bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of AV Delay and VV Delay on Left Atrial Pressure and Waveform in Ambulant Heart Failure Patients: Insights into CRT Optimization
Authors: CHAN W.Y. WANDY , BLOMQVIST ANDREAS , MELTON IAIN C. , NORÉN KJELL , CROZIER IAN G. , BENSER MICHAEL E. , EIGLER NEAL L. , GUTFINGER DAN , TROUGHTON RICHARD W.
Published: February 2014

Background

We hypothesized that left atrial pressure (LAP) obtained by a permanent implantable sensor is sensitive to changes in cardiac resynchronization therapy (CRT) settings and could guide CRT optimization to improve the response rate. We investigated the effect of CRT optimization on LAP and its waveform parameters in ambulant heart failure (HF) patients.

Methods

CRT optimization was performed in eight ambulant HF patients, using echocardiography as reference. LAP waveform was acquired at each of eight atrioventricular (AV) intervals and five inter‐ventricular (VV) intervals. Selected waveform parameters were also evaluated for their sensitivity to CRT changes and agreement with echocardiography‐guided optimal settings.

Results

Optimal AV and VV intervals varied considerably between patients. All patients exhibited significant changes in waveform morphology with AV optimization. Optimal AV delay determined from echocardiography ranged between 140 ms and 225 ms. Mean LAP tended to be lower at optimal setting 14 ± 3 mmHg compared to shorter (<100 ms) or longer (>160 ms) AV settings (P = 0.16). There were clear trends to smaller peak a‐wave (P = 0.11) and gentler positive a‐slope (P = 0.15) and positive v‐slope (P = 0.09) with longer AV delays. Mean LAP and negative v‐wave slope correlated well with echo‐guided optimal setting, r = 0.91 (P = 0.001) and 0.79 (P = 0.03), respectively. No significant effects on LAP or waveform were seen during VV optimization.

Conclusions

LAP and its waveform changes considerably with AV optimization. There is good agreement between echo‐guided optimal setting and LAP. LAP could provide an objective guide to CRT optimization. (Clinical Trial Registry information: URL: http://www.clinicaltrials.gov. Unique Identifier: NCT00632372)
Influences of Acid on Molecular Forms of Fluorescein and Photoinduced Electron Transfer in Fluorescein‐Dispersing Sol–Gel Titania Films
Authors: Nishikiori Hiromasa , Setiawan Rudi Agus , Miyashita Kyohei , Teshima Katsuya , Fujii Tsuneo
Published: February 2014

Fluorescein‐dispersing titania gel films were prepared by the acid‐catalyzed sol–gel reaction using a titanium alkoxide solution containing fluorescein. The molecular forms of fluorescein in the films, depending on its acid–base equilibria, and the complex formation and photoinduced electron transfer process between the dye and titania surface were investigated by fluorescence and photoelectric measurements. The titanium species were coordinated to the carboxylate and phenolate‐like groups of the fluorescein species. The quantum efficiencies of the fluorescence quenching and photoelectric conversion were higher upon excitation of the dianion species interacting with the titania, i.e. the dye–titania complex. This result indicated that the dianion form was the most favorable for formation of the dye–titania complex exhibiting the highest electron transfer efficiency. Using nitric acid as the catalyst, the titania surface bonded to the fluorescein instead of the adsorbed nitrate ion during the steam treatment. The dye–titania complex formation played an important role in the electron injection from the dye to the titania conduction band.
Nitric oxide and hydrogen peroxide are important signals mediating the allelopathic response of Arabidopsis to p‐hydroxybenzoic acid
Authors: Guan Yanlong , Lin Huaming , Ma Lan , Yang Yongping , Hu Xiangyang
Published: February 2014

