cao Liang , Wu Chunchen , Shi Hui , Gong Zuojiong , Zhang Ejuan , Wang Hui , Zhao Kaitao , Liu Shuhui , Li Songxia , Gao Xiuzhu , Wang Yun , Pei Rongjuan , Lu Mengji , Chen Xinwen
Published: May 2014

Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as a reservoir for viral selection under antiviral treatment and the immune response, leading to the acute exacerbation and subsequent development of liver failure. However, there is no clear experimental evidence for a significant role of HBV quasispecies in viral pathogenesis. In the present study, HBV sequences were amplified from a patient with severe liver disease and used for construction of HBV replication-competent plasmids. Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were performed to analyze the expression, secretion, and subcellular localization of viral proteins
in vitro
. Viral replication intermediates were detected by Southern blotting. HBV gene expression and replication and the induction of specific immune responses in an HBV hydrodynamic injection (HI) mouse model were investigated. The results demonstrated that two naturally occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS expressed only a nonexportable hepatitis B virus surface antigen (HBsAg) with abnormal intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4 (SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg were induced in mice by the HBV variants when coapplied by HI. These findings uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses, representing a novel mechanism for the immunopathogenesis of HBV infection.

IMPORTANCE
Hepatitis B virus (HBV) is an important human pathogen. HBV quasispecies with genetically heterogenous variants are thought to play a role in the progression of HBV-associated liver diseases. So far, direct evidence is available in only a few cases to confirm the proposed role of HBV variants in the pathogenesis. We report here that the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased HBV replication and induced significantly stronger intrahepatic cytotoxic T lymphocyte responses and antibody responses specific to HBV surface antigen (HBsAg) in mice. Our discovery uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses and may enhance immune-mediated liver damage under some circumstances, representing a novel mechanism for the immunopathogenesis of HBV infection.
Inhibition of Arenavirus Infection by a Glycoprotein-Derived Peptide with a Novel Mechanism
Authors: Spence Jennifer S. , Melnik Lilia I. , Badani Hussain , Wimley William C. , Garry Robert F.
Published: May 2014

The family
Arenaviridae
includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC
50
) for the peptide is 7 μM, with complete inhibition of viral plaque formation at approximately 20 μM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection.

IMPORTANCE
Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic and prone to treatment failure, we identified a peptide, AVP-p, derived from the fusion glycoprotein of a nonpathogenic model arenavirus, which demonstrates antiviral activity and no acute cytotoxicity. AVP-p is unique among self-derived inhibitory peptides in that it shows broad, specific activity against pseudoviruses bearing Old and New World arenavirus glycoproteins but not against viruses from other families. Further, the peptide's mechanism of action is highly novel. Biochemical assays and cryo-electron microscopy indicate that AVP-p induces premature activation of viral fusion proteins through membrane perturbance. Peptide treatment, however, does not increase the infectivity of cell-bound virus. We hypothesize that prematurely activated virions are less fit for receptor binding and membrane fusion and that AVP-p may represent a viable therapeutic strategy for arenavirus infection.
Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication
Authors: Jung Jaesung , Hwang Seong Gyu , Chwae Yong-Joon , Park Sun , Shin Ho-Joon , Kim Kyongmin
Published: May 2014

Phosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However,
in vivo
phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [
32
P]orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication.

IMPORTANCE
Threonine 162, within the carboxyl-terminal end of the hepatitis B virus (HBV adw) core (C) protein, has long been ignored as a phosphoacceptor, even though it is highly conserved among mammalian hepadnaviruses and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication.