bluuebunny/crossref_metadata_2025 huggingface dataset:
The Tourism of North Korea in the Kim Jong‐un Era: Propaganda, Profitmaking, and Possibilities for Engagement
Authors: Ouellette Dean J.
Published: December 2016

In this article, I explore changes in North Korean tourism, with a focus on the industry since Kim Jong‐un came to power. The study: highlights North Korea's concept of “socialist tourism” and recent developments; investigates Western social media to uncover how non‐mainstream digital media characterizes the country as a unique travel destination; discusses investment and profitmaking with respect to Chinese investors and tourists; and suggests tourism as a possible path to consider for future peaceful engagement approaches.
The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence
Authors: Rinaldo Amy , Gilbert Brian , Boni Rainer , Krattinger Simon G. , Singh Davinder , Park Robert F. , Lagudah Evans , Ayliffe Michael
Published: December 2016

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.
Improved Detection of Changes in Species Richness in High Diversity Microbial Communities
Authors: Willis Amy , Bunge John , Whitman Thea
Published: December 2016

Biodiversity is important for balance and function of a broad variety of ecosystems, and identifying factors that influence biodiversity can assist environmental management and maintenance. However, low abundance taxa are often missing from ecosystem surveys. These rare taxa, which may be critical to the ecosystem function, are not accounted for in existing methods for detecting changes in species richness. We introduce a model for total (observed and unobserved) biodiversity that explicitly accounts for these rare taxa. Our method permits rigorous testing for both heterogeneity and biodiversity changes, and simultaneously improves type I and II error rates compared with existing methods. To estimate model parameters we utilize the well-developed literature of meta-analysis. The problem of substantial low abundance taxa missing from samples is especially pronounced in microbiomes, which are the focus of our case-studies.
Perception and signalling of light and temperature cues in plants
Authors: Legris Martina , Nieto Cristina , Sellaro Romina , Prat Salomé , Casal Jorge J.
Published: December 2016

Light and temperature patterns are often correlated under natural plant growth conditions. In this review, we analyse the perception and signalling mechanisms shared by both these environmental cues and discuss the functional implications of their convergence to control plant growth. The first point of integration is the phytochrome B (phyB) receptor, which senses light and temperature. Downstream of phyB, the signalling core comprises two branches, one involving PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and the other CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and ELONGATED HYPOCOTYL 5 (HY5). The dynamics of accumulation and/or localization of each of these core signalling components depend on light and temperature conditions. These pathways are connected through COP1, which enhances the activity of PIF4. The circadian clock modulates this circuit, since EARLY FLOWERING 3 (ELF3), an essential component of the evening complex (EC), represses expression of the PIF4 gene and PIF4 transcriptional activity. Phytochromes are probably not the only entry point of temperature into this network, but other sensors remain to be established. The sharing of mechanisms of action for two distinct environmental cues is to some extent unexpected, as it renders these responses mutually dependent. There are nonetheless many ecological contexts in which such a mutual influence could be beneficial.
Physiological redox signalling and regulation of ion channels: implications for pulmonary hypertension
Authors: Ward Jeremy P. T.
Published: December 2016

New Findings

What is the topic of this review?

The review concerns the role of reactive oxygen species as physiological second messengers in potentiating G‐protein‐coupled receptor‐mediated vasoconstriction and its potential dysregulation by oxidant stress in pulmonary hypertension.

What advances does it highlight?

The review highlights the concept that physiological signalling by reactive oxygen species must normally be highly compartmentalized to prevent self‐regenerating oxidant stress and promiscuous and uncontrolled signalling, which contribute to the aetiology.

