bluuebunny/crossref_metadata_2025 huggingface dataset:
Neotropical Bats from Costa Rica harbour Diverse Coronaviruses
Authors: Moreira‐Soto A. , Taylor‐Castillo L. , Vargas‐Vargas N. , Rodríguez‐Herrera B. , Jiménez C. , Corrales‐Aguilar E.
Published: February 2015

Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host–species barrier. For analysing coronavirus diversity in a bat species‐rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA‐dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus‐derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α‐CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected.
A New Design Tool for Tire Braking Performance Evaluations
Authors: Sivaramakrishnan Srikanth , Siramdasu Yaswanth , Taheri Saied
Published: February 2015

The objective of this study is to understand the influence of high frequency tire vibrations induced due to road disturbances and brake torque cycling due to anti-lock braking system (ABS) on braking performance. Under these conditions, transient dynamics of the tire play a crucial role in the generation of braking force. To implement this, a dynamic tire model was developed using a rigid ring tire and a tandem elliptical cam design for the enveloping model. This tire model is validated using experimental data obtained from high-speed cleat tests on a fixed axle. The validated tire model is then integrated with a quarter vehicle and a commercial grade rule-based ABS model to evaluate braking performance with and without a road cleat, which can provide a high frequency disturbance. Simulation results show that the presence of a 1 cm cleat causes large variations in wheel slip, consequently increasing the braking distance. The developed tool will help both tire and vehicle manufactures for quick and early evaluation of braking performance without computationally intensive finite element analysis (FEA) tools.
The Flow Behavior of a Biofluid in a Separated and Reattached Flow Region
Authors: Hammad Khaled J.
Published: February 2015

The flow behavior of human blood in a separated and reattached flow region is investigated. Hemorheological data that account for the yield stress and shear-thinning non-Newtonian characteristics of blood are used. The governing mass and momentum conservation equations along with the Herschel–Bulkley constitutive equation are solved numerically using a finite-difference scheme. Two inflow velocity profiles are considered, uniform and fully developed (fd) ones. A parametric study is performed to reveal the impact of inflow velocity profile, upstream flow restriction, and rheology on the recirculation strength and reattachment characteristics of the flow field. Uniform inflow conditions result in larger relative recirculation intensity, in comparison with the fd ones, only for a moderate upstream flow restriction. The separated flow region size in the case of a fd inflow is always larger than the one observed for uniform inflow. Larger separated flow regions with stronger flow recirculation, are predicted by the Newtonian (N) model in comparison with the yield shear-thinning (HB) model for all studied inflow and upstream restriction conditions. The separated flow region size displays a stronger dependency on the inflow velocity profile and upstream flow restriction, in comparison with the observed dependency on the used hemorheological model.
Organometallic exposure dependence on organic–inorganic hybrid material formation in polyethylene terephthalate and polyamide 6 polymer fibers
Authors: Akyildiz Halil I. , Jur Jesse S.
Published: February 2015

The effect of exposure conditions and surface area on hybrid material formation during sequential vapor infiltrations of trimethylaluminum (TMA) into polyamide 6 (PA6) and polyethylene terephthalate (PET) fibers is investigated. Mass gain of the fabric samples after infiltration was examined to elucidate the reaction extent with increasing number of sequential TMA single exposures, defined as the times for a TMA dose and a hold period. An interdependent relationship between dosing time and holding time on the hybrid material formation is observed for TMA exposure PET, exhibited as a linear trend between the mass gain and total exposure (dose time × hold time × number of sequential exposures). Deviation from this linear relationship is only observed under very long dose or hold times. In comparison, amount of hybrid material formed during sequential exposures to PA6 fibers is found to be highly dependent on amount of TMA dosed. Increasing the surface area of the fiber by altering its cross-sectional dimension is shown to have little on the reaction behavior but does allow for improved diffusion of the TMA into the fiber. This work allows for the projection of exposure parameters necessary for future high-throughput hybrid modifications to polymer materials.
Light output measurements and computational models of microcolumnar CsI scintillators for x‐ray imaging
Authors: Nillius Peter , Klamra Wlodek , Sibczynski Pawel , Sharma Diksha , Danielsson Mats , Badano Aldo
Published: February 2015

Purpose:

The authors report on measurements of light output and spatial resolution of microcolumnar CsI:Tl scintillator detectors for x‐ray imaging. In addition, the authors discuss the results of simulations aimed at analyzing the results of synchrotron and sealed‐source exposures with respect to the contributions of light transport to the total light output.