Both nitric oxide (NO) and hydrogen peroxide (H2O2) are important signals that mediate plant response to environmental stimulation. Their role in plants' allelopathic interactions has also been reported, but the underlying mechanism remains little understood. p‐Hydroxybenzoic acid (pHBA) has been proposed to be an allelopathic chemical. Here, we found that pHBA at 0.4 mM efficiently suppressed Arabidopsis growth. Meanwhile, pHBA rapidly induced the accumulation of NO and H2O2, where such effect could be reversed by NO or H2O2 metabolism inhibitors or scavengers. Also, pHBA‐induced NO and H2O2 could be compromised in NO synthesis mutants noa1, nia1 and nia2, or H2O2 metabolism mutant rbohD/F, but suppressing NO accumulation with a NO synthesis inhibitor or using NO synthesis‐related mutants did not reduce pHBA‐induced H2O2 accumulation. Furthermore, we found that the effect of pHBA on allelopathic inhibition of growth was aggravated in NO/H2O2 metabolism‐related mutants or reducing NO/H2O2 by different inhibitors, whereas the addition of an NO/H2O2 donor could partly relieve the inhibitory effect of pHBA on the growth of wild type. However, adding only an NO donor, but not low concentration of H2O2 as the donor, could relieve the inhibitory effect of pHBA on root growth in NO metabolism mutants. On the basis of these results, we propose that both NO and H2O2 are important signals that mediate Arabidopsis response to the allelopathic chemical pHBA, where during this process H2O2 may work upstream of the NO signal.
High‐Throughput Quantitation of Intracellular Trafficking and Organelle Disruption by Flow Cytometry
Authors: Chia Pei Zhi Cheryl , Ramdzan Yasmin M. , Houghton Fiona J. , Hatters Danny M. , Gleeson Paul A.
Published: February 2014

Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry‐based method that circumvents these limitations. The method utilizes fluorescent pulse‐width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse‐width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso‐endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane‐to‐endosomes‐to‐Golgi, by decreasing pulse‐width values. A block in transport upon RNAi‐mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse‐width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.
Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages
Authors: Puissant Emeline , Gilis Florentine , Dogné Sophie , Flamion Bruno , Jadot Michel , Boonen Marielle
Published: February 2014

The hyaluronidase Hyal‐1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal‐1 is synthesized as a glycosylated precursor that is only weakly mannose 6‐phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6‐phosphate‐independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co‐distributed with lysosomal hydrolases in the densest fraction of a self‐forming PercollTM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal‐1 was largely underestimated when assayed by zymography after SDS‐PAGE and subsequent renaturation of the proteins, by contrast to the full‐length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal‐1.
Aid for Trade Effectiveness: Complementarities with Economic Integration
Authors: Vijil Mariana
Published: February 2014

Developing countries are increasingly using regional integration as a main policy lever when pursuing a trade‐led growth strategy, and today, ‘deep’ preferential trade agreements go beyond trade policy negotiations and cover trade facilitation issues. Since aid for trade (AfT) has been recognised as a powerful instrument for increasing developing countries' trade capacity by targeting internal trade costs, this article tests whether complementarities exist between this type of aid and economic integration using a gravity model on panel data for the period 1995–2005. Results indicate that AfT, when combined with economic integration, has been effective in increasing trade flows. Both South–South and North–South trade flows have benefited, and the combination of the two instruments has been particularly effective in expanding the South's exports to the North. Finally, when breaking down AfT into categories, assistance to trade‐related institutions seems to generate the strongest complementarities with economic integration.
Where Do Firms Export, How Much and Why?
Authors: Lawless Martina , Whelan Karl
Published: February 2014

The empirical finding that exporting firms are more productive on average than non‐exporters has provoked a large theoretical literature based on models such as Melitz (), where more productive firms are more likely to overcome costs associated with trade. This paper investigates how closely the productivity heterogeneity framework fits the data from a firm‐level survey that includes information on export destinations and firm characteristics such as productivity. We find a high degree of unpredictable idiosyncratic participation in export markets by firms and a relatively weak positive correlation between the extent of a firm's export market participation and its export sales. We find that a small number of standard gravity variables provide a close fit to the country‐level determinants of trade but that greater variation results in more difficulty in explaining firm‐specific factors driving exporting behaviour. We also illustrate some elements of the dynamics over time in firm exporting patterns by destination. We show that lagged exporting activity has a significant effect on a firm's current exporting profile.
Secreting and Sensing the Same Molecule Allows Cells to Achieve Versatile Social Behaviors
Authors: Youk Hyun , Lim Wendell A.
Published: February 2014