Pulmonary hypertension is associated with oxidant stress and increased generation of reactive oxygen species (ROS) by NADPH oxidases (NOX), mitochondria and other sources. There is considerable evidence that these contribute to the aetiology via promotion of pulmonary vascular remodelling, endothelial dysfunction and enhanced vasoreactivity. However, it is now recognized that ROS act as important signalling mediators and second messengers in normal physiological conditions. Many ion channels and protein kinases crucial to pulmonary vascular function are directly or indirectly affected by redox/ROS, including K+, Ca2+ and non‐selective cation channels and Rho kinase. However, the inherent difficulties in quantifying ROS, particularly in subcellular compartments, make it uncertain whether these reported effects are of relevance in physiological rather than pathological conditions. In an attempt to address such issues, we have focused on the role of physiologically generated ROS in the regulation of G‐protein‐coupled receptor (GPCR)‐activated vasoconstrictor pathways. We have recently reported a novel mechanism whereby low concentrations of GPCR‐linked vasoconstrictors greatly potentiate Ca2+ entry via a NOX1‐ and ROS‐mediated pathway parallel to the classical vasoconstrictor pathways of Ca2+ mobilization and activation of Rho kinase. Our findings imply that ROS signalling is highly compartmentalized in physiological conditions, but that this may be compromised by pathological increases in oxidant production, for example in pulmonary hypertension, leading to promiscuous actions that contribute to the aetiology. This model is consistent with the proposal that targeted antioxidants could prove to be an effective therapy for pulmonary hypertension.
A critical period of corticomuscular and EMG–EMG coherence detection in healthy infants aged 9–25 weeks
Authors: Ritterband‐Rosenbaum Anina , Herskind Anna , Li Xi , Willerslev‐Olsen Maria , Olsen Mikkel Damgaard , Farmer Simon Francis , Nielsen Jens Bo
Published: December 2016

Key points

The early postnatal development of functional corticospinal connections in human infants is not fully clarified.

Corticospinal drive to upper and lower limb muscle shows developmental changes with an increased functional coupling in infants between 9 and 25 weeks in the beta frequency band.

The changes in functional coupling coincide with the developmental period where fidgety movements are present in healthy infants.

Data support a possible sensitive period where functional connections between corticospinal tract fibres and spinal motoneurones undergo activity‐dependent reorganization.

The early postnatal development of functional corticospinal connections in human infants is not fully clarified. We used EEG and EMG to investigate the development of corticomuscular and intramuscular coherence as indicators of functional corticospinal connectivity in healthy infants aged 1–66 weeks. EEG was recorded over leg and hand area of motor cortex. EMG recordings were made from right ankle dorsiflexor and right wrist extensor muscles. Quantification of the amount of corticomuscular coherence in the 20–40 Hz frequency band showed a significantly larger coherence for infants aged 9–25 weeks compared to younger and older infants. Coherence between paired EMG recordings from tibialis anterior muscle in the 20–40 Hz frequency band was also significantly larger for the 9–25 week age group. A low‐amplitude, broad‐duration (40–50 ms) central peak of EMG–EMG synchronization was observed for infants younger than 9 weeks, whereas a short‐lasting (10–20 ms) central peak was observed for EMG–EMG synchronization in older infants. This peak was largest for infants aged 9–25 weeks. These data suggest that the corticospinal drive to lower and upper limb muscles shows significant developmental changes with an increase in functional coupling in infants aged 9–25 weeks, a period which coincides partly with the developmental period of normal fidgety movements. We propose that these neurophysiological findings may reflect the existence of a sensitive period where the functional connections between corticospinal tract fibres and spinal motoneurones undergo activity‐dependent reorganization. This may be relevant for the timing of early therapy interventions in infants with pre‐ and perinatal brain injury.
Non‐chemosensitive parafacial neurons simultaneously regulate active expiration and airway patency under hypercapnia in rats
Authors: de Britto Alan A. , Moraes Davi J. A.
Published: December 2016

Key points

Hypercapnia or parafacial respiratory group (pFRG) disinhibition at normocapnia evokes active expiration in rats by recruitment of pFRG late‐expiratory (late‐E) neurons.

We show that hypercapnia simultaneously evoked active expiration and exaggerated glottal dilatation by late‐E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic expiratory activity in previously silent pFRG late‐E neurons, which did not express the marker of ventral medullary CO2‐sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia.

