bluuebunny/crossref_metadata_2025 huggingface dataset:
National Human Trafficking Initiatives: Dimensions of Policy Diffusion
Authors: Yoo Eun‐hye , Boyle Elizabeth Heger
Published: March 2015

The implementation of criminal law involves formal law enforcement, education, and public outreach aimed at preventing criminal activity and providing services for victims. Historically, quantitative research on global trends has focused on a single policy dimension, potentially masking the unique factors that affect the diffusion of each policy dimension independently. Using an ordered‐probit model to analyze new human trafficking policy data on national prosecution, prevention, and victim‐protection efforts, we find that global ties and domestic interest groups matter more where international law is less defined. Although prosecution, mandated by the Trafficking Protocol, was relatively impervious to global ties and domestic interest groups, both trafficking prevention and victim protection were associated with these factors. Our findings also suggest that fear of repercussions is not a major driver of state actions to combat trafficking—neither ratification of the protocol nor levels of US aid were associated with greater implementation of antitrafficking measures.
Redundancy and diversity of functional reef fish groups of the Mexican Eastern Pacific
Authors: Aguilar‐Medrano Rosalia , Calderon‐Aguilera Luis Eduardo
Published: March 2015

Coral reefs are in crisis worldwide. Fish play a key role in the resilience of reefs because most of the biomass of the ecosystem flows through them. Evidence indicates that the response to stressors will vary markedly depending on the resilience of the ecosystem. In order to assess the functional groups of reef fishes, morphometric and ecomorphological analyses were carried out on the shape and diet of 117 reef fish species from the Mexican Eastern Pacific (MEP) from 23°N to 15°N, spanning the years 2009–2012. Six trophic groups were identified and subdivided into 19 functional groups: (A) detritus and plants consumers, three morphologies; (B) plants and zoobenthos consumers, four morphologies; (C) zoobenthos consumers, two morphologies; (D) zoobenthos and zooplankton consumers, three morphologies; (E) zooplankton and nekton consumers, three morphologies; and (F) nekton consumers, four morphologies. Our main conclusions are: (i) fish communities of the MEP have a high degree of ecological redundancy; (ii) fish diversity is not related to the coral cover; and (iii) the combination of using both morphology and diet produce a more effective classification of functional groups. Future studies such as these will allow a deeper understanding of environment and it will help illuminate the effects of fish morphology and diet on population structure.
A systematic review of motivational interviewing for weight loss among adults in primary care
Authors: Barnes R. D. , Ivezaj V.
Published: March 2015

Motivational interviewing (MI) is a client‐centred method of intervention focused on enhancing intrinsic motivation and behaviour change. A previous review of the literature and meta‐analyses support the effectiveness of MI for weight loss. None of these studies, however, focused on the bourgeoning literature examining MI for weight loss among adults within primary care settings, which confers unique barriers to providing weight loss treatment. Further, the current review includes 19 studies not included in previous reviews or meta‐analyses. We conducted a comprehensive review of PubMed, MI review papers, and citations from relevant papers. A total of 24 adult randomized controlled trials were identified. MI interventions typically were provided individually by a range of clinicians and compared with usual care. Few studies provided adequate information regarding MI treatment fidelity. Nine studies (37.5%) reported significant weight loss at post‐treatment assessment for the MI condition compared with control groups. Thirteen studies (54.2%) reported MI patients achieving at least 5% loss of initial body weight. There is potential for MI to help primary care patients lose weight. Conclusions, however, must be drawn cautiously as more than half of the reviewed studies showed no significant weight loss compared with usual care and few reported MI treatment fidelity.
Unusual histology and morphology of the ribs of mosasaurs (Squamata)
Authors: D'Emic Michael D. , Smith Kathlyn M. , Ansley Zachary T.
Published: March 2015