Methods:

The authors measured light output from a 490‐μm CsI:Tl scintillator screen using two setups. First, the authors used a photomultiplier tube (PMT) to measure the response of the scintillator to sealed‐source exposures. Second, the authors performed imaging experiments with a 27‐keV monoenergetic synchrotron beam and a slit to calculate the total signal generated in terms of optical photons per keV. The results of both methods are compared to simulations obtained with hybridmantis, a coupled x‐ray, electron, and optical photon Monte Carlo transport package. The authors report line response (LR) and light output for a range of linear absorption coefficients and describe a model that fits at the same time the light output and the blur measurements. Comparing the experimental results with the simulations, the authors obtained an estimate of the absorption coefficient for the model that provides good agreement with the experimentally measured LR. Finally, the authors report light output simulation results and their dependence on scintillator thickness and reflectivity of the backing surface.

Results:

The slit images from the synchrotron were analyzed to obtain a total light output of 48 keV−1 while measurements using the fast PMT instrument setup and sealed‐sources reported a light output of 28 keV−1. The authors attribute the difference in light output estimates between the two methods to the difference in time constants between the camera and PMT measurements. Simulation structures were designed to match the light output measured with the camera while providing good agreement with the measured LR resulting in a bulk absorption coefficient of 5 × 10−5 μm−1.

Conclusions:

The combination of experimental measurements for microcolumnar CsI:Tl scintillators using sealed‐sources and synchrotron exposures with results obtained via simulation suggests that the time course of the emission might play a role in experimental estimates. The procedure yielded an experimentally derived linear absorption coefficient for microcolumnar Cs:Tl of 5 × 10−5 μm−1. To the author's knowledge, this is the first time this parameter has been validated against experimental observations. The measurements also offer insight into the relative role of optical transport on the effective optical yield of the scintillator with microcolumnar structure.
Combinatorial labeling of single cells for gene expression cytometry
Authors: Fan H. Christina , Fu Glenn K. , Fodor Stephen P. A.
Published: February 2015

Single-cell expression analysis on a large scale

To understand why cells differ from each other, we need to understand which genes are transcribed at a single-cell level. Several methods measure messenger RNA (mRNA) expression in single cells, but most are limited to relatively low numbers of cells or genes. Fan
et al.
labeled each mRNA molecule in a cell with both a cellular barcode and a molecular barcode. Further analysis did not then require single-cell technologies. Instead, the labeled mRNA from all cells was pooled, amplified, and sequenced, and the gene expression profile of individual cells was reconstructed based on the barcodes. The technique successfully revealed heterogeneity across several thousand blood cells.

Science
, this issue
10.1126/science.1258367
The coordination chemistry of nanocrystal surfaces
Authors: Owen Jonathan
Published: February 2015

The luminescence and electronic properties of inorganic nanocrystals depends on surface-layer structure
A rational strategy for the realization of chain-growth supramolecular polymerization
Authors: Kang Jiheong , Miyajima Daigo , Mori Tadashi , Inoue Yoshihisa , Itoh Yoshimitsu , Aida Takuzo
Published: February 2015

Popping open one by one into polymers

We rarely board airplanes by joining the back of a single well-ordered line. More often, we jostle around in one of several bulging crowds that merge haphazardly near the gate. Roughly speaking, these processes are analogous to the chain growth and step growth mechanisms of polymer assembly at the molecular level. Kang
et al.
present a strategy to link molecular building blocks through hydrogen bonding in accord with the well-controlled chain growth model. The molecules start out curled inward, as they engage in internal hydrogen bonding, until an initiator pulls one open; that molecule is then in the right conformation to pull a partner into the growing chain, poising it to pull in yet another, and so forth down the line.