Hypercapnia‐evoked active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation.

Hypercapnia produces disinhibition of non‐chemosensitive pFRG late‐E neurons to evoke active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency.

Hypercapnia produces active expiration in rats and the recruitment of late‐expiratory (late‐E) neurons located in the parafacial respiratory group (pFRG) of the ventral medullary brainstem. We tested the hypothesis that hypercapnia produces active expiration and concomitant cranial respiratory motor responses controlling the oropharyngeal and upper airway patency by disinhibition of pFRG late‐E neurons, but not via synaptic excitation. Phrenic nerve, abdominal nerve (AbN), cranial respiratory motor nerves, subglottal pressure, and medullary and spinal neurons/motoneurons were recorded in in situ preparations of juvenile rats. Hypercapnia evoked AbN active expiration, exaggerated late‐E discharges in cranial respiratory motor outflows, and glottal dilatation via late‐E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic late‐E activity in previously silent pFRG neurons, which did not express the marker of ventral medullary CO2‐sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. In addition, hypercapnia‐evoked AbN active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. On the other hand, pFRG inhibition did not affect either hypercapnia‐induced inspiratory increases in respiratory motor outflows or CO2 sensitivity of the more medial Phox2b‐positive neurons in the retrotrapezoid nucleus (RTN). Our data suggest that neither RTN Phox2b‐positive nor other CO2‐sensitive brainstem neurons activate Phox2b‐negative pFRG late‐E neurons under hypercapnia to produce AbN active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia produces disinhibition of non‐chemosensitive pFRG late‐E neurons in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.
Effect of Strain, Region, and Tissue Composition on Glucose Partitioning in Meniscus Fibrocartilage
Authors: Kleinhans Kelsey L. , Jackson Alicia R.
Published: December 2016

A nearly avascular tissue, the knee meniscus relies on diffusive transport for nutritional supply to cells. Nutrient transport depends on solute partitioning in the tissue, which governs the amount of nutrients that can enter a tissue. The purpose of the present study was to investigate the effects of mechanical strain, tissue region, and tissue composition on the partition coefficient of glucose in meniscus fibrocartilage. A simple partitioning experiment was employed to measure glucose partitioning in porcine meniscus tissues from two regions (horn and central), from both meniscal components (medial and lateral), and at three levels of compression (0%, 10%, and 20%). Partition coefficient values were correlated to strain level, water volume fraction, and glycosaminoglycan (GAG) content of tissue specimens. Partition coefficient values ranged from 0.47 to 0.91 (n = 48). Results show that glucose partition coefficient is significantly (p < 0.001) affected by compression, decreasing with increasing strain. Furthermore, we did not find a statistically significant effect of tissue when comparing medial versus lateral (p = 0.181) or when comparing central and horn regions (p = 0.837). There were significant positive correlations between tissue water volume fraction and glucose partitioning for all groups. However, the correlation between GAG content and partitioning was only significant in the lateral horn group. Determining how glucose partitioning is affected by tissue composition and loading is necessary for understanding nutrient availability and related tissue health and/or degeneration. Therefore, this study is important for better understanding the transport and nutrition-related mechanisms of meniscal degeneration.
A Monolithic Force-Balanced Oscillator
Authors: Weeke Sybren L. , Tolou Nima , Semon Guy , Herder Just L.
Published: December 2016