The Message in the Medium

What is the point of autocrine signaling in which a cell produces a signal that activates receptors on its own cell surface? An internal signal seems simpler, unless there is value to allowing neighboring cells to know what other cells are up to.
Youk and Lim
(p.
10.1126/science.1242782
; see the Perspective by

Lee and You

) explored the broad range of signaling outcomes that can result in a system in which some yeast cells could secrete and sense a signal whereas others could only sense signals from their neighbors. The cells were engineered so that the response of the two cell types could be distinguished from one another. Experiments and mathematical modeling showed that depending on how circuits were constructed—for example, how much receptor was present, how the signal molecule was degraded, the presence of feedback, the density of the cell culture, and so on—a range of behaviors was possible: Some conditions favored activation of one type of cell over another. Others altered the timing or consistency of the response within a population. The principles revealed could also be used in other biological contexts or in the design of synthetic biological cell systems with desired regulatory properties.
Interchromosomal Communication Coordinates Intrinsically Stochastic Expression Between Alleles
Authors: Johnston Robert J. , Desplan Claude
Published: February 2014

Stochasticity and Cell Fate

Stochastic mechanisms can diversify cell fates in nervous systems. In the
Drosophila
retina, the stochastic distribution of color-sensing photoreceptors is controlled by the random, On/Off expression of the Spineless transcriptional regulator.
Johnston and Desplan
(p.
661
) found that each allele of
spineless
makes an intrinsically random expression choice controlled by an enhancer and two silencer DNA elements acting at long range.
spineless
alleles communicate between chromosomes using activating and repressing mechanisms to determine the frequency of expression and coordinate expression state. These findings suggest critical roles for intrinsically random expression decisions and interchromosomal communication in stochastic cell fate specification.
Elastic Instability of a Crystal Growing on a Curved Surface
Authors: Meng Guangnan , Paulose Jayson , Nelson David R. , Manoharan Vinothan N.
Published: February 2014

Curving Crystals

When a material with a different set of lattice parameters is grown on the surface of a crystal of a second material, the stresses at the interface can affect the growing crystal.

Meng
et al.

(p.
634
) studied the growth of colloidal crystals on top of a curved water droplet. Owing to the elastic stress caused by the bending of the crystal, strong distortions occurred in the growing crystal, but, nonetheless, large single-crystalline domains with no topological defects were formed.
Scientific Diversity Interventions
Authors: Moss-Racusin Corinne A. , van der Toorn Jojanneke , Dovidio John F. , Brescoll Victoria L. , Graham Mark J. , Handelsman Jo
Published: February 2014

Fair treatment of other scientists is an essential aspect of scientific integrity, warranting diversity interventions.
Enabling Visual Discovery
Medical Illuminations
Using Evidence, Visualizations and Statistical Thinking to Improve Healthcare

by Howard Wainer

Oxford University Press, Oxford, 2014. 191 pp. $35, £19.99. ISBN 9780199668793.
Authors: Shneiderman Ben
Published: February 2014

Wainer presents 13 biomedical examples to illustrate advantages of statistical thinking and data visualization.
Limits of Soil Production?
Authors: Heimsath Arjun M.
Published: February 2014

Steep mountain regions can weather faster and produce soil more quickly than previously thought.

[Also see Report by

Larsen
et al.

]
Could Autism Be Treated Prenatally?
Authors: Zimmerman Andrew W. , Connors Susan L.
Published: February 2014

Treatment of rodent models of autism spectrum disorder with a drug that alters the function of a neurotransmitter ameliorates autistic-like behavior in offspring.

[Also see Report by

Tyzio
et al.

