bluuebunny/crossref_metadata_2025 huggingface dataset:
The hand hygiene compliance of student nurses during clinical placements
Authors: Sundal Jorun Sætre , Aune Anne Grethe , Storvig Eline , Aasland Jenny Kristin , Fjeldsæter Kaja Linn , Torjuul Kirsti
Published: March 2017

Aims and objectives

To observe student nurses’ overall and moment‐specific hand hygiene compliance during clinical placement.

Background

Hand hygiene is the single most important measure to prevent healthcare‐associated infections. However, research has shown low compliance among healthcare workers. During clinical placements, student nurses perform various nursing tasks and procedures to a large number of patients, requiring extensive patient contact. It is crucial that they practice correct hand hygiene to prevent healthcare‐associated infections.

Design

Open, standardised and nonparticipating observations.

Methods

Twenty‐nine student nurses were observed three times for 20 ± 10 min during clinical placement in a Norwegian university hospital. To measure compliance, we used WHO's Hand Hygiene Observation tool, based on the model “My five moments for hand hygiene”.

Results

Overall hand hygiene compliance in the student group was 83.5%. Highest moment‐specific compliance was after touching patient surroundings, after touching patients and after body fluid exposure risk. Lowest moment‐specific compliance was recorded before touching patients or patient surroundings, and before clean/aseptic procedures.

Conclusions

Nurse education needs to be improved both theoretically and during clinical placements in order to advance and sustain compliance among student nurses.

Relevance to clinical practice

Increasing healthcare workers’ compliance with hand hygiene guidelines remains a challenge to the clinical community. In order to reduce healthcare‐associated infections, it is important to educate student nurses to comply with the guidelines during clinical placements. Identifying student nurses’ hand hygiene performance is the first step towards developing teaching methods to improve and sustain their overall and moment‐specific compliance. As a measure to ensure student compliance during clinical placements, mentors should be aware of their influence on students’ performance, act as hand hygiene ambassadors, encourage students to comply with established guidelines and provide regular feedback.
Spiritual aspects of living with infertility: A synthesis of qualitative studies
Authors: Romeiro Joana , Caldeira Sílvia , Brady Vivienne , Timmins Fiona , Hall Jenny
Published: March 2017

Aims and objectives

To identify the spiritual aspects of patients experiencing infertility and seek a deeper and broader meaning of the involuntary childlessness experience.

Background

Infertility can be the cause for a spiritual crisis among some couples. Those who endure this involuntary childlessness condition frequently experience contradictory feelings and needs. In this context, core aspects of spirituality such as meaning and purpose in life are often questioned.

Design

A review and synthesis of qualitative empirical research was undertaken to seek a deeper understanding of the spiritual aspects of patients’ experiences of infertility.

Methods

An aggregative synthesis was conducted according to Saini and Shlonsky (Systematic synthesis of qualitative research, 2012, Oxford University Press, Oxford), using thematic analysis.

Results

A total of 26 studies included female, male and couples. Settings revealed interviewees in different infertility phases such as diagnosis, assisted reproductive technologies and following fertility treatments. Two main themes emerged: spiritual needs and spirituality as a coping resource for infertility.

Conclusion

Infertility affects the holistic existence of the couples. This adversity awakens spiritual needs along with unmet needs of parenthood. Coping strategies incorporating spirituality can enhance the ability of couples to overcome childlessness and suffering.

Relevance to clinical practice

Infertile couples’ experiences of infertility may offer an opportunity for spiritual care particularly related to the assessment of spiritual needs and the promotion of spiritual coping strategies. Effective holistic care should support couples in overcoming and finding meaning in this life and health condition.
Maternal vernalization and vernalization‐pathway genes influence progeny seed germination
Authors: Auge Gabriela A. , Blair Logan K. , Neville Hannah , Donohue Kathleen
Published: March 2017

Different life stages frequently respond to the same environmental cue to regulate development so that each life stage is matched to its appropriate season. We investigated how independently each life stage can respond to shared environmental cues, focusing on vernalization, in Arabidopsis thaliana plants.

We first tested whether effects of rosette vernalization persisted to influence seed germination. To test whether genes in the vernalization flowering pathway also influence germination, we assessed germination of functional and nonfunctional alleles of these genes and measured their level of expression at different life stages in response to rosette vernalization.