Science
, this issue p.
646
Our skewed sense of space
Authors: Buzsáki György
Published: February 2015

The distribution of neuron activity reveals an organization that supports the brain's spatial mapping capacity
Skin and Mucosal Human Papillomavirus Seroprevalence in Persons with Fanconi Anemia
Authors: Katzenellenbogen Rachel A. , Carter Joseph J. , Stern Joshua E. , Butsch Kovacic Melinda S. , Mehta Parinda A. , Sauter Sharon L. , Galloway Denise A. , Winer Rachel L.
Published: February 2015

Persons with Fanconi anemia (FA) are at risk for human papillomavirus (HPV)-associated cancers; however, their natural HPV exposure and infection rates are unknown as is the adequacy with which they mount antibodies to HPV vaccination. This study aimed to determine, in 62 persons with FA, the seroprevalence of skin and mucosal HPV types, the seroprevalence in individuals self-reporting a history of HPV vaccination, and the factors associated with HPV seropositivity. A bead Luminex assay was used to determine seropositivity for HPV1, -2, and -4 (low-risk skin), -6 and -11 (low-risk mucosal, included in one HPV vaccine), -16 and -18 (high-risk mucosal, included in both HPV vaccines), and -52 and -58 (high-risk mucosal). Health- and behavior-related questionnaires were completed. Type-specific seroprevalence estimates and participant characteristics associated with seroprevalence were calculated; 48% reported HPV vaccination. Type-specific seropositivity in unvaccinated persons ranged from 7 to 21% for skin HPV types and 7 to 38% for mucosal HPV types. Among the unvaccinated participants, adults versus children demonstrated increased HPV1, -6, -16, and -58 seroprevalence of 45% versus 6%, 64% versus 22%, 64% versus 17%, and 36% versus 0%, respectively (allP< 0.05). The vaccinated participants versus the nonvaccinated participants demonstrated increased seroprevalence of HPV6, -11, -16, and -18 of 92% versus 38%, 92% versus 24%, 96% versus 34%, and 75% versus 7%, respectively (allP< 0.0001). Our data demonstrate that the unvaccinated participants had serologic evidence of prior skin and mucosal HPV infections and that seroprevalence increased among adults; in self-reported vaccinees, seroprevalence of HPV vaccine types was 75 to 96%.
Lack of Clinical Utility of Urine Gram Stain for Suspected Urinary Tract Infection in Pediatric Patients
Authors: Cantey Joseph B. , Gaviria-Agudelo Claudia , McElvania TeKippe Erin , Doern Christopher D.
Published: February 2015

Urinary tract infection (UTI) is one of the most common infections in children. Urine culture remains the gold standard for diagnosis, but the utility of urine Gram stain relative to urinalysis (UA) is unclear. We reviewed 312 pediatric patients with suspected UTI who had urine culture, UA, and urine Gram stain performed from a single urine specimen. UA was considered positive if ≥10 leukocytes per oil immersion field were seen or if either nitrates or leukocyte esterase testing was positive. Urine Gram stain was considered positive if any organisms were seen. Sensitivity, specificity, and positive and negative predictive values were calculated using urine culture as the gold standard. Thirty-seven (12%) patients had a culture-proven UTI. Compared to urine Gram stain, UA had equal sensitivity (97.3% versus 97.5%) and higher specificity (85% versus 74%). Empirical therapy was prescribed before the Gram stain result was known in 40 (49%) patients and after in 42 (51%) patients. The antibiotics chosen did not differ between the two groups (
P
= 0.81), nor did they differ for patients with Gram-negative rods on urine Gram stain compared to those with Gram-positive cocci (
P
= 0.67). From these data, we conclude that UA has excellent negative predictive value that is not enhanced by urine Gram stain and that antibiotic selection did not vary based on the urine Gram stain result. In conclusion, the clinical utility of urine Gram stain does not warrant the time or cost it requires.
A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR
Authors: Belaz Sorya , Gangneux Jean-Pierre , Dupretz Peggy , Guiguen Claude , Robert-Gangneux Florence
Published: February 2015

