bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus
Authors: Piret Jocelyne , Goyette Nathalie , Boivin Guy 
Published: June 2016

ABSTRACT

The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC
50
s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC
50
s of acyclovir and foscarnet against a reference wild-type (WT) HSV-1 strain in Vero cells were, respectively, 0.5 μM and 32.6 μM by PRA and 0.8 μM and 93.6 μM by RTCA. The EC
50
ratios for acyclovir against several HSV-1 thymidine kinase (TK) mutants were 101.8×, 73.4×, 28.8×, and 35.4× (PRA) and 18.0×, 52.0×, 5.5×, and 87.8× (RTCA) compared to those for the WT. The EC
50
ratios for acyclovir and foscarnet against the HSV-1 TK/DNA polymerase mutant were 182.8× and 9.7× (PRA) and >125.0× and 10.8× (RTCA) compared to the WT. The EC
50
s of ganciclovir and foscarnet against WT HCMV strain AD169 in fibroblasts were, respectively, 1.6 μM and 27.8 μM by PRA and 5.0 μM and 111.4 μM by RTCA. The EC
50
ratios of ganciclovir against the HCMV UL97 mutant were 3.8× (PRA) and 8.2× (RTCA) compared to those for the WT. The EC
50
ratios of ganciclovir and foscarnet against the HCMV UL97/DNA polymerase mutant were 17.1× and 12.1× (PRA) and 14.7× and 4.6× (RTCA) compared to those for the WT. RTCA allows objective drug susceptibility testing of HSV and HCMV and could permit high-throughput screening of new antivirals.
Interaction between Nonstructural Proteins NS4B and NS5A Is Essential for Proper NS5A Localization and Hepatitis C Virus RNA Replication
Authors: Biswas Avik , Treadaway Jason , Tellinghuisen Timothy L. 
Published: June 2016

ABSTRACT
The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in-plane into the outer endoplasmic reticulum (ER)-derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy, we identified a number of essential residues for replication on the charged face of the NS5A anchor and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy-terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant (D1979K/D1982K) and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions.

IMPORTANCE
Information about the functional role of the cytosolic face of the NS5A anchoring helix remains obscure. In this study, we show that while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays a key role during hepatitis C virus (HCV) replication by mediating the interaction of NS5A with other HCV nonstructural proteins via NS4B. Such an interaction determines the subcellular localization of NS5A by engaging NS5A in the HCV replication process during the formation of a functional HCV replication complex. Thus, collectively, it can be stated that the findings in the present study provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide a platform to gain more insights about the molecular architecture of HCV replication complexes.
Mutagenesis of
S
-Adenosyl-
l
-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity
Authors: Case James Brett , Ashbrook Alison W. , Dermody Terence S. , Denison Mark R. 
Published: June 2016

ABSTRACT

Eukaryotic mRNAs possess a methylated 5′-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5′ caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved
S
-adenosyl-
l
-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity
in vitro
. However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-β, and nsp14 G332A genomes were translated less efficiently both
in vitro
and during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-β compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ
in vitro
and during virus infection.

IMPORTANCE
Human coronaviruses, most notably severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that substitution of alanine at G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.
Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis
Authors: Cai Hui , Zhang Yu , Lu Mijia , Liang Xueya , Jennings Ryan , Niewiesk Stefan , Li Jianrong 
Published: June 2016

ABSTRACT

Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis
in vivo
.