Rosette vernalization increased seed germination in diverse ecotypes. Genes in the vernalization flowering pathway also influenced seed germination. In the Columbia accession, functional alleles of most of these genes opposed the germination response observed in the ecotypes. Some genes influenced germination in a manner consistent with their known effects on FLOWERING LOCUS C gene regulation during the transition to flowering. Others did not, suggesting functional divergence across life stages.

Despite persistent effects of environmental conditions across life stages, and despite pleiotropy of genes that affect both flowering and germination, the function of these genes can differ across life stages, potentially mitigating pleiotropic constraints and enabling independent environmental regulation of different life stages.
Intraspecific chemical communication in microalgae
Authors: Venuleo Marianna , Raven John A. , Giordano Mario
Published: March 2017

Contents

Summary
516

I.
Introduction
516

II.
What does ‘intraspecific’ mean in the case of microalgae?
518

III.
Modulation of intraspecific chemical communication
518

IV.
Genetic and phenotypic bases for intraspecific communication
521

V.
Possible evolutionary trajectories of intraspecific chemical communication
523

VI.
Processes entailing intraspecific communication
523

VII.
Conclusions
526

Acknowledgements
526

References
526

The relevance of infochemicals in the relationships between organisms is emerging as a fundamental aspect of aquatic ecology. Exchanges of chemical cues are likely to occur not only between organisms of different species, but also between conspecific individuals. Especially intriguing is the investigation of chemical communication in microalgae, because of the relevance of these organisms for global primary production and their key role in trophic webs. Intraspecific communication between algae has been investigated mostly in relation to sexuality and mating. The literature also contains information on other types of intraspecific chemical communication that have not always been explicitly tagged as ways to communicate to conspecifics. However, the proposed role of certain compounds as intraspecific infochemicals appears questionable. In this article, we make use of this plethora of information to describe the various instances of intraspecific chemical communication between conspecific microalgae and to identify the common traits and ecological significance of intraspecific communication. We also discuss the evolutionary implications of intraspecific chemical communication and the mechanisms by which it can be inherited. A special focus is the genetic diversity among conspecific algae, including the possibility that genetic diversity is an absolute requirement for intraspecific chemical communication.
Effect of Tempol on the prevention of irradiation‐induced mucositis in miniature pigs
Authors: Hu L , Wang Y , Cotrim AP , Zhu Z , Gao R , Zheng C , Goldsmith CM , Jin L , Zhang C , Mitchell JB , Baum BJ , Wang S
Published: March 2017

Objective

The goals of this study were to (i) establish a useful miniature pig (minipig) model for irradiation‐induced oral mucositis and (ii) evaluate the effect of Tempol to prevent its development.

Methods and Materials

Minipigs were irradiated with 6 Gy for five consecutive days targeting the entire oral cavity. To prevent radiation damage, minipigs were treated with 30 mg kg−1 Tempol 10 min before irradiation (n = 4), while the radiation‐alone group was similarly injected with saline (n = 4). Lesions were graded using an oral mucositis score and visual inspection every 3 days, and biopsy of multiple sites was performed at day 18. Weight and chest and abdominal circumferences were measured every 3 days.

Results

Lesions began about 12 days after the first irradiation fraction and healed about 30 days after irradiation. Epithelial thickness was calculated on the lingual and buccal mucosa on the 18th day after the first irradiation fraction. Tempol provided modest protection from ulceration after irradiation using this treatment strategy.

Conclusions

This study established a useful large animal model for irradiation‐induced oral mucositis and showed modest beneficial effects of Tempol in limiting tissue damage. The latter finding may be potentially valuable in preventing oral mucositis in patients receiving irradiation for head and neck cancers.
A bibliometric analysis of pediatric liver transplantation publications
Authors: McDowell Dermot T. , Darani Alexandre , Shun Albert , Thomas Gordon , Holland Andrew J. A.
Published: March 2017

Citation counts can identify landmark papers. The aim of this study was to identify and characterize the top‐cited articles in the pediatric liver transplantation literature. A search strategy for the Scopus® database was designed for pediatric liver transplantation publications from 1945 to 2014. The 50 top‐cited articles were analyzed. Author co‐citation analysis was performed using VOSviewer techniques. There were 2896 articles published between 1969 and 2015. The mean citation count of the top 50 cited articles was 166 (range 95‐635). There were three case reports in this top‐cited list. There were 15 collaborations in this top‐cited list with nine being international. The top‐cited publications originated in 12 countries, with the USA and the UK contributing 31 and seven articles, respectively. There were 14 authors with four or more publications in this list. There was a single author with nine publications in the top‐cited list. These top‐cited papers were found in 16 journals, with three journals collectively publishing over 50% of these publications. Pediatric liver transplantation research is an evolving entity. Surgical techniques and case reports are influential articles. Collaborations at a national and international level produce highly cited articles, which are found in influential journals.
Imaging for Vascular Calcification
Authors: Raggi Paolo , O'Neill W. Charles
Published: March 2017