This study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (

C
T

) obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained with REP-529 qPCR (
P
< 0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean
Toxoplasma
quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay.
Community-Onset Pyomyositis Caused by a Salmonella enterica Serovar Enteritidis Sequence Type 11 Strain Producing CTX-M-15 Extended-Spectrum β-Lactamase
Authors: Hwang Jeong-Hwan , Shin Gee-Wook , Lee Chang-Seop
Published: February 2015

We report a case of community-onset pyomyositis due to
Salmonella enterica
serovar Enteritidis in South Korea. The isolated strain was resistant to extended-spectrum cephalosporins and harbored sequence type 11 coproducing CTX-M-15 extended-spectrum β-lactamase (ESBL). Physicians should be alert for early diagnosis and appropriate treatment since ESBL-producing nontyphoidal
Salmonella
infections are difficult to treat without initiation of appropriate empirical antibiotics.
First Report of Chronic Pulmonary Infection by KPC-3-Producing and Colistin-Resistant Klebsiella pneumoniae Sequence Type 258 (ST258) in an Adult Patient with Cystic Fibrosis
Authors: Delfino Emanuele , Giacobbe Daniele Roberto , Del Bono Valerio , Coppo Erika , Marchese Anna , Manno Graziana , Morelli Patrizia , Minicucci Laura , Viscoli Claudio
Published: February 2015

The spread of
Klebsiella pneumoniae
carbapenemase (KPC)-producing
K. pneumoniae
continues to increase, and the possible development of KPC-producing
K. pneumoniae
infections in cystic fibrosis (CF) patients is a matter of concern. Here, we describe the establishment of a chronic lung infection due to a colistin-resistant KPC-producing
K. pneumoniae
isolate in an Italian CF patient.
Evaluation of a New Phenotypic OXA-48 Disk Test for Differentiation of OXA-48 Carbapenemase-Producing Enterobacteriaceae Clinical Isolates
Authors: Tsakris Athanassios , Poulou Aggeliki , Bogaerts Pierre , Dimitroulia Evangelia , Pournaras Spyros , Glupczynski Youri
Published: February 2015

The current phenotypic methods for detecting carbapenemase-producing
Enterobacteriaceae
(CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing
Enterobacteriaceae
isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative
Enterobacteriaceae
isolates (57
Klebsiella pneumoniae
carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing
Enterobacteriaceae
. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in
Enterobacteriaceae
species and provide important infection control information.
Monitoring of Cytomegalovirus Viral Loads by Two Molecular Assays in Whole-Blood and Plasma Samples from Hematopoietic Stem Cell Transplant Recipients
Authors: Babady N. Esther , Cheng Cindy , Cumberbatch Evelyn , Stiles Jeffrey , Papanicolaou Genovefa , Tang Yi-Wei
Published: February 2015

Cytomegalovirus (CMV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Roche Cobas AmpliPrep/Cobas TaqMan CMV test (Cobas CMV) has recently been cleared by the FDA for the monitoring of CMV viral loads in plasma samples from transplant patients. In this study, we compare and correlate the viral loads obtained by a laboratory-developed test (LC CMV) (using Roche analyte-specific reagents [ASR] on the LightCycler 2.0) on whole-blood specimens with those obtained on corresponding plasma and whole-blood specimens by the Cobas CMV assay. Testing was performed on 773 archived patient specimens. The strength of the agreement was good for the two assays performed on whole blood (κ = 0.6; 95% confidence interval [CI], 0.51 to 0.7) and moderate when the tests were performed on different sample types (κ = 0.54; 95% CI, 0.47 to 0.62 for the LC CMV whole blood [WB] assay versus Cobas plasma [PL], and κ = 0.57; 95% CI, 0.5 to 0.65 for the Cobas WB assay versus Cobas PL), although the difference was not statistically significant. Using a combination gold standard (i.e., a true positive was a specimen that was positive by two or more methods), the sensitivity and specificity of the assays were 78.8% and 99.3% for the LC CMV assay, 85.2% and 98.1% for the Cobas CMV WB assay, and 100% and 90.5% for Cobas CMV PL assay, respectively. A comparison of the CMV viral load trends in both plasma and whole blood from a few patients with multiple positive successive samples showed similar slopes, with differences in the slope ranging from 0.01 to 0.22. However, the absolute value for individual viral load differed markedly with whole-blood viral loads, being on average 0.5- to 1.22-log higher than those in plasma. The Cobas CMV assay provides a valid option for the monitoring of viral loads in transplant patients. Due to its increased sensitivity, the detection of CMV DNA in patients with low viral loads (i.e., those below limit of quantification [LOQ]) is increased with the Cobas CMV assay in plasma specimens. Longitudinal prospective studies will be needed to examine the clinical significance of these low-level viral loads.
MicroRNA miR-24-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication through Suppression of Heme Oxygenase-1 Expression
Authors: Xiao Shuqi , Wang Xue , Ni Huaibao , Li Na , Zhang Angke , Liu Hongliang , Pu Fengxing , Xu Lele , Gao Jiming , Zhao Qin , Mu Yang , Wang Chengbao , Sun Yani , Du Taofeng , Xu Xingang , Zhang Gaiping , Hiscox Julian A. , Goodfellow Ian G. , Zhou En-Min
Published: February 2015