]
Cells Listen to Their Inner Voice
Authors: Lee Anna Jisu , You Lingchong
Published: February 2014

A signaling circuit that controls the release and detection of the same signaling molecule can trigger diverse behaviors in a cell population.

[Also see Research Article by
Youk and Lim
]
The Mountaintop Witness
Authors: Stokstad Erik
Published: February 2014

Margaret Palmer started out just studying streams. She's ended up in court, passionately defending them from coal mining.
Peering Into Peer Review
Authors: Mervis Jeffrey
Published: February 2014

Why don't proposals given better scores by the National Institutes of Health lead to more important research outcomes?
Making Every Scientist a Research Funder
Authors: Mervis Jeffrey
Published: February 2014

A radical proposal to revamp peer review would give scientists an even bigger role in deciding how to distribute U.S. research dollars—at a fraction of the current cost.
Evaluation of the Borrelia burgdorferi BBA64 Protein as a Protective Immunogen in Mice
Authors: Brandt Kevin S. , Patton Toni G. , Allard Anna S. , Caimano Melissa J. , Radolf Justin D. , Gilmore Robert D.
Published: February 2014

The
Borrelia burgdorferi
bba64
gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against
B. burgdorferi
challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to
B. burgdorferi
challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in
Escherichia coli
was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from
B. burgdorferi
challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by
in vitro
borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying
B. burgdorferi
proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.
Four Cases of Bacteremia Caused by Oscillibacter ruminantium, a Newly Described Species
Authors: Sydenham Thomas V. , Arpi Magnus , Klein Kasper , Justesen Ulrik S.
Published: February 2014

The genus
Oscillibacter
has been known since 2007, but no association to human infection has been reported. Here, we present four cases of
Oscillibacter ruminantium
bacteremia from hospitals across Denmark from 2001 to 2010. Correct identification is now possible, as the 16S rRNA gene sequence was recently made publicly available.
Universal Influenza B Virus Genomic Amplification Facilitates Sequencing, Diagnostics, and Reverse Genetics
Authors: Zhou Bin , Lin Xudong , Wang Wei , Halpin Rebecca A. , Bera Jayati , Stockwell Timothy B. , Barr Ian G. , Wentworth David E.
Published: February 2014

Although human influenza B virus (IBV) is a significant human pathogen, its great genetic diversity has limited our ability to universally amplify the entire genome for subsequent sequencing or vaccine production. The generation of sequence data via next-generation approaches and the rapid cloning of viral genes are critical for basic research, diagnostics, antiviral drugs, and vaccines to combat IBV. To overcome the difficulty of amplifying the diverse and ever-changing IBV genome, we developed and optimized techniques that amplify the complete segmented negative-sense RNA genome from any IBV strain in a single tube/well (IBV genomic amplification [IBV-GA]). Amplicons for >1,000 diverse IBV genomes from different sample types (e.g., clinical specimens) were generated and sequenced using this robust technology. These approaches are sensitive, robust, and sequence independent (i.e., universally amplify past, present, and future IBVs), which facilitates next-generation sequencing and advanced genomic diagnostics. Importantly, special terminal sequences engineered into the optimized IBV-GA2 products also enable ligation-free cloning to rapidly generate reverse-genetics plasmids, which can be used for the rescue of recombinant viruses and/or the creation of vaccine seed stock.
Bloodstream Infections Caused by Pseudomonas spp.: How To Detect Carbapenemase Producers Directly from Blood Cultures
Authors: Dortet Laurent , Boulanger Anne , Poirel Laurent , Nordmann Patrice
Published: February 2014

The Carba NP test has been evaluated to detect carbapenemase-producing
Pseudomonas
spp. directly from blood cultures. This rapid and cost-effective test permits an early identification of carbapenemase-producing
Pseudomonas
spp. directly from blood cultures with excellent sensitivity and specificity. Results may be useful in particular for guiding the first-line therapy and epidemiological purposes.
Cutaneous Manifestations of a Zoonotic Onchocerca Species in an Adult Male, Acquired in Nova Scotia, Canada
Authors: Lai Jonathan H. , Walsh Noreen M. G. , Pritt Bobbi S. , Sloan Lynne , Gibson Lawrence E. , Desormeau Leon , Haldane David J. M.
Published: February 2014