Chronic decline in renal function is accompanied by deterioration of bone structure and function and progressive calcification of the vascular system. Both disease states have been linked with increased morbidity and mortality in chronic kidney disease. The severe alterations of mineral metabolism inherent with loss of renal function have an impact on vascular calcification development and progression, and several investigators have focused on ways to reduce their impact on vascular health. Imaging has contributed an important role in the assessment of vascular calcification, and the impact of various interventions aimed at curbing their progression.
Early structural and functional signature of 3‐day human skeletal muscle disuse using the dry immersion model
Authors: Demangel Rémi , Treffel Loïc , Py Guillaume , Brioche Thomas , Pagano Allan F. , Bareille Marie‐Pierre , Beck Arnaud , Pessemesse Laurence , Candau Robin , Gharib Claude , Chopard Angèle , Millet Catherine
Published: March 2017

Key points

Our study contributes to the characterization of muscle loss and weakness processes induced by a sedentary life style, chronic hypoactivity, clinical bed rest, immobilization and microgravity.

This study, by bringing together integrated and cellular evaluation of muscle structure and function, identifies the early functional markers and biomarkers of muscle deconditioning.

Three days of muscle disuse in healthy adult subjects is sufficient to significantly decrease muscle mass, tone and force, and to induce changes in function relating to a weakness in aerobic metabolism and muscle fibre denervation.

The outcomes of this study should be considered in the development of an early muscle loss prevention programme and/or the development of pre‐conditioning programmes required before clinical bed rest, immobilization and spaceflight travel.

Microgravity and hypoactivity are associated with skeletal muscle deconditioning. The decrease of muscle mass follows an exponential decay, with major changes in the first days. The purpose of the study was to dissect out the effects of a short‐term 3‐day dry immersion (DI) on human quadriceps muscle function and structure. The DI model, by suppressing all support zones, accurately reproduces the effects of microgravity. Twelve healthy volunteers (32 ± 5 years) completed 3 days of DI. Muscle function was investigated through maximal voluntary contraction (MVC) tests and muscle viscoelasticity. Structural experiments were performed using MRI analysis and invasive experiments on muscle fibres. Our results indicated a significant 9.1% decrease of the normalized MVC constant (P = 0.048). Contraction and relaxation modelization kinetics reported modifications related to torque generation (kACT = −29%; P = 0.014) and to the relaxation phase (kREL = +34%; P = 0.040) after 3 days of DI. Muscle viscoelasticity was also altered. From day one, rectus femoris stiffness and tone decreased by, respectively, 7.3% (P = 0.002) and 10.2% (P = 0.002), and rectus femoris elasticity decreased by 31.5% (P = 0.004) after 3 days of DI. At the cellular level, 3 days of DI translated into a significant atrophy of type I muscle fibres (−10.6 ± 12.1%, P = 0.027) and an increased proportion of hybrid, type I/IIX fibre co‐expression. Finally, we report an increase (6‐fold; P = 0.002) in NCAM+ muscle fibres, showing an early denervation process. This study is the first to report experiments performed in Europe investigating human short‐term DI‐induced muscle adaptations, and contributes to deciphering the early changes and biomarkers of skeletal muscle deconditioning.
Particulate Matter Emission Suppression Strategies in a Turbocharged Gasoline Direct-Injection Engine
Authors: Ming Zhang , Jun Zhong , Stefano Capelli , Luigi Lubrano
Published: March 2017