IMPORTANCE
The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis
in vivo
. Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses.
A shale-hosted Cr isotope record of low atmospheric oxygen during the Proterozoic
Authors: Cole Devon B. , Reinhard Christopher T. , Wang Xiangli , Gueguen Bleuenn , Halverson Galen P. , Gibson Timothy , Hodgskiss Malcolm S.W. , McKenzie N. Ryan , Lyons Timothy W. , Planavsky Noah J. 
Published: June 2016

Abstract
The emergence and expansion of animal life on Earth represents a dramatic shift in the structure and complexity of the biosphere. A lack of firm constraints on surface oxygen levels during the mid-Proterozoic has resulted in heated debate as to whether the rise and earliest diversification of animals was directly linked to a change in environmental oxygen levels or, instead, simply reflects the timing of innovations in gene expression and developmental regulation and was independent of a direct environmental trigger. Here, we present chromium (Cr) isotope data from marine black shales that provide evidence for minimal Cr oxidation throughout the mid-Proterozoic leading up to the diversification of eukaryotes and the rise of animals during the late Neoproterozoic. This observation requires very low background oxygen levels (&lt;1% of present atmospheric levels). Accepting previously proposed estimates of pO2 levels needed to induce Cr isotope fractionation, our data provide support for the persistence of an Earth system in which baseline atmospheric pO2 would have been low enough to inhibit the diversification of animals until ca. 800 Ma. More generally, evidence for a delayed rise of atmospheric oxygen strongly suggests that environmental factors have played a fundamental role in controlling the emergence and expansion of complex life on Earth.
Anoxic oxidation of chromium
Authors: Oze Christopher , Sleep Norman H. , Coleman Robert G. , Fendorf Scott 
Published: June 2016

Abstract
Naturally occurring Cr(VI) has been ascribed to terrestrial Cr(III) oxidation by Mn (di)oxides, generated through reaction of Mn(II) with molecular oxygen (O2). However, hydrogen peroxide (H2O2) is a potential oxidant of Cr(III) that may form in serpentinization (high H2, low O2) systems where chromite [i.e., the main mineralogical source of Cr(III)] is abundant. Accordingly, here we evaluate H2O2 and chromite interactions in serpentinization systems to determine pathways of Cr(III) oxidation that alters the current paradigm of O2-dependent oxidation. Field observations support that metastable H2O2 and Cr(VI) are present in serpentinization-related fluids relatively absent of O2. Further, laboratory experiments demonstrate and support that H2O2 is a kinetically facile oxidant of chromite, especially under alkaline conditions, which provides a variety of alternative means by which Cr(VI) may be generated and supplied to the oceans not directly linked to atmospheric O2. Thus, Cr(III) oxidation pathways, and their influence on the Cr isotopic record, must account for anoxic Cr(III) oxidation in serpentinization systems as well as a variety of H2O2-induced Cr(III) oxidation pathways that may occur in both terrestrial and marine systems.
Critical components of neuromuscular training to reduce ACL injury risk in female athletes: meta-regression analysis
Authors: Sugimoto Dai , Myer Gregory D , Barber Foss Kim D , Pepin Michael J , Micheli Lyle J , Hewett Timothy E 
Published: June 2016

ObjectiveThe aim of this study was to determine key components in neuromuscular training that optimise ACL injury reduction in female athletes using meta-regression analyses.DesignSystematic review and meta-regression.Data sourcesThe literature search was performed in PubMed and EBSCO.Eligibility criteriaInclusion criteria for the current analysis were: (1) documented the number of ACL injuries, (2) employed a neuromuscular training intervention that aimed to reduce ACL injuries, (3) had a comparison group, (4) used a prospective control study design and (5) recruited female athletes as participants. Two independent reviewers extracted studies which met the inclusion criteria. Methodological quality of included study and strength of recommendation were evaluated. Number of ACL injuries and participants in control and intervention groups, age of participants, dosage of neuromuscular training, exercise variations within neuromuscular training and status of verbal feedback were extracted.ResultsThe meta-regression analyses identified age of participants, dosage of neuromuscular training, exercise variations within neuromuscular training and utilisation of verbal feedback as significant predictors of ACL injury reduction (p=0.01 in fixed-effects model, p=0.03 in random-effects model). Inclusion of 1 of the 4 components in neuromuscular training could reduce ACL injury risk by 17.2–17.7% in female athletes. No significant heterogeneity and publication bias effects were detected. Strength of recommendation was rated as A (recommendation based on consistent and good-quality patient-oriented study evidence).ConclusionsAge of participants, dosage of neuromuscular training, exercise variations within neuromuscular training and utilisation of verbal feedback are predictors that influence the optimisation of prophylactic effects of neuromuscular training and the resultant ACL injury reduction in female athletes.
Community-based participatory research to improve life quality and clinical outcomes of patients with breast cancer (DianaWeb in Umbria pilot study)
Authors: Villarini Milena , Lanari Chiara , Nucci Daniele , Gianfredi Vincenza , Marzulli Tiziana , Berrino Franco , Borgo Alessandra , Bruno Eleonora , Gargano Giuliana , Moretti Massimo , Villarini Anna 
Published: June 2016