We report the presence of two previously unrecognized features in the dorsal ribs of mosasaurs: first, the presence of extremely dense, pervasive extrinsic fibres (anchoring soft tissue to bone, sometimes called Sharpey's fibres); and second, high intraspecific variation in costal bone compactness. Extensive extrinsic fibres are developed in the dorsal ribs of the mosasaurs Tylosaurus proriger and Eonatator sternbergi. The dorsal ribs of these mosasaurs are also characterized by a longitudinally ridged texture that almost completely covers the bone. Pervasive extrinsic fibres and ridged textures are absent in the mosasaur Selmasaurus russelli as well as the dorsal ribs of extant semi‐aquatic reptiles (e.g. crocodyliforms) and mosasaurs' close extant relatives and analogues (e.g. snakes and varanids). Similar ridged textures characterize the dorsal ribs of several other mosasaur taxa but are developed to a lesser extent (e.g. Mosasaurus, Clidastes, Platecarpus and Ectenosaurus), but in no other taxa have pervasive extrinsic fibres been reported. We interpret these osteohistological features in T. proriger and E. sternbergi as evidence of tendinous attachment of extensive and highly differentiated axial musculature capable of producing great stresses, most likely related to stabilization of the trunk relative to contralateral movements of the tail during carangiform locomotion. We also report the compactness indices (percentage of space occupied by bone rather than cavities) for these large mosasaur ribs, which are much higher than previously reported. This suggests high intraspecific variation in bone compactness that complicates its use in reconstructing mosasaur palaeoecology.
Widely Metastatic Squamous Cell Carcinoma Originating from Malignant Transformation of Hypertrophic Lichen Planus in a 24‐Year‐Old Woman: Case Report and Review of the Literature
Authors: Tong Lana X. , Weinstock Matthew J. , Drews Reed , Sheikine Yuri , Kim Caroline C.
Published: March 2015

Hypertrophic lichen planus (HLP) is a T‐cell‐mediated process typically presenting with hypertrophic or verrucous plaques on the lower limbs. We report the case of a 24‐year‐old woman with a history of HLP since age 3 years presenting with rapid malignant transformation of one lesion into a large squamous cell carcinoma (SCC). Subsequent examination revealed progressive, widespread metastatic involvement, and the patient ultimately died from her disease. SCC associated with HLP is rare, with a review of the literature revealing fewer than 50 cases. This case highlights the need to be aware of suspicious changes in HLP and to educate patients as to when to be reevaluated.
Development of a Fusarium oxysporum f. sp. melonis functional GFP fluorescence tool to assist melon resistance breeding programmes
Authors: Ramos B. , López G. , Molina A.
Published: March 2015

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (Fom), is one of the most widespread and devastating melon diseases. This vascular disease is caused by the colonization of melon xylem vessels by any of the four Fom races reported (r0, r1, r2 and r1,2, subdivided into r1,2w and r1,2y). The macroscopic evaluation of disease symptoms (disease rating, DR) at several days post‐inoculation (dpi) with Fom spores has been the traditional method to determine the resistance of melon accessions to this fungal pathogen. In this study, one isolate from each Fom race was transformed by Agrobacterium tumefaciens to constitutively express the green fluorescent protein (GFP). Fom‐GFP transformants, as virulent as the corresponding wildtype races, were selected to develop an inoculation assay based on the non‐invasive evaluation of the fluorescence emitted by Fom‐GFP. It was determined that melon root neck was the appropriate area to follow Fom‐GFP and a fluorescence signal rating (FSR) was established in parallel to DR determination. This method allowed the evaluation of GFP signal in the root neck of inoculated melon seedlings at 11–15 dpi. The GFP signal was scored in 62 melon accessions/breeding lines inoculated with different Fom‐GFP, followed by evaluation of the macroscopic DR in the aerial part of melon seedlings at 20–28 dpi. Correlation analysis demonstrated a direct and significant relationship between FSR and DR. This method has shown to be an effective and reliable tool that can assist Fom resistance breeding programmes in melon.
Impact of traditional soil burning (guie) on Planosol properties and land‐use intensification in south‐western Ethiopia
Authors: Mertens K. , Tolossa A. R. , Verdoodt A. , Dumon M. , Deckers J. , Van Ranst E.
Published: March 2015

In the Gilgel Gibe catchment in Ethiopia, local farmers intensify land use on Planosols by adjusting a traditional soil burning practice known as guie. The burning practice used to be applied in a cycle of shifting cultivation. However, more recently, farmers burn small plots to make fertile seedbeds for Eucalyptus seedlings in the first year before these trees are transplanted to larger plots. The purpose of this research was to assess the physico‐chemical properties of Planosols that have been subjected to burning over the last 10 yrs and evaluate the contribution of guie to land‐use intensification of these soils. Transect studies and interviews of local farmers, followed by chemical, physical and micromorphological analyses of samples from selected plots were used to compare the soil properties of recently (0–2 yrs) and formerly (3–10 yrs) burnt Planosols with those of unburnt Planosols. The analytical results show that the burning practice improved nutrient availability in the first 2 yrs after guie. Increased amounts of exchangeable aluminium (Al) were reported in the long term. Charge fingerprints illustrate that the nutrient‐buffering capacity of the soil was high shortly after the practice but subsequently decreased with time. Given the population pressure on the formerly extensively used Planosols, it is argued that the current application of guie on small, localized plots for raising Eucalyptus seedlings is well adapted to the local socio‐economic context and promotes land‐use intensification on the Planosols. The increased exchangeable Al content of former Eucalyptus seedbeds merits further in‐depth research into the biophysical sustainability of the burning practice.
Sympathetic activity during passive heat stress in healthy aged humans
Authors: Gagnon Daniel , Schlader Zachary J. , Crandall Craig G.
Published: March 2015