The development process of a down-sized turbocharged gasoline direct-injection (GDI) engine/vehicle was partially introduced with the focus on particulate matter (PM)/particle number (PN) emission reduction. To achieve this goal, the injection system was upgraded to obtain higher injection pressure. Two types of prototype injectors were designed and compared under critical test conditions. Combined numerical and experimental analysis was made to select the right injector in terms of particle emission. With the selected injector, the effect of injection parameters calibration (injection pressure, start of injection (SOI) timing, number of injection pulses, etc.) on PM/PN emission was illustrated. The number of fuel injection pulses, SOI timing, and injection pressure were found playing the leading role in terms of the particle emission suppression. With single-injection strategy, the injection pressure and SOI timing were found to be a dominant factor to reduce particle emission in warm-up condition and cold condition, respectively; a fine combination of injection timing and injection pressure is generally able to decrease up to 50% of PM emission in a wide range of the engine map. While with multiple injection, up to an order of magnitude PM emission reduction can be achieved. Several New European Driving Cycle (NEDC) emission cycles were arranged on a demo vehicle to evaluate the effect of the injection system upgrade and adjusted calibration. This work will provide a guide for the emission control of GDI engines/vehicles fulfilling future emission legislation.
Social sounds produced by franciscana dolphins, Pontoporia blainvillei (Cetartiodactyla, Pontoporiidae)
Authors: Cremer Marta Jussara , Holz Annelise Colin , Bordino Pablo , Wells Randall S. , Simões-Lopes Paulo César
Published: March 2017

Franciscana dolphin (Pontoporia blainvillei) whistles were documented for the first time during 2003–2013 in Babitonga Bay estuary, South Brazil, together with burst pulses. Recordings were made from small boats under good sea conditions, and recording equipment that allowed analysis of sounds up to 96 kHz. The recordings were made in the presence of 2–31 franciscana dolphins. During 23 h and 53 min, 90 whistles and 51 burst pulse series were recorded. Although Guiana dolphins (Sotalia guianensis) inhabit nearby waters, none were observed in the area during the recordings. The authors recorded ten types of whistles. The initial frequency varied between 1.6 and 94.6 kHz, and the final frequency varied between 0.7 and 94.5 kHz; the authors were not able to determine if dolphin whistles exceeded the 96 kHz recording limit of the authors' equipment, although that is likely, especially because some whistles showed harmonics. Whistle duration varied between 0.008 and 0.361 s. Burst pulses had initial frequencies between 69 and 82.1 kHz (77 ± 3.81). These results showed that P. blainvillei produces whistles and burst pulses, although they seem to be produced infrequently.
Strength and wave parameters for sound propagation in random media
Authors: Ostashev Vladimir E. , Wilson D. Keith
Published: March 2017

Line-of-sight sound propagation of plane and spherical waves in a statistically isotropic, random moving medium is considered. The variances of the phase and log-amplitude fluctuations of these waves are expressed in terms of the strength and wave parameters for arbitrary spectra of temperature and velocity fluctuations, and results are then derived specifically for the Gaussian and generalized von Kármán spectra. This representation of the variances reduces significantly the number of independent parameters of the problem and enables better understanding of sound scattering by plane and spherical waves, and due to temperature and velocity fluctuations. Using this representation, the boundary between the weak and strong scattering regimes is determined in terms of the strength and wave parameters. The results obtained are compared with the Λ – Φ diagram adopted in ocean acoustics. Other statistical moments of plane and spherical waves in a medium with arbitrary spectra of temperature and velocity fluctuations such as the mean sound field, the spatial and temporal mutual coherence functions, the coherence bandwidth, and the variance of the angle-of-arrival fluctuations are expressed in terms of the strength parameter and length scale of the fluctuations.
Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip
Authors: Yeh Erh-Chia , Fu Chi-Cheng , Hu Lucy , Thakur Rohan , Feng Jeffrey , Lee Luke P.
Published: March 2017

Using lab-on-chip technology, bulky lab equipment is shrunk into a simple, portable, and quantitative microfluidic chip for DNA diagnostics.
Terrestrial support of lake food webs: Synthesis reveals controls over cross-ecosystem resource use
Authors: Tanentzap Andrew J. , Kielstra Brian W. , Wilkinson Grace M. , Berggren Martin , Craig Nicola , del Giorgio Paul A. , Grey Jonathan , Gunn John M. , Jones Stuart E. , Karlsson Jan , Solomon Christopher T. , Pace Michael L.
Published: March 2017

There is widespread evidence that aquatic consumers use terrestrial resources depending on the features of surrounding catchments.
Lysosomal cholesterol activates mTORC1 via an SLC38A9–Niemann-Pick C1 signaling complex
Authors: Castellano Brian M. , Thelen Ashley M. , Moldavski Ofer , Feltes McKenna , van der Welle Reini E. N. , Mydock-McGrane Laurel , Jiang Xuntian , van Eijkeren Robert J , Davis Oliver B. , Louie Sharon M. , Perera Rushika M. , Covey Douglas F. , Nomura Daniel K. , Ory Daniel S. , Zoncu Roberto
Published: March 2017