A 65-year-old male with known hypertension and hypercholesterolemia sought medical attention because of a 3-month history of skin swelling on his upper back. Histopathology and molecular techniques were employed and identified an organism in the
Onchocerca
genus. This represents a very uncommon example of cutaneous infection by a zoonotic
Onchocerca
species.
Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material
Authors: de Beer Jessica L. , Akkerman Onno W. , Schürch Anita C. , Mulder Arnout , van der Werf Tjip S. , van der Zanden Adri G. M. , van Ingen Jakko , van Soolingen Dick
Published: February 2014

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of
Mycobacterium tuberculosis
complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of
M. tuberculosis
in bronchoalveolar lavage fluid. A 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities.
Identification of Aerobic Gram-Positive Bacilli by Use of Vitek MS
Authors: Navas Maria , Pincus David H. , Wilkey Kathy , Sercia Linda , LaSalvia Margaret , Wilson Deborah , Procop Gary W. , Richter Sandra S.
Published: February 2014

The accuracy of Vitek MS mass spectrometric identifications was assessed for 206 clinically significant isolates of aerobic Gram-positive bacilli representing 20 genera and 38 species. The Vitek MS identifications were correct for 85% of the isolates (56.3% to the species level, 28.6% limited to the genus level), with misidentifications occurring for 7.3% of the isolates.
Prospective Evaluation of Diagnostic Accuracy of Dried Blood Spots from Finger Prick Samples for Determination of HIV-1 Load with the NucliSENS Easy-Q HIV-1 Version 2.0 Assay in Malawi
Authors: Fajardo Emmanuel , Metcalf Carol A. , Chaillet Pascale , Aleixo Lucia , Pannus Pieter , Panunzi Isabella , Triviño Laura , Ellman Tom , Likaka Andrew , Mwenda Reuben
Published: February 2014

HIV-1 viral load (VL) testing is not widely available in resource-limited settings. The use of finger prick dried blood spot (FP-DBS) samples could remove barriers related to sample collection and transport. Measurement of VL using DBS from EDTA venous blood (VB-DBS) in place of plasma has previously been validated using the NucliSENS Easy-Q HIV-1 v2.0 assay, but information on the accuracy of FP-DBS samples for measuring VL is limited. This prospective study, conducted at Thyolo District Hospital in southern Malawi, compared VL levels measured on FP-DBS samples and plasma using the NucliSENS Easy-Q HIV-1 v2.0 assay. Comparability was assessed by means of agreement and correlation (131 patients with VLs of ≥100 copies/ml), sensitivity, and specificity (612 patients on antiretroviral treatment [ART]). Samples of EDTA venous blood and FP-DBS from 1,009 HIV-infected individuals were collected and prepared in the laboratory. Bland-Altman analysis found good agreement between plasma and FP-DBS VL levels, with a mean difference of −0.35 log10, and 95% limits of agreement from −1.26 to 0.55 log10. FP-DBS had a sensitivity of 88.7% (95% confidence interval [CI], 81.1 to 94.4%) and a specificity of 97.8% (95% CI, 96.1 to 98.9%) using a 1,000-copies/ml cut point and a sensitivity of 83.0% (95% CI, 73.4 to 90.1%) and a specificity of 100% (95% CI, 99.3 to 100%) using a 5,000-copies/ml cut point. This study shows that FP-DBS is an acceptable alternative to plasma for measuring VL using the NucliSENS Easy-Q HIV-1 v2.0. We are conducting a second study to assess the proficiency of health workers at preparing FP-DBS in primary health care clinics.
Identification of Herpesvirus Proteins That Contribute to G
1
/S Arrest
Authors: Paladino Patrick , Marcon Edyta , Greenblatt Jack , Frappier Lori
Published: February 2014

Lytic infection by herpesviruses induces cell cycle arrest at the G
1
/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G
1
/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G
1
/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G
1
/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G
1
/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G
1
/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G
1
/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP.