Key points

Cardiovascular adjustments to heat stress are attenuated in healthy aged individuals, which could contribute to their greater prevalence of heat‐related illnesses and deaths during heat waves.

The attenuated cardiovascular adjustments in the aged could be due to lower increases in sympathetic nerve activity during heat stress.

We examined muscle sympathetic nerve activity (MSNA) and plasma catecholamine concentrations in healthy young and aged individuals during whole‐body passive heat stress.

The main finding of this study is that increases in MSNA and plasma catecholamine concentrations did not differ between young and aged healthy individuals during passive heating. Furthermore, the increase in these variables did not differ when a cold pressor test and lower body negative pressure were superimposed upon heating.

These findings suggest that attenuated cardiovascular adjustments to heat stress in healthy aged individuals are unlikely to be related to attenuated increases in sympathetic activity.

Cardiovascular adjustments during heat stress are generally attenuated in healthy aged humans, which could be due to lower increases in sympathetic activity compared to the young. We compared muscle sympathetic nerve activity (MSNA) between 11 young (Y: 28 ± 4 years) and 10 aged (A: 70 ± 5 years) subjects prior to and during passive heating. Furthermore, MSNA responses were compared when a cold pressor test (CPT) and lower body negative pressure (LBNP) were superimposed upon heating. Baseline MSNA burst frequency (Y: 15 ± 4 vs. A: 31 ± 3 bursts min−1, P ≤ 0.01) and burst incidence (Y: 26 ± 8 vs. A: 50 ± 7 bursts (100 cardiac cycles (CC))−1, P ≤ 0.01) were greater in the aged. Heat stress increased core temperature to a similar extent in both groups (Y: +1.2 ± 0.1 vs. A: +1.2 ± 0.0°C, P = 0.99). Absolute levels of MSNA remained greater in the aged during heat stress (burst frequency: Y: 47 ± 6 vs. A: 63 ± 11 bursts min−1, P ≤ 0.01; burst incidence: Y: 48 ± 8 vs. A: 67 ± 9 bursts (100 CC)−1, P ≤ 0.01); however, the increase in both variables was similar between groups (both P ≥ 0.1). The CPT and LBNP further increased MSNA burst frequency and burst incidence, although the magnitude of increase was similar between groups (both P ≥ 0.07). These results suggest that increases in sympathetic activity during heat stress are not attenuated in healthy aged humans.
Numerical Evaluation of the Optical Properties of Encapsulated Phase Change Particles for Thermotropic Materials
Authors: Gladen Adam , Mantell Susan , Davidson Jane
Published: March 2015

Phase change thermotropic materials have been proposed as a low cost method to provide passive overheat protection for polymer solar thermal absorbers. One challenge to their development is control of the size of the phase change particles dispersed within the matrix. Here we explore encapsulation as a means to resolve this challenge with a focus on the selection of materials, including the encapsulating shell, to achieve desirable optical behavior. Hydroxystearic acid (HSA) particles in a matrix of poly(methyl methacrylate) (PMMA) is down selected from candidate materials based on its optical properties and the melt temperature of the dispersed phase. The optical properties (normal-hemispherical transmittance, reflectance, and absorptance) as a function of the properties of the encapsulation shell and the particle volume fraction are predicted at a wavelength of 589 nm using a Monte Carlo ray tracing model. A range of shell relative refractive indices, from 0.95 to 1, and thicknesses, up to 35 nm, can be employed to achieve greater than 80% transmittance in the clear state and greater than 50% reflectance in the translucent state.
Numerical Modeling of Multiple Bubbles Condensation in Subcooled Flow Boiling
Authors: Liu Zhenyu , Sunden Bengt , Wu Huiying
Published: March 2015