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV downregulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, postinfection and that overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression and, by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP; a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection.

IMPORTANCE
MicroRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host genes at the posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses, such as Ebola virus, hepatitis C virus, human immunodeficiency virus, and our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here, we show that PRRSV infection induces host miRNA miR-24-3p expression and that miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection. Therefore, our findings provide new insights into the pathogenesis of PRRSV.
Inhibition of Hepatitis B Virus Gene Expression and Replication by Hepatocyte Nuclear Factor 6
Authors: Hao Ruidong , He Jing , Liu Xing , Gao Guozhen , Liu Dan , Cui Lei , Yu Guojun , Yu Wenhui , Chen Yu , Guo Deyin
Published: February 2015

Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanistically, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a
cis
element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms.

IMPORTANCE
HBV is a major human pathogen whose replication is regulated by host factors. Liver-enriched transcription factors are critical for many liver functions, including metabolism, development, and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this study, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.
X-Ray Structure of the Pestivirus NS3 Helicase and Its Conformation in Solution
Authors: Tortorici M. Alejandra , Duquerroy Stéphane , Kwok Jane , Vonrhein Clemens , Perez Javier , Lamp Benjamin , Bricogne Gerard , Rümenapf Till , Vachette Patrice , Rey Félix A.
Published: February 2015

Pestiviruses form a genus in the
Flaviviridae
family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the
Flaviviridae
family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication.

IMPORTANCE
Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior—a characteristic probably shared with NS3 helicases from all
Flaviviridae
members—that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function.
ATP1A1-Mediated Src Signaling Inhibits Coronavirus Entry into Host Cells
Authors: Burkard Christine , Verheije Monique H. , Haagmans Bart L. , van Kuppeveld Frank J. , Rottier Peter J. M. , Bosch Berend-Jan , de Haan Cornelis A. M.
Published: February 2015

In addition to transporting ions, the multisubunit Na
+
,K
+
-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na
+
,K
+
-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na
+
,K
+
-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.

IMPORTANCE
Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na
+
,K
+
-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na
+
,K
+
-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.
Analysis by Live Imaging of Effects of the Adenovirus E4orf4 Protein on Passage through Mitosis of H1299 Tumor Cells
Authors: Sriskandarajah Neera , Blanchette Paola , Kucharski Thomas J. , Teodoro Jose G. , Branton Philip E.
Published: February 2015

The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G
1
tetraploids in the population of dying E4orf4-expressing cells.
Dolutegravir Resistance Mutation R263K Cannot Coexist in Combination with Many Classical Integrase Inhibitor Resistance Substitutions
Authors: Anstett Kaitlin , Mesplede Thibault , Oliveira Maureen , Cutillas Vincent , Wainberg Mark A.
Published: February 2015

The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not.
A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40
Authors: Inoue Takamasa , Tsai Billy
Published: February 2015

The nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs.