A cholesterol-mTORC1 axis may play a role in metabolic homeostasis in normal and disease states.
Vertically extensive and unstable magmatic systems: A unified view of igneous processes
Authors: Cashman Katharine V. , Sparks R. Stephen J. , Blundy Jonathan D.
Published: March 2017

Making magma chambers from mush

Shallow magma chambers either erupt as volcanoes or solidify as intrusive magma bodies. These magma bodies are traditionally considered to be long-lived and dominated by melt. Cashman
et al.
review the evidence that shallow magma chambers are actually assembled quickly from much larger, crystal-rich transcrustal magmatic systems. This paradigm helps explain many geophysical and geochemical features of volcanic systems. It also presents challenges for understanding the evolution of magma and provides insight into how and why volcanoes erupt.

Science
, this issue p.
eaag3055
PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D
Authors: Toska Eneda , Osmanbeyoglu Hatice U. , Castel Pau , Chan Carmen , Hendrickson Ronald C. , Elkabets Moshe , Dickler Maura N. , Scaltriti Maurizio , Leslie Christina S. , Armstrong Scott A. , Baselga José
Published: March 2017

Tumor cells develop resistance to a drug used to treat breast cancer through a chromatin remodeling mechanism.
[C
ii
] 158-μm emission from the host galaxies of damped Lyman-alpha systems
Authors: Neeleman Marcel , Kanekar Nissim , Prochaska J. Xavier , Rafelski Marc , Carilli Chris L. , Wolfe Arthur M.
Published: March 2017

Gas emission lines from damped Lyman-α systems probe galaxy evolution in the early universe.
Transition from turbulent to coherent flows in confined three-dimensional active fluids
Authors: Wu Kun-Ta , Hishamunda Jean Bernard , Chen Daniel T. N. , DeCamp Stephen J. , Chang Ya-Wen , Fernández-Nieves Alberto , Fraden Seth , Dogic Zvonimir
Published: March 2017

Go with the changing flow

The transport of ordinary fluids tends to be driven by pressure differentials, whereas for active or biological matter, transport may be isotropic or governed by the presence of specific chemical gradients. Wu
et al.
analyzed the emergence of spontaneous directional flows in active fluids containing a suspension of microtubules and clusters of the molecular motor kinesin, confined in a variety of microfluidic geometries (see the Perspective by Morozov). When confined in periodic toroidal channels and cylindrical domains, the flow was organized and persisted in a unidirectional motion, either clockwise or counterclockwise. Oddly, this behavior was independent of scale; as long as the aspect ratio of the geometry was chosen appropriately, flows were observed for a wide range of system dimensions.

Science
, this issue p.
eaal1979
; see also p.
1262
Grain boundary stability governs hardening and softening in extremely fine nanograined metals
Authors: Hu J. , Shi Y. N. , Sauvage X. , Sha G. , Lu K.
Published: March 2017

Stabilized nanograin boundaries in nickel-molybdenum alloys result in increased hardness with decreasing grain size.
Using fire to promote biodiversity
Authors: Kelly L. T. , Brotons L.
Published: March 2017

Biodiversity can benefit from fires tailored to suit particular ecosystems and species
Macrophage, a long-distance middleman
Authors: Guilliams Martin
Published: March 2017

An immune cell organizes pigment cells during zebrafish stripe development
Dientamoeba fragilis, a Commensal in Children in Danish Day Care Centers
Authors: Jokelainen Pikka , Hebbelstrup Jensen Betina , Andreassen Bente Utoft , Petersen Andreas Munk , Röser Dennis , Krogfelt Karen A. , Nielsen Henrik V. , Stensvold Christen R.
Published: March 2017