The understanding of multiple bubbles condensation is of significant importance in developing continuum models for the large-scale subcooled flow boiling. The computational fluid dynamics (CFD) modeling for multiple bubbles condensation is developed with the volume of fluid (VOF) method in this work. An explicit transient simulation is performed to solve the governing equations including the source terms for heat and mass transfer due to condensation. The geometric reconstruction scheme, which is a piecewise linear interface calculation (PLIC) method, is employed to keep the interface sharp. The surface tension is modeled by the continuum surface force (CSF) approach, which is taken into account in the numerical model. Numerical simulations predict the dynamical behavior of the actual condensing bubbles. The results show that the condensation rate of a single bubble is influenced by the velocity of the fluid flow and the temperature difference between the bubble and fluid. For multiple bubbles, the effect of bubble–bubble interaction on their condensation process is analyzed based on the numerical predictions. The condensation rate of lower bubbles increases due to the random perturbation induced by other bubbles. The influence of other bubbles on the condensation rate can be neglected if the distances between the bubbles are large enough.
Experimental Investigation of Residual Stress in Minimum Quantity Lubrication Grinding of AISI 1018 Steel
Authors: Shao Yamin , Fergani Omar , Ding Zishan , Li Beizhi , Liang Steven Y.
Published: March 2015

Minimum quantity lubrication (MQL) is a promising alternative to conventional flood cooling to substantially reduce lubricant cost and converse energy. Literature survey shows lack of investigation on the influence of MQL on the residual stress profile in grinding process. Residual stress is an important attribute of machined components for its notable influences on fatigue life, corrosion resistance, and fracture strength. This study has presented a thorough experimental investigation on grinding force, temperature, surface roughness, and residual stress behavior in grinding of AISI 1018 steel under MQL, dry, and flood cooling conditions.
Overview of atomic layer etching in the semiconductor industry
Authors: Kanarik Keren J. , Lill Thorsten , Hudson Eric A. , Sriraman Saravanapriyan , Tan Samantha , Marks Jeffrey , Vahedi Vahid , Gottscho Richard A.
Published: March 2015

Atomic layer etching (ALE) is a technique for removing thin layers of material using sequential reaction steps that are self-limiting. ALE has been studied in the laboratory for more than 25 years. Today, it is being driven by the semiconductor industry as an alternative to continuous etching and is viewed as an essential counterpart to atomic layer deposition. As we enter the era of atomic-scale dimensions, there is need to unify the ALE field through increased effectiveness of collaboration between academia and industry, and to help enable the transition from lab to fab. With this in mind, this article provides defining criteria for ALE, along with clarification of some of the terminology and assumptions of this field. To increase understanding of the process, the mechanistic understanding is described for the silicon ALE case study, including the advantages of plasma-assisted processing. A historical overview spanning more than 25 years is provided for silicon, as well as ALE studies on oxides, III–V compounds, and other materials. Together, these processes encompass a variety of implementations, all following the same ALE principles. While the focus is on directional etching, isotropic ALE is also included. As part of this review, the authors also address the role of power pulsing as a predecessor to ALE and examine the outlook of ALE in the manufacturing of advanced semiconductor devices.
Directional cell elongation through filopodia-steered lamellipodial extension on patterned silk fibroin films
Authors: You Renchuan , Li Xiufang , Luo Zuwei , Qu Jing , Li Mingzhong
Published: March 2015

Micropatterned biomaterials have been used to direct cell alignment for specific tissue engineering applications. However, the understanding of how cells respond to guidance cues remains limited. Plasticity in protrusion formation has been proposed to enable cells to adapt their motility mode to microenvironment. In this study, the authors investigated the key role of protrusion response in cell guidance on patterned silk fibroin films. The results revealed that the ability to transform between filopodia and small lamellipodia played important roles in directional cell guidance. Filopodia did not show directional extension on patterned substrates prior to spreading, but they transduced topographical cues to the cell to trigger the formation of small lamellipodia along the direction of a microgrooved or parallel nanofiber pattern. The polar lamellipodia formation provided not only a path with directionality, but a driving force for directional cell elongation. Moreover, aligned nanofibers coating provided better mechanical support for the traction of filopodia and lamellipodia, promoting cell attachment, spreading, and migration. This study provides new insight into how cells respond to guidance cues and how filopodia and lamellipodia control cell contact guidance on micropatterned biomaterial surfaces.
Structure of the Get3 targeting factor in complex with its membrane protein cargo
Authors: Mateja Agnieszka , Paduch Marcin , Chang Hsin-Yang , Szydlowska Anna , Kossiakoff Anthony A. , Hegde Ramanujan S. , Keenan Robert J.
Published: March 2015