IMPORTANCE
PyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.
Reduction of Virion-Associated σ1 Fibers on Oncolytic Reovirus Variants Promotes Adaptation toward Tumorigenic Cells
Authors: Mohamed Adil , Teicher Carmit , Haefliger Sarah , Shmulevitz Maya
Published: February 2015

Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity
in vivo
. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved
in vivo
oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of σ1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ∼2- and 4-fold-lower levels of σ1 fiber than did T3wt virions. Importantly, using RNA interference to reduce σ1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of σ1 per virion. As σ1 levels were reduced, virion infectivity increased by 2- to 5-fold per cell-bound particle, demonstrating a causal relationship between virion σ1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins σ3 and μ1C at similar rates. However, having started with fewer σ1 molecules, a complete loss of σ1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid σ3 and μ1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and σ1 levels.

IMPORTANCE
Wild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity
in vitro
and
in vivo
now show that distinct reovirus structures promote adaptation toward cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of the cell attachment protein σ1 became more infectious toward transformed cells. Reduced σ1 levels conferred a benefit to incoming particles only, resulting in an earlier depletion of σ1 and a higher probability of establishing productive infection. Conversely, reovirus variants with fewer σ1 molecules showed reduced stability and infectivity and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure and provide a molecular rationale for considering alternative reovirus structures during oncolytic therapy.
Origin and Proliferation of Multiple-Drug Resistance in Bacterial Pathogens
Authors: Chang Hsiao-Han , Cohen Ted , Grad Yonatan H. , Hanage William P. , O'Brien Thomas F. , Lipsitch Marc
Published: February 2015

Many studies report the high prevalence of multiply drug-resistant (MDR) strains. Because MDR infections are often significantly harder and more expensive to treat, they represent a growing public health threat. However, for different pathogens, different underlying mechanisms are traditionally used to explain these observations, and it is unclear whether each bacterial taxon has its own mechanism(s) for multidrug resistance or whether there are common mechanisms between distantly related pathogens. In this review, we provide a systematic overview of the causes of the excess of MDR infections and define testable predictions made by each hypothetical mechanism, including experimental, epidemiological, population genomic, and other tests of these hypotheses. Better understanding the cause(s) of the excess of MDR is the first step to rational design of more effective interventions to prevent the origin and/or proliferation of MDR.
Fifteen-minute consultation: rheumatic fever
Authors: Markham Rebecca , Tulloh Robert
Published: February 2015

Objective

To present a structured approach for an outpatient consultation or inpatient assessment of a child with possible rheumatic fever.

Method

Review of literature and description of diagnostic and therapeutic approach.

Conclusions

A focused history and examination is key to establish the cause and draw a management plan for rheumatic fever.
The effect of Chinese herbal medicine Jian Ling Decoction for the treatment of essential hypertension: a systematic review
Authors: Xiong Xingjiang , Wang Pengqian , Li Xiaoke , Zhang Yuqing
Published: February 2015

Objectives

Jian Ling Decoction (JLD) is often prescribed to improve hypertension-related symptoms in China. However, this treatment has not been systematically reviewed for its efficacy against essential hypertension (EH). This review aims to assess the current clinical evidence of JLD in the treatment of EH.

Design

Seven electronic databases, including the Cochrane Central Register of Controlled Trials, PubMed, EMBASE, the Chinese National Knowledge Infrastructure (CNKI), the Chinese Scientific Journal Database (VIP), the Chinese Biomedical Literature Database (CBM) and the Wanfang Database, were searched up to March 2014. Randomised control trials (RCTs) comparing JLD or combined with antihypertensive drugs versus antihypertensive drugs were included. We assessed the methodological quality, extracted the valid data and conducted the meta-analysis according to criteria from the Cochrane group. The primary outcome was categorical or continuous blood pressure (BP), and the secondary outcome was quality of life (QOL).

Results

Ten trials (655 patients) with unclear-to-high risk of bias were identified. Meta-analysis showed that JLD used alone showed no BP reduction effect; however, improvement on QOL was found in the JLD group compared to antihypertensive drugs. A significant reduction in systolic and diastolic BP was observed for JLD plus antihypertensive drugs when compared with antihypertensive drugs alone. No serious adverse effects were reported.