Usage of compliant micromechanical oscillators has increased in recent years, due to their reliable performance despite the growing demand for miniaturization. However, ambient vibrations affect the momentum of such oscillators, causing inaccuracy, malfunction, or even failure. Therefore, this paper presents a compliant force-balanced mechanism based on rectilinear motion, enabling usage of prismatic oscillators in translational accelerating environments. The proposed mechanism is based on the opposite motion of two coplanar prismatic joints along noncollinear axes via a shape-optimized linkage system. Rigid-body replacement with shape optimized X-bob, Q-LITF, and LITF joints yielded a harmonic (R > 0.999), low frequency (f=27 Hz) single piece force-balanced micromechanical oscillator (∅ 35 mm). The experimental evaluation of large-scale prototypes showed a low ratio of the center of mass (CoM) shift compared to the stroke of the device (≈ 0.01) and proper decoupling of the mechanism from the base, as the oscillating frequency of the balanced devices during ambient disturbances was unaffected, whereas unbalanced devices had frequency deviations up to 1.6%. Moreover, the balanced device reduced the resultant inertial forces transmitted to the base by 95%.
pneuRIPTM: A Novel Respiratory Inductance Plethysmography Monitor
Authors: Rahman Tariq , Page Ralph , Page Christopher , Bonnefoy Jean-Remy , Cox Tim , Shaffer Thomas H.
Published: December 2016

Objective pulmonary function (PF) evaluation is essential for the diagnosis, monitoring, and management of many pediatric respiratory diseases as seen in the emergency room, intensive care, and outpatient settings. In this paper, the development and testing of a new noninvasive PF instrument, pneuRIPTM, which utilizes respiratory inductance plethysmography (RIP) are discussed. The pneuRIPTM hardware includes a small circuit board that connects to the RIP bands and measures and wirelessly transmits the band inductance data to any designated wirelessly connected tablet. The software provides indices of respiratory work presented instantaneously in a user-friendly graphical user interface on the tablet. The system was tested with ten normal children and compared with an existing system, Respitrace (Sensormedics, Yorba Linda, CA), under normal and loaded breathing conditions. Under normal breathing, the percentage differences between the two systems were 2.9% for labored breathing index (LBI), 31.8% for phase angle (Φ), 4.8% for percentage rib cage (RC%), and 26.7% for respiratory rate (BPM). Under loaded breathing, the percentage differences between the two systems were 1.6% for LBI, 4.1% for Φ, 8.5% for RC%, and 52.7% for BPM. For LBI, Φ, and RC%, the two systems were in general agreement. For BPM the pneuRIPTM is shown to be more accurate than the respitrace when compared to manually counting the breaths: 13.2% versus 36.4% accuracy for normal breathing and 16.9% versus 60.7% accuracy for breathing under load, respectively.
New blood tests make strides in detecting prion disease
Authors: Servick Kelly
Published: December 2016

Long after the European mad cow outbreak, researchers hope to screen blood supplies for misfolded proteins.
Like birds, insects may travel in sync with the seasons
Authors: Pennisi Elizabeth
Published: December 2016

Long-term radar studies reveal masses of winged migrants.
The cosmos aquiver
Authors: Cho Adrian
Published: December 2016

Science
has named the detection of gravitational waves as the Breakthrough of the Year. The discovery fulfilled a 100-year-old prediction by Albert Einstein and foreshadows a new way to eavesdrop on the most violent events in the cosmos.
Majorana bound state in a coupled quantum-dot hybrid-nanowire system
Authors: Deng M. T. , Vaitiekėnas S. , Hansen E. B. , Danon J. , Leijnse M. , Flensberg K. , Nygård J. , Krogstrup P. , Marcus C. M.
Published: December 2016

Watching Majorana bound states form

Majorana bound states (MBSs) are peculiar quasiparticles that may one day become the cornerstone of topological quantum computing. To engineer these states, physicists have used semiconductor nanowires in contact with a superconductor. Although many of the observed properties align with theoretical predictions, a closer look into the creation of MBSs is desirable. Deng
et al.
fabricated nanowires with a quantum dot at one end that served as a spectrometer for the states that formed inside the superconducting gap of the nanowire. Using this setup, topologically trivial bound states were seen to coalesce into MBSs as the magnetic field was varied.