IMPORTANCE
Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins from three different herpesviruses that contribute to this block. Several of the proteins we identified had previously unknown functions or were structural components of the virion. Subsets of these proteins from Epstein-Barr virus were studied for their effects on the cell cycle regulatory proteins p53 and p21, thereby identifying two proteins that induce p53 and one that induces p21 (BGLF2). We identified interactions of BGLF2 with two human proteins, both of which regulate p21, suggesting that BGLF2 induces p21 by interfering with the functions of these two host proteins. Our study indicates that multiple herpesvirus proteins contribute to the cell proliferation block, including components of the incoming virions.
Identification of a Broad-Spectrum Antiviral Small Molecule against Severe Acute Respiratory Syndrome Coronavirus and Ebola, Hendra, and Nipah Viruses by Using a Novel High-Throughput Screening Assay
Authors: Elshabrawy Hatem A. , Fan Jilao , Haddad Christine S. , Ratia Kiira , Broder Christopher C. , Caffrey Michael , Prabhakar Bellur S.
Published: February 2014

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Ebola, Hendra, and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage, rendering them active for fusion with the host cell membrane. In this study, we developed a novel high-throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein-derived peptide (pro-neuropeptide Y). The small molecule inhibited the entry of all pseudotyped viruses
in vitro
and the cleavage of SARS-CoV spike glycoprotein in an
in vitro
cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell-based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV and Ebola, Hendra, and Nipah viruses.

IMPORTANCE
We developed a novel high-throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV and Ebola, Hendra, and Nipah viruses that are required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV or Ebola, Hendra, or Nipah virus. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug.
Emergence of a Highly Pathogenic Avian Influenza Virus from a Low-Pathogenic Progenitor
Authors: Monne Isabella , Fusaro Alice , Nelson Martha I. , Bonfanti Lebana , Mulatti Paolo , Hughes Joseph , Murcia Pablo R. , Schivo Alessia , Valastro Viviana , Moreno Ana , Holmes Edward C. , Cattoli Giovanni
Published: February 2014

Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low-pathogenic (LPAI) progenitor viruses currently is poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultradeep-sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high- and low-pathogenicity viruses undertaken to date and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties.

IMPORTANCE
The Italian H7 AI epidemic of 1999 to 2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP forms from LP precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data, and ultradeep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low-frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.
Short-Chain Fatty Acids from Periodontal Pathogens Suppress Histone Deacetylases, EZH2, and SUV39H1 To Promote Kaposi's Sarcoma-Associated Herpesvirus Replication
Authors: Yu Xiaolan , Shahir Abdel-Malek , Sha Jingfeng , Feng Zhimin , Eapen Betty , Nithianantham Stanley , Das Biswajit , Karn Jonathan , Weinberg Aaron , Bissada Nabil F. , Ye Fengchun
Published: February 2014

Periodontal pathogens such as
Porphyromonas gingivalis
and
Fusobacterium nucleatum
produce five different short-chain fatty acids (SCFAs) as metabolic by-products. We detect significantly higher levels of SCFAs in the saliva of patients with severe periodontal disease. The different SCFAs stimulate lytic gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) dose dependently and synergistically. SCFAs inhibit class-1/2 histone deacetylases (HDACs) and downregulate expression of silent information regulator-1 (SIRT1). SCFAs also downregulate expression of enhancer of zeste homolog2 (EZH2) and suppressor of variegation 3-9 homolog1 (SUV39H1), which are two histone N-lysine methyltransferases (HLMTs). By suppressing the different components of host epigenetic regulatory machinery, SCFAs increase histone acetylation and decrease repressive histone trimethylations to transactivate the viral chromatin. These new findings provide mechanistic support that SCFAs from periodontal pathogens stimulate KSHV replication and infection in the oral cavity and are potential risk factors for development of oral Kaposi's sarcoma (KS).