Conclusions

Owing to insufficient clinical data, it is difficult to draw a definite conclusion regarding the effectiveness and safety of JLD for EH, and better trials are needed.
Can certified health professionals treat obesity in a community-based programme? A quasi-experimental study
Authors: Miedema Baukje , Reading Stacey A , Hamilton Ryan A , Morrison Katherine S , Thompson Ashley E
Published: February 2015

Objective

To test the effectiveness of a non-pharmaceutical programme for obese participants in a rural Eastern Canadian Province using certified health professionals.

Design

A prospective quasi-experimental design with repeated premeasure and postmeasure.

Participants

146 participants with obesity (body mass index >30 kg/m2) from rural and urban communities in an Eastern Canadian Province were divided into four groups.

Intervention

A 6-month intensive active community-based lifestyle intervention (InI) delivered by Certified Exercise Physiologists, Certified Personal Trainers and Registered Dietitians, followed by 6 months of self-management. A second intervention (InII) was nested in InI and consisted of group-mediated cognitive–behavioral intervention (GMCBI) delivered by an exercise psychologist to two of the four InI groups.

Outcomes

(1) Improving health outcomes among the participants’ preactive and postactive 6-month intervention and self-management period, (2) Documenting the impact of InII (GMCBI) and location of the intervention (urban vs rural).

Results

The 6-month active InI significantly improved cardiovascular health for participants who completed the intervention. InII (GMCBI) significantly lowered the attrition rate among the participants. The self-management period was challenging for the participants and they did not make further gains; however, most were able to maintain the gains achieved during the active intervention. The location of the intervention, urban or rural, had little impact on outcomes.

Conclusions

A community-based programme utilising healthcare professionals other than physicians to treat obese patients was effective based on premeasure and postmeasure. During the self-management phase, the participants were able to maintain the gains. Psychological support is essential to participant retention.
Over-reassurance and undersupport after a ‘false alarm’: a systematic review of the impact on subsequent cancer symptom attribution and help seeking
Authors: Renzi Cristina , Whitaker Katriina L , Wardle Jane
Published: February 2015

Objectives

This literature review examined research into the impact of a previous ‘all-clear’ or non-cancer diagnosis following symptomatic presentation (‘false alarm’) on symptom attribution and delays in help seeking for subsequent possible cancer symptoms.

Design and setting

The comprehensive literature review included original research based on quantitative, qualitative and mixed data collection methods. We used a combination of search strategies, including in-depth searches of electronic databases (PubMed, EMBASE, PsychInfo), searching key authors and articles listed as ‘related’ in PubMed, and reference lists. We performed a narrative synthesis of key themes shared across studies.

Participants

The review included studies published after 1990 and before February 2014 reporting information on adult patients having experienced a false alarm following symptomatic presentation. We excluded false alarms in the context of screening.

Primary and secondary outcome measures

We evaluated the effect of a ‘false alarm’ on symptom attribution and help seeking for new or recurrent possible cancer symptoms.

Results

Overall, 1442 papers were screened and 121 retrieved for full-text evaluation. Among them, 19 reported on false alarms and subsequent symptom attribution or help seeking. They used qualitative (n=14), quantitative (n=3) and mixed methods (n=2). Breast (n=7), gynaecological (n=3), colorectal (n=2), testicular (n=2), and head and neck cancers (n=2) were the most studied. Two broad themes emerged underlying delays in help seeking: (1) over-reassurance from the previous ‘all-clear’ diagnosis leading to subsequent symptoms being interpreted as benign, and (2) unsupportive healthcare experiences in which symptoms were dismissed, leaving patients concerned about appearing hypochondriacal or uncertain about the appropriate next actions. The evidence suggested that the effect of a false alarm can persist for months and even years.

Conclusions

In conclusion, over-reassurance and undersupport of patients after a false alarm can undermine help seeking in the case of new or recurrent potential cancer symptoms, highlighting the need for appropriate patient information when investigations rule out cancer.