How to GET to the right membrane

Membrane proteins with a hydrophobic transmembrane domain (TMD) play critical roles in virtually all aspects of cell physiology. After it has been synthesized in the cytosol, this TMD must be targeted to and inserted into the correct membrane. The GET pathway is one of two targeting pathways to the endoplasmic reticulum conserved across all eukaryotes. It is not clear how the central targeting factor, Get3, recognizes a TMD to shield it from aggregation until it is successfully inserted into the membrane. Now, Mateja
et al.
show that the functional targeting complex comprises a Get3 dimer bound to a single TMD. The helical hydrophobic TMD binds deep within a large hydrophobic groove in the Get3 dimer. This groove closes slightly upon TMD binding, forming a dynamic “lid” over the mouth of the groove.

Science
, this issue p.
1152
On the endogeneity of political preferences: Evidence from individual experience with democracy
Authors: Fuchs-Schündeln Nicola , Schündeln Matthias
Published: March 2015

Political preferences provide economic capital

Longer periods of democratic government favor economic growth, which in turn stabilizes democracy. But is this relationship a given? Fuchs-Schundeln and Schundeln collected individual-level data from more than 100 countries over two decades. Support for democracy did indeed increase as the length of time lived in a democratic system increased.

Science
, this issue p.
1145
Early
Homo
at 2.8 Ma from Ledi-Geraru, Afar, Ethiopia
Authors: Villmoare Brian , Kimbel William H. , Seyoum Chalachew , Campisano Christopher J. , DiMaggio Erin N. , Rowan John , Braun David R. , Arrowsmith J Ramón , Reed Kaye E.
Published: March 2015

Finding
Homo
nearly 3 million years ago

The fossil record of humans is notoriously patchy and incomplete. Even so, skeletal remains and artifacts unearthed in Africa in recent decades have done much to illuminate human evolution. But what is the origin of the genus
Homo
? Villmoare
et al.
found a fossil mandible and teeth from the Afar region in Ethiopia. The find extends the record of recognizable
Homo
by at least half a million years, to almost 2.8 million years ago. The morphological traits of the fossil align more closely with
Homo
than with any other hominid genus. DiMaggio
et al.
confirm the ancient date of the site and suggest that these early humans lived in a setting that was more open and arid than previously thought.

Science
, this issue p.
1352
, p.
1355
Being smart about SMART environmental targets
Authors: Maxwell S. L. , Milner-Gulland E. J. , Jones J. P. G. , Knight A. T. , Bunnefeld N. , Nuno A. , Bal P. , Earle S. , Watson J. E. M. , Rhodes J. R.
Published: March 2015

Focus on the negotiation process, not just the end target
A sustainable model for antibiotics
Authors: Perros Manos
Published: March 2015

How can we foster the development of novel drugs against resistant bacteria?

[Also see Perspective by
Baker
]
Isolation and Characterization of Porcine Deltacoronavirus from Pigs with Diarrhea in the United States
Authors: Hu Hui , Jung Kwonil , Vlasova Anastasia N. , Chepngeno Juliet , Lu Zhongyan , Wang Qiuhong , Saif Linda J.
Published: March 2015

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Following its first detection in the United States in February 2014, additional PDCoV strains have been identified in the United States and Canada. Currently, no treatments or vaccines for PDCoV are available. In this study, U.S. PDCoV strain OH-FD22 from intestinal contents of a diarrheic pig from Ohio was isolated in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures by using various medium additives. We also isolated PDCoV [OH-FD22(DC44) strain] in LLC-PK cells from intestinal contents of PDCoV OH-FD22 strain-inoculated gnotobiotic (Gn) pigs. Cell culture isolation and propagation were optimized, and the isolates were serially propagated in cell culture for >20 passages. The full-length S and N genes were sequenced to study PDCoV genetic changes after passage in Gn pigs and cell culture (passage 11 [P11] and P20). Genetically, the S and N genes of the PDCoV isolates were relatively stable during the first 20 passages in cell culture, with only 5 nucleotide changes, each corresponding to an amino acid change. The S and N genes of our sequenced strains were genetically closely related to each other and to other U.S. PDCoV strains, with the highest sequence similarity to South Korean strain KNU14-04. This is the first report describing cell culture isolation, serial propagation, and biological and genetic characterization of cell-adapted PDCoV strains. The information presented in this study is important for the development of diagnostic reagents, assays, and potential vaccines against emergent PDCoV strains.
Antimicrobial Resistance and Virulence Characterization among Escherichia coli Clinical Isolates Causing Severe Obstetric Infections in Pregnant Women
Authors: Guiral Elisabet , Sáez-López Emma , Bosch Jordi , Goncé Anna , López Marta , Sanz Sergi , Vila Jordi , Soto Sara M.
Published: March 2015