IntroductionBreast cancer (BC) is the most frequent cancer in Europe and the International Agency for Research on Cancer (IARC) has estimated over 460 000 incident cases per year. Survival among patients with BC has increased in the past decades and EUROCARE-5 has estimated a 5-year relative survival rate of 82% for patients diagnosed in 2000–2007. There is growing evidence that lifestyle (such as a diet based on Mediterranean principles associated with moderate physical activity) may influence prognosis of BC; however, this information is not currently available to patients and is not considered in oncology protocols. Only a few epidemiological studies have investigated the role of diet in BC recurrence and metastasis.Methods and analysisDianaWeb is a community-based participatory research dedicated to patients with BC and represents a collaborative effort between participants and research institutions to determine if specified changes in lifestyle would result in improved outcomes in terms of quality of life or survival. The aim of the study is to recruit a large number of participants, to monitor their lifestyle and health status over time, to provide them tips to encourage sustainable lifestyle changes, to analyse clinical outcomes as a function of baseline risk factors and subsequent changes, and to share with patients methodologies and results. DianaWeb uses a specific interactive website (http://www.dianaweb.org/) and, with very few exceptions, all communications will be made through the web. In this paper we describe the pilot study, namely DianaWeb in Umbria.Ethics and disseminationDianaWeb does not interfere with prescribed oncological treatments; rather, it recommends that participants should follow the received prescriptions. The results will be used to plan guidelines for nutrition and physical activity for patients with BC. The pilot study was approved by the ethics committee of the University of Perugia (reference number 2015-002), and is supported by Fondazione Cassa di Risparmio di Perugia (2013.0185 021).
Evolution of target organ damage and haemodynamic parameters over 4 years in patients with increased insulin resistance: the LOD-DIABETES prospective observational study
Authors: Gómez-Marcos Manuel Ángel , Recio-Rodríguez José Ignacio , Patino-Alonso María Carmen , Agudo-Conde Cristina , Rodríguez-Sanchez Emiliano , Maderuelo-Fernandez Jose Angel , Gómez-Sánchez Leticia , Gomez-Sanchez Marta , García-Ortiz Luís ,   LOD-DIABETES Group
Published: June 2016

ObjectivesWe prospectively examined the impact of type 2 diabetes compared with metabolic syndrome (MetS) on the development of vascular disease over 4 years as determined by anatomic and functional markers of vascular disease. By comparing the vascular outcomes of the 2 disorders, we seek to determine the independent effect of elevated glucose levels on vascular disease.Setting2 primary care centres in Salamanca, Spain.ParticipantsWe performed a prospective observational study involving 112 patients (68 with type 2 diabetes and 44 with MetS) who were followed for 4 years.Primary and secondary outcome measuresMeasurements included blood pressure, blood glucose, lipids, smoking, body mass index, waist circumference, Homeostasis Model Assessment Insulin Resistance (HOMA-IR), hs-c-reactive protein and fibrinogen levels. We also evaluated vascular, carotid intima media thickness (IMT), pulse wave velocity (PWV) and ankle/brachial index, heart and renal target organ damage (TOD). The haemodynamic parameters were central (CAIx) and peripheral (PAIx) augmentation indices.ResultsIn year 4, participants with type 2 diabetes had increased IMT thickness. These patients had more plaques and an IMT>0.90 mm. In participants with MetS, we only found an increase in the number of plaques. We found no changes in PWV, CAIx and PAIx. The patients with diabetes had a greater frequency of vascular TOD. There were no differences neither in renal nor cardiac percentage of TOD in the patients with MetS or diabetes mellitus type 2.ConclusionsThis prospective study showed that the evolution of vascular TOD is different in participants with type 2 diabetes compared with those with MetS. While IMT and PWV increased in type 2 diabetes, these were not modified in MetS. The renal and cardiac TOD evolution, as well as the PAIx and CAIx, did not change in either group.Trial registration numberNCT01065155; Results.
K-shell photoionization of Li, Be+ and B2+
Authors: Li Jun , Liu Jian Dang , Zhang Song Bin , Ye Bang Jiao 
Published: June 2016