Dientamoeba fragilis
is an intestinal protozoan of debated clinical significance. Here, we present cross-sectional and longitudinal observations on
D. fragilis
in children aged 0 to 6 years from a 1-year multi-day-care-center cohort study set in Copenhagen, Denmark. The inclusion period for the cohort was 2009 through 2012. Stool samples collected from the children were accompanied by questionnaires completed by the parents or guardians of the children. Using real-time PCR,
D. fragilis
was detected in the first stool sample from 97 of 142 (68.3%) children. We evaluated the associations between seven plausible risk factors (age, sex, having siblings, having domestic animals at home, having had infant colic, recent history of intake of antibiotics, and recent history of travel abroad) as well as six reported symptoms (lack of appetite, nausea, vomiting, abdominal pain, weight loss, and diarrhea) and testing positive for
D. fragilis
. The final multivariable model identified being >3 years old and having a history of recent travel abroad as risk factors for testing positive for
D. fragilis
. Moreover, univariable analyses indicated that having siblings was a risk factor. There was no statistical association between a recent history of gastrointestinal symptoms and testing positive for
D. fragilis
. Among the 108 children who were represented by ≥2 samples and thus included in the longitudinal analysis, 32 tested negative on the first sample and positive later, and the last sample from each of the 108 children was positive. The results are in support of
D. fragilis
being a common enteric commensal in this population.
Predicting the Ability of Preclinical Diagnosis To Improve Control of Farm-to-Farm Foot-and-Mouth Disease Transmission in Cattle
Authors: Nelson Noel , Paton David J. , Gubbins Simon , Colenutt Claire , Brown Emma , Hodgson Sophia , Gonzales Jose L.
Published: March 2017

Foot-and-mouth disease (FMD) can cause large disruptive epidemics in livestock. Current eradication measures rely on the rapid clinical detection and removal of infected herds. Here, we evaluated the potential for preclinical diagnosis during reactive surveillance to reduce the risk of between-farm transmission. We used data from transmission experiments in cattle where both samples from individual animals, such as blood, probang samples, and saliva and nasal swabs, and herd-level samples, such as air samples, were taken daily during the course of infection. The sensitivity of each of these sample types for the detection of infected cattle during different phases of the early infection period was quantified. The results were incorporated into a mathematical model for FMD, in a cattle herd, to evaluate the impact of the early detection and culling of an infected herd on the infectious output. The latter was expressed as the between-herd reproduction ratio,

R
h

, where an effective surveillance approach would lead to a reduction in the

R
h

value to <1. Applying weekly surveillance, clinical inspection alone was found to be ineffective at blocking transmission. This was in contrast to the impact of weekly random sampling (i.e., using saliva swabs) of at least 10 animals per farm or daily air sampling (housed cattle), both of which were shown to reduce the

R
h

to <1. In conclusion, preclinical detection during outbreaks has the potential to allow earlier culling of infected herds and thereby reduce transmission and aid the control of epidemics.
In Vitro
Susceptibility Testing of Tedizolid against Nontuberculous Mycobacteria
Authors: Brown-Elliott Barbara A. , Wallace Richard J.
Published: March 2017

Tedizolid is a new oxazolidinone with improved
in vitro
and intracellular potency against
Mycobacterium tuberculosis
, including multidrug-resistant strains, and some species of nontuberculous mycobacteria (NTM) compared with that of linezolid. Using the current Clinical and Laboratory Standards Institute (CLSI)-recommended method of broth microdilution, susceptibility testing of 170 isolates of rapidly growing mycobacteria showed equivalent or lower (1- to 8-fold) MIC
50
and/or MIC
90
values for tedizolid compared with that for linezolid. The tedizolid MIC
90
values for 81 isolates of
M. abscessus
subsp.
abscessus
and 12 isolates of
M. abscessus
subsp.
massiliense
were 8 μg/ml and 4 μg/ml, respectively, compared with linezolid MIC
90
values of 32 μg/ml for both. The MIC
90
values for 20 isolates of
M. fortuitum
were 2 μg/ml for tedizolid and 4 μg/ml for linezolid. Twenty-two isolates of
M. chelonae
had tedizolid and linezolid MIC
90
s of 2 μg/ml and 16 μg/ml, respectively. One hundred forty-two slowly growing NTM, including 7/7
M. marinum
, 7/7
M. kansasii
, and 7/11 of other less commonly isolated species, had tedizolid MICs of ≤1 μg/ml and linezolid MICs of ≤4 μg/ml. One hundred isolates of
Mycobacterium avium
complex and eight
M. simiae
isolates had tedizolid MIC
50
s of 8 μg/ml and linezolid MIC
50
s 32 and 64 μg/ml, respectively. Nine
M. arupense
isolates had MIC
50
s of 4 μg/ml and 16 μg/ml for tedizolid and linezolid, respectively. These findings demonstrate a greater
in vitro
potency of tedizolid than linezolid against NTM and suggest that an evaluation of tedizolid as a potential treatment agent for infections caused by selected NTM is warranted.
Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species
Authors: Lee Justin S. , Mackie Ryan S. , Harrison Thomas , Shariat Basir , Kind Trey , Kehl Timo , Löchelt Martin , Boucher Christina , VandeWoude Sue
Published: March 2017

Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.
Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis
Authors: Anthwal Divya , Gupta Rakesh Kumar , Bhalla Manpreet , Bhatnagar Shinjini , Tyagi Jaya Sivaswami , Haldar Sagarika
Published: March 2017

Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in
Mycobacterium tuberculosis
in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of
rpoB
,
katG
, and an
inhA
promoter and used as positive controls in HRM. The assay was first validated in 25 MDR
M. tuberculosis
clinical isolates. The assay was evaluated on DNA isolated from 99
M. tuberculosis
culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-
katG
, and INH-
inhA
assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.
Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis
Authors: Adkins P. R. F. , Middleton J. R. , Calcutt M. J. , Stewart G. C. , Fox L. K.
Published: March 2017

Staphylococcus hyicusandStaphylococcus agnetisare two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positiveS. hyicusbased on phenotypic species identification methods and to develop a PCR-based method for differentiatingS. hyicus,S. agnetis, andStaphylococcus aureus. Isolates (n= 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiateS. hyicus,S. agnetis, andS. aureuswas developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be eitherS. agnetis(n= 43) orS. hyicus(n= 1). Overall, 88% (37/42) of coagulase-positiveS. agnetisisolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positiveS. agnetisranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest thatS. agnetisis likely more prevalent on dairy farms thanS. hyicus. Also, someS. agnetisisolates in this study appeared to be contagious and associated with persistent infections.
Usefulness of Two Aspergillus PCR Assays and Aspergillus Galactomannan and β-
d
-Glucan Testing of Bronchoalveolar Lavage Fluid for Diagnosis of Chronic Pulmonary Aspergillosis
Authors: Urabe Naohisa , Sakamoto Susumu , Sano Go , Suzuki Junko , Hebisawa Akira , Nakamura Yasuhiko , Koyama Kazuya , Ishii Yoshikazu , Tateda Kazuhiro , Homma Sakae
Published: March 2017

We evaluated the usefulness of an
Aspergillus
galactomannan (GM) test, a β-
d
-glucan (βDG) test, and two different
Aspergillus
PCR assays of bronchoalveolar lavage fluid (BALF) samples for the diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 30 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for the diagnosis of CPA were 0.5 and 100 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. A comparison of the PCR assays showed that PCR assay 1 had a better sensitivity, a better negative predictive value, and a better negative likelihood ratio and PCR assay 2 had a better specificity, a better positive predictive value, and a better positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests on BALF was more useful than any single test for diagnosing CPA.
Heterologous Packaging Signals on Segment 4, but Not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment
Authors: White Maria C. , Steel John , Lowen Anice C.
Published: March 2017

Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment and strain specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous versus heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when segment 4 (HA) of coinfecting parental viruses was modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when segment 6 (NA) or segment 8 (NS) carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment dependent.

IMPORTANCE
Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment.
Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses
Authors: Kwon Hyeok-Il , Kim Young-Il , Park Su-Jin , Song Min-Suk , Kim Eun-Ha , Kim Se Mi , Si Young-Jae , Lee In-Won , Song Byung-Min , Lee Youn-Jeong , Yun Seok Joong , Kim Wun-Jae , Choi Young Ki
Published: March 2017

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%).
In vivo
immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses.

IMPORTANCE
Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.
Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory
Authors: Chen Christopher Phillip , Lyu Yuanzhi , Chuang Frank , Nakano Kazushi , Izumiya Chie , Jin Di , Campbell Mel , Izumiya Yoshihiro
Published: March 2017

Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence
in situ
RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.

IMPORTANCE
B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.
Structural Characterization of Human Coronavirus NL63 N Protein
Authors: Szelazek Bozena , Kabala Wojciech , Kus Krzysztof , Zdzalik Michal , Twarda-Clapa Aleksandra , Golik Przemyslaw , Burmistrz Michal , Florek Dominik , Wladyka Benedykt , Pyrc Krzysztof , Dubin Grzegorz
Published: March 2017

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.

IMPORTANCE
Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.