The virulence markers and the antimicrobial resistance profiles of 78
Escherichia coli
isolates causing obstetric infections accompanied by sepsis or not were studied. Adhesion-related virulence factors were the most prevalent markers. Low rates of resistance to the antimicrobial agents used as first-line therapy suggest their correct implementation in stewardship guidelines.
Matrix-Assisted Laser Desorption Ionization–Time of Flight and Comparative Genomic Analysis of M-18 Group A Streptococcus Strains Associated with an Acute Rheumatic Fever Outbreak in Northeast Italy in 2012 and 2013
Authors: Gaibani Paolo , Scaltriti Erika , Foschi Claudio , Baggio Enrico , Tamburini Maria Vittoria , Creti Roberta , Pascucci Maria Grazia , Fagioni Marco , Ambretti Simone , Comandatore Francesco , Pongolini Stefano , Landini Maria Paola
Published: March 2015

Acute rheumatic fever (ARF) is a postsuppurative sequela caused by
Streptococcus pyogenes
infections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A
Streptococcus
(GAS) strains belonged to the M-18 serotype, including subtypes
emm18.29
and
emm18.32
. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different
S. pyogenes
lineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that
emm18.29
and
emm18.32
GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between
emm18.29
and
emm18.32
strains that differed from noninvasive
emm18.0
strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of
emm18.29
and
emm18.32
strains. Notably, phage ΦSPBO.1 carried erythrogenic toxin A gene (
speA1
) in six ARF-related M-18 GAS strains but not in
emm18.0
strains. In addition, a phage-encoded hyaluronidase gene (
hylP.2
) presented different variants among M-18 GAS strains by showing internal deletions located in the α-helical and TSβH regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.
Assessing the Public Health Risk of Shiga Toxin-Producing Escherichia coli by Use of a Rapid Diagnostic Screening Algorithm
Authors: de Boer Richard F. , Ferdous Mithila , Ott Alewijn , Scheper Henk R. , Wisselink Guido J. , Heck Max E. , Rossen John W. , Kooistra-Smid Anna M. D.
Published: March 2015

Shiga toxin-producing
Escherichia coli
(STEC) is an enteropathogen of public health concern because of its ability to cause serious illness and outbreaks. In this prospective study, a diagnostic screening algorithm to categorize STEC infections into risk groups was evaluated. The algorithm consists of prescreening stool specimens with real-time PCR (qPCR) for the presence of
stx
genes. The qPCR-positive stool samples were cultured in enrichment broth and again screened for
stx
genes and additional virulence factors (
escV
,
aggR
,
aat
,
bfpA
) and O serogroups (O26, O103, O104, O111, O121, O145, O157). Also, PCR-guided culture was performed with sorbitol MacConkey agar (SMAC) and CHROMagar STEC medium. The presence of virulence factors and O serogroups was used for presumptive pathotype (PT) categorization in four PT groups. The potential risk for severe disease was categorized from high risk for PT group I to low risk for PT group III, whereas PT group IV consists of unconfirmed
stx
qPCR-positive samples. In total, 5,022 stool samples of patients with gastrointestinal symptoms were included. The qPCR detected
stx
genes in 1.8% of samples. Extensive screening for virulence factors and O serogroups was performed on 73 samples. After enrichment, the presence of
stx
genes was confirmed in 65 samples (89%). By culture on selective media, STEC was isolated in 36% (26/73 samples). Threshold cycle (