K-shell photoionization (PI) of Li, Be[Formula: see text] and B[Formula: see text] from ground state [Formula: see text] have been studied by using the [Formula: see text]-matrix method with pseudostates. The K-shell PI process is featured with the contributions from the core-excited metastable states or dominated by the Auger states 2Po. The resonant parameters of the Auger states 2Po and the PI cross-sections have been calculated and compared with the available experimental and theoretical works. Our results agree very well with that of the published works. It is worth noting that compared with previous theoretical calculations, our results of B[Formula: see text] show better agreements with the latest high-resolution advanced light source measurements [A. Müller et al., J. Phys. B 43 (2010) 135602].
Image Analysis–based Assessment of PD-L1 and Tumor-Associated Immune Cells Density Supports Distinct Intratumoral Microenvironment Groups in Non–small Cell Lung Carcinoma Patients
Authors: Parra Edwin R. , Behrens Carmen , Rodriguez-Canales Jaime , Lin Heather , Mino Barbara , Blando Jorge , Zhang Jianjun , Gibbons Don L. , Heymach John V. , Sepesi Boris , Swisher Stephen G. , Weissferdt Annikka , Kalhor Neda , Izzo Julie , Kadara Humam , Moran Cesar , Lee Jack J. , Wistuba Ignacio I. 
Published: June 2016

Abstract
Purpose: We investigated the correlation between immunohistochemical PD-L1 expression and tumor-associated immune cells (TAICs) density in non–small cell lung carcinoma (NSCLC) and correlated them with clinicopathologic variables.
Experimental Design: Tumor tissue specimens from 254 stage I–III NSCLCs [146 adenocarcinomas; 108 squamous cell carcinomas (SCCs)] were examined. PD-L1 expression in malignant cells and macrophages and the density of TAICs expressing CD3, CD4, CD8, CD57, granzyme B, CD45RO, PD-1, FOXP3, and CD68 were evaluated using immunohistochemistry and image analysis.
Results: Malignant cells PD-L1 H-score &gt; 5 was detected in 23% of adenocarcinomas and 31% of SCCs, and no significant differences were detected comparing both histologies; the median H-score in macrophages was significantly higher in SCC than in adenocarcinoma (P &lt; 0.001). In adenocarcinoma, high malignant cells PD-L1 expression and high TAIC density correlated with solid histology, smoking history, and airflow limitation. Multivariate analysis demonstrated that high CD57-positive cell density correlated with better recurrence-free survival (RFS; P = 0.0236; HR, 0.457) and overall survival (OS; P = 0.0261; HR, 0.481) rates for SCC. High CD68-positive cell density in intratumoral compartment correlated with better RFS (P = 0.0436; HR, 0.553) for adenocarcinoma. The combination of low CD4/CD8/C68-positive cell density and PD-L1 H-score &gt;5 in malignant cells identified small subset of adenocarcinomas with worse outcomes (RFS: P = 0.036; HR, 4.299; OS: P = 0.00034; HR, 5.632).
Conclusions: We detected different PD-L1 expression and TAIC density patterns in NSCLC. Distinct groups of tumor microenvironment correlated with NSCLC clinicopathologic features, including outcome. Clin Cancer Res; 22(24); 6278–89. ©2016 AACR.