bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and functional partitioning of bread wheat chromosome 3B
Authors: Choulet Frédéric , Alberti Adriana , Theil Sébastien , Glover Natasha , Barbe Valérie , Daron Josquin , Pingault Lise , Sourdille Pierre , Couloux Arnaud , Paux Etienne , Leroy Philippe , Mangenot Sophie , Guilhot Nicolas , Le Gouis Jacques , Balfourier Francois , Alaux Michael , Jamilloux Véronique , Poulain Julie , Durand Céline , Bellec Arnaud , Gaspin Christine , Safar Jan , Dolezel Jaroslav , Rogers Jane , Vandepoele Klaas , Aury Jean-Marc , Mayer Klaus , Berges Hélène , Quesneville Hadi , Wincker Patrick , Feuillet Catherine
Published: July 2014

We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we assembled a sequence of 774 megabases carrying 5326 protein-coding genes, 1938 pseudogenes, and 85% of transposable elements. The distribution of structural and functional features along the chromosome revealed partitioning correlated with meiotic recombination. Comparative analyses indicated high wheat-specific inter- and intrachromosomal gene duplication activities that are potential sources of variability for adaption. In addition to providing a better understanding of the organization, function, and evolution of a large and polyploid genome, the availability of a high-quality sequence anchored to genetic maps will accelerate the identification of genes underlying important agronomic traits.
Ancient hybridizations among the ancestral genomes of bread wheat
Authors: Marcussen Thomas , Sandve Simen R. , Heier Lise , Spannagl Manuel , Pfeifer Matthias ,   The International Wheat Genome Sequencing Consortium,, Jakobsen Kjetill S. , Wulff Brande B. H. , Steuernagel Burkhard , Mayer Klaus F. X. , Olsen Odd-Arne , Rogers Jane , Doležel Jaroslav , Pozniak Curtis , Eversole Kellye , Feuillet Catherine , Gill Bikram , Friebe Bernd , Lukaszewski Adam J. , Sourdille Pierre , Endo Takashi R. , Kubaláková Marie , Číhalíková Jarmila , Dubská Zdeňka , Vrána Jan , Šperková Romana , Šimková Hana , Febrer Melanie , Clissold Leah , McLay Kirsten , Singh Kuldeep , Chhuneja Parveen , Singh Nagendra K. , Khurana Jitendra , Akhunov Eduard , Choulet Frédéric , Alberti Adriana , Barbe Valérie , Wincker Patrick , Kanamori Hiroyuki , Kobayashi Fuminori , Itoh Takeshi , Matsumoto Takashi , Sakai Hiroaki , Tanaka Tsuyoshi , Wu Jianzhong , Ogihara Yasunari , Handa Hirokazu , Maclachlan P. Ron , Sharpe Andrew , Klassen Darrin , Edwards David , Batley Jacqueline , Lien Sigbjørn , Caccamo Mario , Ayling Sarah , Ramirez-Gonzalez Ricardo H. , Clavijo Bernardo J. , Wright Jonathan , Martis Mihaela M. , Mascher Martin , Chapman Jarrod , Poland Jesse A. , Scholz Uwe , Barry Kerrie , Waugh Robbie , Rokhsar Daniel S. , Muehlbauer Gary J. , Stein Nils , Gundlach Heidrun , Zytnicki Matthias , Jamilloux Véronique , Quesneville Hadi , Wicker Thomas , Faccioli Primetta , Colaiacovo Moreno , Stanca Antonio Michele , Budak Hikmet , Cattivelli Luigi , Glover Natasha , Pingault Lise , Paux Etienne , Sharma Sapna , Appels Rudi , Bellgard Matthew , Chapman Brett , Nussbaumer Thomas , Bader Kai Christian , Rimbert Hélène , Wang Shichen , Knox Ron , Kilian Andrzej , Alaux Michael , Alfama Françoise , Couderc Loïc , Guilhot Nicolas , Viseux Claire , Loaec Mikaël , Keller Beat , Praud Sebastien
Published: July 2014

The allohexaploid bread wheat genome consists of three closely related subgenomes (A, B, and D), but a clear understanding of their phylogenetic history has been lacking. We used genome assemblies of bread wheat and five diploid relatives to analyze genome-wide samples of gene trees, as well as to estimate evolutionary relatedness and divergence times. We show that the A and B genomes diverged from a common ancestor ~7 million years ago and that these genomes gave rise to the D genome through homoploid hybrid speciation 1 to 2 million years later. Our findings imply that the present-day bread wheat genome is a product of multiple rounds of hybrid speciation (homoploid and polyploid) and lay the foundation for a new framework for understanding the wheat genome as a multilevel phylogenetic mosaic.
Role of synaptic phosphatidylinositol 3-kinase in a behavioral learning response in
C. elegans
Authors: Ohno Hayao , Kato Shinya , Naito Yasuki , Kunitomo Hirofumi , Tomioka Masahiro , Iino Yuichi
Published: July 2014

How the worm changes its tastes

In associative learning, you link potentially unrelated things because you are exposed to them at the same time. Ohno
et al.
studied a simple associative learning task in the nematode worm
Caenorhabditis elegans
. They presented the worms with a taste substance while withholding food. After starving in the presence of the taste substance, the animals switched their behavior from being attracted to the taste to finding it aversive. A specific isoform of the insulin receptor is critical for this type of associative learning—at least in worms.