Science
, this issue p.
1557
Expand innovation finance via crowdfunding
Authors: Sorenson Olav , Assenova Valentina , Li Guan-Cheng , Boada Jason , Fleming Lee
Published: December 2016

Crowdfunding attracts venture capital to new regions
Genetic identification of familial hypercholesterolemia within a single U.S. health care system
Authors: Abul-Husn Noura S. , Manickam Kandamurugu , Jones Laney K. , Wright Eric A. , Hartzel Dustin N. , Gonzaga-Jauregui Claudia , O’Dushlaine Colm , Leader Joseph B. , Lester Kirchner H. , Lindbuchler D’Andra M. , Barr Marci L. , Giovanni Monica A. , Ritchie Marylyn D. , Overton John D. , Reid Jeffrey G. , Metpally Raghu P. R. , Wardeh Amr H. , Borecki Ingrid B. , Yancopoulos George D. , Baras Aris , Shuldiner Alan R. , Gottesman Omri , Ledbetter David H. , Carey David J. , Dewey Frederick E. , Murray Michael F.
Published: December 2016

Unleashing the power of precision medicine

Precision medicine promises the ability to identify risks and treat patients on the basis of pathogenic genetic variation. Two studies combined exome sequencing results for over 50,000 people with their electronic health records. Dewey
et al.
found that ∼3.5% of individuals in their cohort had clinically actionable genetic variants. Many of these variants affected blood lipid levels that could influence cardiovascular health. Abul-Husn
et al.
extended these findings to investigate the genetics and treatment of familial hypercholesterolemia, a risk factor for cardiovascular disease, within their patient pool. Genetic screening helped identify at-risk patients who could benefit from increased treatment.

Science
, this issue p.
10.1126/science.aaf6814
, p.
10.1126/science.aaf7000
Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweat versus hair fate decision
Authors: Lu Catherine P. , Polak Lisa , Keyes Brice E. , Fuchs Elaine
Published: December 2016

How to grow hair or sweat glands

Unlike other mammals that must pant or seek shade or water when overheated, humans are able to self-cool to tolerate extreme heat. Sweat glands, which enable humans to run in marathons, are instrumental for this remarkable feat. Lu
et al.
investigated skin appendage diversity during development of the furry backs and sweaty paws of mice (see the Perspective by Lai and Chuong). They also examined human skin, which is capable of making both hairs and sweat glands in the same area of the body. Epithelialmesenchymal interactions, with varied signaling pathways that act at specific times in development, are key to producing different skin appendages for adaptation to the environment.

Science
, this issue p.
10.1126/science.aah6102
; see also p.
1533
Tomorrow's arsenal
Predator Empire Drone Warfare and Full Spectrum Dominance
Ian G. R. Shaw
University of Minnesota Press, 2016. 334 pp.
Ethics and Cyber Warfare The Quest for Responsible Security in the Age of Digital Warfare
George Lucas
Oxford University Press, 2016. 201 pp.
Authors: Al-Rodhan Nayef
Published: December 2016

Two authors probe the technologies transforming warfare
A stitch in time saves nine…billion
Authors: Fulton Elizabeth A.
Published: December 2016

Acting now to limit global temperature change will help to ensure global food security
The “tao” of integuments
Authors: Lai Yung Chih , Chuong Cheng-Ming
Published: December 2016

Hair follicle and sweat gland fates can be switched by morphogens at specific skin regions or developmental stages
“Pheno”menal value for human health
Authors: Rader Daniel J. , Damrauer Scott M.
Published: December 2016

Rare genetic variants are linked to electronic health record phenotypes at a population scale
LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313
Authors: Wilson Charlotte M. , Klingeman Dawn M. , Schlachter Caleb , Syed Mustafa H. , Wu Chia-wei , Guss Adam M. , Brown Steven D.
Published: December 2016

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions.
Clostridium thermocellum
expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in
C. thermocellum
ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the three
C. thermocellum
LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313
lacI
genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a Δ
glyR1
strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a Δ
glyR2
strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the Δ
glyR3
strain, upregulation of the two genes adjacent to
glyR3
in the
celC-glyR3-licA
operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.