C
T

) values for
stx
genes were significantly lower after enrichment compared to direct qPCR (
P
< 0.001). In total, 11 (15%), 19 (26%), 35 (48%), and 8 (11%) samples were categorized into PT groups I, II, III, and IV, respectively. Several virulence factors (
stx
2
,
stx
2a
,
stx
2f
,
toxB
,
eae
,
efa1
,
cif
,
espA
,
tccP
,
espP
,
nleA
and/or
nleB
,
tir
cluster) were associated with PT groups I and II, while others (
stx
1
,
eaaA
,
mch
cluster,
ireA
) were associated with PT group III. Furthermore, the number of virulence factors differed between PT groups (analysis of variance,
P
< 0.0001). In conclusion, a diagnostic algorithm enables fast discrimination of STEC infections associated with a high to moderate risk for severe disease (PT groups I and II) from less-virulent STEC (PT group III).
Detection of Enterovirus D68 in Canadian Laboratories
Authors: Hatchette Todd F. , Drews Steven J. , Grudeski Elsie , Booth Tim , Martineau Christine , Dust Kerry , Garceau Richard , Gubbay Jonathan , Karnauchow Tim , Krajden Mel , Levett Paul N. , Mazzulli Tony , McDonald Ryan R. , McNabb Alan , Mubareka Samira , Needle Robert , Petrich Astrid , Richardson Susan , Rutherford Candy , Smieja Marek , Tellier Raymond , Tipples Graham , LeBlanc Jason J.
Published: March 2015

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68.
A Metagenomics and Case-Control Study To Identify Viruses Associated with Bovine Respiratory Disease
Authors: Ng Terry Fei Fan , Kondov Nikola O. , Deng Xutao , Van Eenennaam Alison , Neibergs Holly L. , Delwart Eric
Published: March 2015

Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing,
de novo
assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirty-eight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (
P
< 0.0001), bovine rhinitis A virus (
P
= 0.005), and the recently described bovine influenza D virus (
P
= 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and real-time PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies.

IMPORTANCE
Bovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact.
Increased Levels of Macrophage Inflammatory Proteins Result in Resistance to R5-Tropic HIV-1 in a Subset of Elite Controllers
Authors: Walker Wendy E. , Kurscheid Sebastian , Joshi Samit , Lopez Charlie A. , Goh Gerald , Choi Murim , Barakat Lydia , Francis John , Fisher Ann , Kozal Michael , Zapata Heidi , Shaw Albert , Lifton Richard , Sutton Richard E. , Fikrig Erol
Published: March 2015

Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4
+
T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4
+
T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1α (MIP-1α),
CCL3
and
CCL3L1
, were found to be upregulated. The MIP-1α, MIP-1β, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1α and/or MIP-1β at the protein level. The supernatant from resistant EC cells contained MIP-1α and MIP-1β and was sufficient to confer R5-tropic resistance to susceptible CD4
+
T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry.

IMPORTANCE
HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4
+
T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals.
Retroviral Cyclin Controls Cyclin-Dependent Kinase 8-Mediated Transcription Elongation and Reinitiation
Authors: Birkenheuer Claire H. , Brewster Connie D. , Quackenbush Sandra L. , Rovnak Joel
Published: March 2015

Walleye dermal sarcoma virus (WDSV) infection is associated with the seasonal development and regression of walleye dermal sarcoma. Previous work showed that the retroviral cyclin (RV-cyclin), encoded by WDSV, has separable cyclin box and transcription activation domains. It binds to cyclin-dependent kinase 8 (CDK8) and enhances its kinase activity. CDK8 is evolutionarily conserved and is frequently overexpressed in human cancers. It is normally activated by cyclin C and is required for transcription elongation of the serum response genes (immediate early genes [IEGs])
FOS
,
EGR1
, and
cJUN
. The IEGs drive cell proliferation, and their expression is brief and highly regulated. Here we show that constitutive expression of RV-cyclin in the HCT116 colon cancer cell line significantly increases the level of IEG expression in response to serum stimulation. Quantitative reverse transcription-PCR (RT-PCR) and nuclear run-on assays provide evidence that RV-cyclin does not alter the initiation of IEG transcription but does enhance the overall rate of transcription elongation and maintains transcription reinitiation. RV-cyclin does not increase activating phosphorylation events in the mitogen-activated protein kinase pathway and does not inhibit decay of IEG mRNAs. At the
EGR1
gene locus, RV-cyclin increases and maintains RNA polymerase II (Pol II) occupancy after serum stimulation, in conjunction with increased and extended
EGR1
gene expression. The RV-cyclin increases CDK8 occupancy at the
EGR1
gene locus before and after serum stimulation. Both of RV-cyclin's functional domains, i.e., the cyclin box and the activation domain, are necessary for the overall enhancement of IEG expression. RV-cyclin presents a novel and ancient mechanism of retrovirus-induced oncogenesis.