IMPORTANCE
About 20% of KS patients develop KS lesions first in the oral cavity, while other patients never develop oral KS. It is not known if the oral microenvironment plays a role in oral KS tumor development. In this work, we demonstrate that a group of metabolic by-products, namely, short-chain fatty acids, from bacteria that cause periodontal disease promote lytic replication of KSHV, the etiological agent associated with KS. These new findings provide mechanistic support that periodontal pathogens create a unique microenvironment in the oral cavity that contributes to KSHV replication and development of oral KS.
Flow Cytometry Reveals that H5N1 Vaccination Elicits Cross-Reactive Stem-Directed Antibodies from Multiple Ig Heavy-Chain Lineages
Authors: Whittle James R. R. , Wheatley Adam K. , Wu Lan , Lingwood Daniel , Kanekiyo Masaru , Ma Steven S. , Narpala Sandeep R. , Yassine Hadi M. , Frank Gregory M. , Yewdell Jonathan W. , Ledgerwood Julie E. , Wei Chih-Jen , McDermott Adrian B. , Graham Barney S. , Koup Richard A. , Nabel Gary J.
Published: February 2014

An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles.

IMPORTANCE
Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.
Rotaviruses Reach Late Endosomes and Require the Cation-Dependent Mannose-6-Phosphate Receptor and the Activity of Cathepsin Proteases To Enter the Cell
Authors: Díaz-Salinas Marco A. , Silva-Ayala Daniela , López Susana , Arias Carlos F.
Published: February 2014

Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the
trans
-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention.

IMPORTANCE
Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.
Human Papillomavirus 45 Genetic Variation and Cervical Cancer Risk Worldwide
Authors: Chen Alyce A. , Heideman Daniëlle A. M. , Boon Debby , Gheit Tarik , Snijders Peter J. F. , Tommasino Massimo , Franceschi Silvia , Clifford Gary M.
Published: February 2014

Human papillomavirus 45 (HPV45) is a member of the HPV18-related alpha-7 species and accounts for approximately 5% of all cervical cancer cases worldwide. This study evaluated the genetic diversity of HPV45 and the association of HPV45 variants with the risk of cervical cancer by sequencing the entire E6 and E7 open reading frames of 300 HPV45-positive cervical samples from 36 countries. A total of 43 HPV45 sequence variants were identified that formed 5 phylogenetic sublineages, A1, A2, A3, B1, and B2, the distribution of which varied by geographical region. Among 192 cases of cervical cancer and 101 controls, the B2 sublineage was significantly overrepresented in cervical cancer, both overall and in Africa and Europe separately. We show that the sequence analysis of E6 and E7 allows the classification of HPV45 variants and that the risk of cervical cancer may differ by HPV45 variant sublineage.

IMPORTANCE
This work describes the largest study to date of human papillomavirus 45 (HPV45)-positive cervical samples and provides a comprehensive reference for phylogenetic classification for use in epidemiological studies of the carcinogenicity of HPV45 genetic variants, particularly as our findings suggest that the B2 sublineage of HPV45 is associated with a higher risk of cervical cancer.
Flexibility in Surface-Exposed Loops in a Virus Capsid Mediates Escape from Antibody Neutralization
Authors: Kolawole Abimbola O. , Li Ming , Xia Chunsheng , Fischer Audrey E. , Giacobbi Nicholas S. , Rippinger Christine M. , Proescher Jody B. G. , Wu Susan K. , Bessling Seneca L. , Gamez Monica , Yu Chenchen , Zhang Rebecca , Mehoke Thomas S. , Pipas James M. , Wolfe Joshua T. , Lin Jeffrey S. , Feldman Andrew B. , Smith Thomas J. , Wobus Christiane E.
Published: February 2014

New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A′B′ and E′F′ loops. Six mutations in the E′F′ loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding.

IMPORTANCE
Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid: one with a superior fit and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody-virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.