IMPORTANCE
Understanding
C. thermocellum
gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific
C. thermocellum
LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in
C. thermocellum
.
Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe
Authors: Fogg Paul C. M. , Haley Joshua A. , Stark W. Marshall , Smith Margaret C. M.
Published: December 2016

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In
Streptomyces
, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new
Streptomyces
phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest
Streptomyces
phage cluster (R4-like) and encodes a serine integrase. The
attB
site from
Streptomyces venezuelae
was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe
int-attP
locus. The
attB
site for ϕJoe integrase was occupied in several
Streptomyces
genomes, including that of
S. coelicolor
, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed
in vivo
. Both the integrase and RDF were active in
in vitro
recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox.

IMPORTANCE
Streptomyces
spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the
Streptomyces
chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated
Streptomyces
phage, ϕJoe. ϕJoe integrase is active
in vitro
and
in vivo
. The specific recognition site for integration is present in a wide range of different actinobacteria, including
Streptomyces venezuelae
, an emerging model bacterium in
Streptomyces
research.
New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations
Authors: Guinard Jérémy , Latreille Anne , Guérin Fabien , Poussier Stéphane , Wicker Emmanuel
Published: December 2016

Bacterial wilt caused by the
Ralstonia solanacearum
species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to
R. pseudosolanacearum
phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580
R. pseudosolanacearum
phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of
R. pseudosolanacearum
phylotype I populations.

IMPORTANCE
Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne
Ralstonia solanacearum
species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-evolving loci, may be a tool of choice for field experimental evolution and spatial genetics studies.
Challenges in Interpretation of Diagnostic Test Results in a Mumps Outbreak in a Highly Vaccinated Population
Authors: Trotz-Williams L. A. , Mercer N. J. , Paphitis K. , Walters J. M. , Wallace D. , Kristjanson E. , Gubbay J. , Mazzulli T.
Published: December 2016

In spite of a greatly reduced incidence rate due to vaccination, mumps outbreaks continue to occur in several areas of the world, sometimes in vaccinated populations. This article describes an outbreak in a highly vaccinated population in southwestern Ontario, Canada, and the challenges encountered in interpreting the results of diagnostic tests used in the outbreak. During the outbreak, patients were interviewed and classified according to the outbreak case definition, and specimens were collected for diagnostic testing according to Ontario guidelines. Twenty-seven individuals were classified as confirmed cases (
n
= 19) or suspect cases (
n
= 8) according to the case definition, only 9 of which were laboratory-confirmed cases: 7 confirmed by reverse transcriptase PCR (RT-PCR) and 2 by IgM serology. All 19 confirmed cases represented patients who were associated with secondary schools in the local area and had been vaccinated against mumps with one (
n
= 2) or two (
n
= 17) doses of the measles-mumps-rubella (MMR) vaccine. This is the first published report of an outbreak of mumps in Ontario in which all confirmed cases had been vaccinated against the disease. It highlights the limitations of and difficulties in interpreting current mumps diagnostic tests when used in vaccinated individuals.
Evaluation of Pyrrolidonyl Arylamidase Activity in Staphylococcus delphini
Authors: Compton Samantha T. , Kania Stephen A. , Robertson Amy E. , Lawhon Sara D. , Jenkins Stephen G. , Westblade Lars F. , Bemis David A.
Published: December 2016

Clinical reference textbooks lack data for pyrrolidonyl arylamidase (PYR) activity in
Staphylococcus delphini
. This study evaluated PYR activities of 21
S. delphini
strains by reference broth, rapid disc, and rapid slide methods. Species and subgroup identifications were confirmed by nucleic acid-based methods and included nine group A and 12 group B strains. Testing by rapid PYR methods with products from four manufacturers was performed at two testing locations, and, with the exception of one strain tested at one location using reagents from one manufacturer, each
S. delphini
strain tested positive for PYR activity. Therefore, PYR may be a useful single-test adjunct for distinguishing
Staphylococcus aureus
from
S. delphini
and other members of the
Staphylococcus intermedius
group.
Adenovirus Serotype 5 Vaccination Results in Suboptimal CD4 T Helper 1 Responses in Mice
Authors: Lee Junghwa , Hashimoto Masao , Im Se Jin , Araki Koichi , Jin Hyun-Tak , Davis Carl W. , Konieczny Bogumila T. , Spies Gregory A. , McElrath M. Juliana , Ahmed Rafi
Published: December 2016

Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling.