IMPORTANCE
The data reported here are important to both virology and cancer biology. The novel mechanism pinpoints CDK8 in the development of walleye dermal sarcoma and sheds light on CDK8's role in many human cancers. CDK8 controls expression from highly regulated genes, including the interferon-stimulated genes. Its function is likely the target of many viral interferon-resistance mechanisms. CDK8 also controls cellular responses to metabolic stimuli, stress, and hypoxia, in addition to the serum response. The retroviral cyclin (RV-cyclin) represents a highly selected probe of CDK8 function. RV-cyclin does not control CDK8 specificity but instead enhances CDK8's effects on regulated genes, an important distinction for its use to delineate natural CDK8 targets. The outcomes of this research are applicable to investigations of normal and abnormal CDK8 functions. The mechanisms defined here will contribute directly to the dermal sarcoma model in fish and clarify an important path for oncogenesis and innate resistance to viruses.
Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle
Authors: McGivern David R. , Masaki Takahiro , Lovell William , Hamlett Chris , Saalau-Bethell Susanne , Graham Brent
Published: March 2015

Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing.

IMPORTANCE
The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.
Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA
Authors: Dueck Kevin J. , Hu YuanShen (Sandy) , Chen Peter , Deschambault Yvon , Lee Jocelyn , Varga Jessie , Cao Jingxin
Published: March 2015

Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity.

IMPORTANCE
dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs.
Flexibility of Bunyavirus Genomes: Creation of an Orthobunyavirus with an Ambisense S Segment
Authors: van Knippenberg Ingeborg , Elliott Richard M.
Published: March 2015

The Bunyamwera (BUNV) orthobunyavirus NSs protein has proven a challenge to study in the context of viral infection. NSs is encoded in a reading frame that overlaps that of the viral nucleocapsid (N) protein thus limiting options for mutagenesis. In addition, NSs is poorly immunogenic, and antibodies only work in certain techniques while the protein itself is subject to proteasomal degradation. In order to generate a virus that expresses NSs independently of N, an ambisense S RNA segment was designed by mutating the 5′- and 3′-terminal nucleotide sequences. These mutations were previously shown to alter promoter activity so that both replication and transcription were promoted from both the genome and the antigenome RNAs (J. N. Barr et al., J. Virol.
79:
12602–12607, 2005). As proof of principle, a recombinant BUNV was created that expressed green fluorescent protein (GFP) in the ambisense orientation. GFP expression was detected throughout at least 10 passages. Recombinant BUNV encoding epitope-tagged versions of NSs in the ambisense orientation expressed NSs via a subgenomic mRNA, and two viruses grew to titers only modestly lower than parental rBUNdelNSs2 virus. The ambisense viruses were temperature sensitive, and NSs was shown to localize to both the nucleus and the cytoplasm during infection. These viruses will be useful in further studies on structure-function relationships of the orthobunyavirus NSs protein.

IMPORTANCE
Bunyamwera virus (BUNV) is the type species and model system for both the family
Bunyaviridae
and the genus
Orthobunyavirus
, a group that includes many significant human and animal pathogens. Studying the basic molecular biology of these viruses is of great importance to underpin research into vaccines and antivirals. We demonstrate here the plasticity of the BUNV genome by generating recombinant viruses where the normal negative-sense S segment has been converted into an ambisense segment, allowing independent expression of either a foreign gene (green fluorescent protein) or the viral nonstructural NSs protein. These new reagents will allow detailed investigation of NSs, the orthobunyavirus interferon antagonist.
Prosthodontic rehabilitation of a patient with aggressive periodontitis
Authors: Livingstone David , Murthy Varsha , Reddy Vineela Katam , Pillai Ajay
Published: March 2015

Aggressive periodontitis previously termed as juvenile periodontitis is characterised by rapid destruction of the periodontium at a relatively young age. Rehabilitation of these patients is often challenging and difficult. Controlling the disease and restoring periodontal health is essential for successful prosthodontic rehabilitation. This clinical report describes an interdisciplinary approach in the rehabilitation of a young adult patient with generalised aggressive periodontitis. Treatment objectives included plaque control, prevention of further attachment loss, reduction/elimination of pockets, and prosthetic rehabilitation to enhance aesthetics and restore masticatory function. One year recall evaluation revealed stable periodontal support with no further loss of attachment and no other complications.