IMPORTANCE
During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.
Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff
Authors: Dai Aimei , Cao Shuai , Dhungel Pragyesh , Luan Yizhao , Liu Yizhi , Xie Zhi , Yang Zhilong
Published: December 2016

Vaccinia virus infection causes a host shutoff that is marked by global inhibition of host protein synthesis. Though the host shutoff may facilitate reallocation of cellular resources for viral replication and evasion of host antiviral immune responses, it poses a challenge for continuous synthesis of cellular proteins that are important for viral replication. It is, however, unclear whether and how certain cellular proteins may be selectively synthesized during the vaccinia virus-induced host shutoff. Using simultaneous RNA sequencing and ribosome profiling, two techniques quantifying genome-wide levels of mRNA and active protein translation, respectively, we analyzed the responses of host cells to vaccinia virus infection at both the transcriptional and translational levels. The analyses showed that cellular mRNA depletion played a dominant role in the shutoff of host protein synthesis. Though the cellular mRNAs were significantly reduced, the relative translation efficiency of a subset of cellular mRNAs increased, particularly those involved in oxidative phosphorylation that are responsible for cellular energy production. Further experiments demonstrated that the protein levels and activities of oxidative phosphorylation increased during vaccinia virus infection, while inhibition of the cellular oxidative phosphorylation function significantly suppressed vaccinia virus replication. Moreover, the short 5′ untranslated region of the oxidative phosphorylation mRNAs contributed to the translational upregulation. These results provide evidence of a mechanism that couples translational control and energy metabolism, two processes that all viruses depend on host cells to provide, to support vaccinia virus replication during a host shutoff.

IMPORTANCE
Many viral infections cause global host protein synthesis shutoff. While host protein synthesis shutoff benefits the virus by relocating cellular resources to viral replication, it also poses a challenge to the maintenance of cellular functions necessary for viral replication if continuous protein synthesis is required. Here we measured the host mRNA translation rate during a vaccinia virus-induced host shutoff by analyzing total and actively translating mRNAs in a genome-wide manner. This study revealed that oxidative phosphorylation mRNAs were translationally upregulated during vaccinia virus-induced host protein synthesis shutoff. Oxidative phosphorylation is the major cellular energy-producing pathway, and we further showed that maintenance of its function is important for vaccinia virus replication. This study highlights the fact that vaccinia virus infection can enhance cellular energy production through translational upregulation in the context of an overall host protein synthesis shutoff to meet energy expenditure.
Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency
Authors: Wille Coral K. , Li Yangguang , Rui Lixin , Johannsen Eric C. , Kenney Shannon C.
Published: December 2016

Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters.

IMPORTANCE
EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency.
Potential Role for a β-Catenin Coactivator (High-Mobility Group AT–Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1
Authors: Zhu Liqian , Workman Aspen , Jones Clinton
Published: December 2016

The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to mediate important steps during the latency-reactivation cycle because a mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency in calves. We recently found that the Wnt/β-catenin signaling pathway is regulated during the BoHV-1 latency-reactivation cycle (Y. Liu, M. Hancock, A. Workman, A. Doster, and C. Jones, J Virol 90:3148–3159, 2016). In the present study, a β-catenin coactivator, high-mobility group AT–hook 1 protein (HMGA1), was detected in significantly more neurons in the trigeminal ganglia of latently infected calves than in those of uninfected calves. Consequently, we hypothesized that HMGA1 cooperates with ORF2 and β-catenin to maintain latency. In support of this hypothesis, coimmunoprecipitation studies demonstrated that ORF2 stably interacts with a complex containing β-catenin and/or HMGA1 in transfected mouse neuroblastoma (Neuro-2A) cells. Confocal microscopy provided evidence that ORF2 was relocalized by HMGA1 and β-catenin in Neuro-2A cells. ORF2 consistently enhanced the ability of HMGA1 to stimulate β-catenin-dependent transcription, suggesting that interactions between ORF2 and a complex containing β-catenin and HMGA1 have functional significance. An ORF2 stop codon mutant, an ORF2 nuclear localization mutant, or a mutant lacking the 5 protein kinase A or C phosphorylation sites interfered with its ability to stimulate β-catenin-dependent transcription. Since the canonical Wnt/β-catenin signaling pathway promotes neurogenesis (synapse formation and remodeling) and inhibits neurodegeneration, interactions between ORF2, HMGA1, and β-catenin may be important for certain aspects of the latency-reactivation cycle.

IMPORTANCE
The lifelong latency of bovine herpesvirus 1 (BoHV-1) requires that significant numbers of infected sensory neurons survive infection and maintain normal functions. Consequently, we hypothesize that viral products expressed during latency cooperate with neuronal factors to maintain latency. Our studies revealed that a β-catenin coactivator, high-mobility group AT–hook 1 protein (HMGA1), was readily detected in a subset of trigeminal ganglion neurons in latently infected calves but not in uninfected calves. A viral protein (ORF2) expressed in latently infected neurons interacted with β-catenin and HMGA1 in transfected cells, which resulted in the nuclear localization of β-catenin. This interaction correlated with the ability of ORF2 to stimulate the coactivator functions of HMGA1. These findings are significant because the canonical Wnt/β-catenin signaling pathway promotes neurogenesis and inhibits neurodegeneration.
Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses
Authors: Yang Lei , Zhu Wenfei , Li Xiaodan , Bo Hong , Zhang Ye , Zou Shumei , Gao Rongbao , Dong Jie , Zhao Xiang , Chen Wenbing , Dong Libo , Zou Xiaohui , Xing Yongcai , Wang Dayan , Shu Yuelong
Published: December 2016

Clade 2.3.4.4 highly pathogenic avian influenza viruses (H5Nx) have spread from Asia to other parts of the world. Since 2014, human infections with clade 2.3.4.4 highly pathogenic avian influenza H5N6 viruses have been continuously reported in China. To investigate the genesis of the virus, we analyzed 123 H5 or N6 environmental viruses sampled from live-poultry markets or farms from 2012 to 2015 in Mainland China. Our results indicated that clade 2.3.4.4 H5N2/N6/N8 viruses shared the same hemagglutinin gene as originated in early 2009. From 2012 to 2015, the genesis of highly pathogenic avian influenza H5N6 viruses occurred via two independent pathways. Three major reassortant H5N6 viruses (reassortants A, B, and C) were generated. Internal genes of reassortant A and B viruses and reassortant C viruses derived from clade 2.3.2.1c H5N1 and H9N2 viruses, respectively. Many mammalian adaption mutations and antigenic variations were detected among the three reassortant viruses. Considering their wide circulation and dynamic reassortment in poultry, we highly recommend close monitoring of the viruses in poultry and humans.

IMPORTANCE
Since 2014, clade 2.3.4.4 highly pathogenic avian influenza (H5Nx) viruses have caused many outbreaks in both wild and domestic birds globally. Severe human cases with novel H5N6 viruses in this group were also reported in China in 2014 and 2015. To investigate the genesis of the genetic diversity of these H5N6 viruses, we sequenced 123 H5 or N6 environmental viruses sampled from 2012 to 2015 in China. Sequence analysis indicated that three major reassortants of these H5N6 viruses had been generated by two independent evolutionary pathways. The H5N6 reassortant viruses had been detected in most provinces of southern China and neighboring countries. Considering the mammalian adaption mutations and antigenic variation detected, the spread of these viruses should be monitored carefully due to their pandemic potential.