Science
, this issue p.
313
A hole-conductor–free, fully printable mesoscopic perovskite solar cell with high stability
Authors: Mei Anyi , Li Xiong , Liu Linfeng , Ku Zhiliang , Liu Tongfa , Rong Yaoguang , Xu Mi , Hu Min , Chen Jiangzhao , Yang Ying , Grätzel Michael , Han Hongwei
Published: July 2014

Improved perovskite photovoltaic performance

A recent entry in the solar cell race is perovskite cells, named for the structure adopted by salt made from metal halides and organic cations that absorb the light and generate charges. The charges generated have to be transferred to a metal oxide (typically titanium oxide), and some of these charge carriers are lost in the transfer. Mei
et al.
made this process more efficient by growing a more crystalline perovskite with fewer defects inside porous versions of titanium and zirconium oxide. They added a second organic cation that stuck to the pore walls and directed the growth of the perovskite crystals. The improved solar cells operated for more than 1000 hours under full sunlight.

Science
, this issue p.
295
High-turnover hypoiodite catalysis for asymmetric synthesis of tocopherols
Authors: Uyanik Muhammet , Hayashi Hiroki , Ishihara Kazuaki
Published: July 2014

Iodine blooms as an oxidation catalyst

Most catalysts for organic oxidation chemistry—whether biochemical or artificial—contain a transition metal like iron or palladium. Uyanik
et al.
now show that iodine can take the place of a metal in catalyzing efficient oxidative ring closures to make chromans—hexagonal rings incorporating oxygen that are perhaps best known as a constituent of the vitamin E structure (see the Perspective by Nachtsheim). The iodine is added as a salt with a chiral cation, which directs the reaction to form just one of two possible mirror-image variants of the product. Key to the success of the system was the addition of a base, which maintained the viability of an unstable, partially oxidized iodine intermediate critical to the reaction cycle. The results bode well for more general use of iodine salts as asymmetric oxidation-reduction catalysts.

Science
, this issue p.
291
; see also p.
270
The elusive heart fix
Authors: Couzin-Frankel Jennifer
Published: July 2014

After years of hope and hype, researchers still don't agree whether cell infusions rejuvenate the human heart.
SAMBA HIV Semiquantitative Test, a New Point-of-Care Viral-Load-Monitoring Assay for Resource-Limited Settings
Authors: Ritchie Allyson V. , Ushiro-Lumb Ines , Edemaga Daniel , Joshi Hrishikesh A. , De Ruiter Annemiek , Szumilin Elisabeth , Jendrulek Isabelle , McGuire Megan , Goel Neha , Sharma Pia I. , Allain Jean-Pierre , Lee Helen H.
Published: July 2014

Routine viral-load (VL) testing of HIV-infected individuals on antiretroviral therapy (ART) is used to monitor treatment efficacy. However, due to logistical challenges, implementation of VL has been difficult in resource-limited settings. The aim of this study was to evaluate the performance of the SAMBA semi-Q (simple amplification-based assay semiquantitative test for HIV-1) in London, Malawi, and Uganda. The SAMBA semi-Q can distinguish between patients with VLs above and below 1,000 copies/ml. The SAMBA semi-Q was validated with diluted clinical samples and blinded plasma samples collected from HIV-1-positive individuals. SAMBA semi-Q results were compared with results from the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test, v2.0. Testing of 96 2- to 10-fold dilutions of four samples containing HIV-1 subtype C as well as 488 samples from patients in the United Kingdom, Malawi, and Uganda yielded an overall accuracy for the SAMBA semi-Q of 99% (95% confidence interval [CI], 93.8 to 99.9%) and 96.9% (95% CI 94.9 to 98.3%), respectively, compared to to the Roche test. Analysis of VL data from patients in Malawi and Uganda showed that the SAMBA cutoff of 1,000 copies/ml appropriately distinguished treated from untreated individuals. Furthermore, analysis of the viral loads of 232 patients on ART in Malawi and Uganda revealed similar patterns for virological control, defined as either <1,000 copies/ml (SAMBA cutoff) or <5,000 copies/ml (WHO 2010 criterion; WHO,
Antiretroviral Therapy for HIV Infection in Adults and Adolescents: Recommendations for a Public Health Approach
, 2010). This study suggests that the SAMBA semi-Q has adequate concurrency with the gold standard measurements for viral load. This test can allow VL monitoring of patients on ART at the point of care in resource-limited settings.
Evaluation of Performance and Acceptability of Two Rapid Oral Fluid Tests for HIV Detection in Mozambique
Authors: Semá Baltazar Cynthia , Raposo Cristina , Jani Ilesh V. , Shodell Daniel , Correia Della , Gonçalves da Silva Cristiane , Kalou Mireille , Patel Hetal , Parekh Bharat
Published: July 2014

Simplified HIV testing based on oral fluid (OF) may allow the expansion of HIV infection counseling and testing (CT) while reducing the risk due to exposure to needles and blood collection. This study evaluated the performance and acceptability of two OF tests (the OraQuick Advance Rapid HIV-1/2 and the Chembio DPP HIV-1/2) from May to September 2009 in two CT sites in Maputo City, Mozambique, compared with results for the national testing algorithm. OF testing was conducted in parallel with whole blood-based testing according to the national HIV algorithm. Blood samples were collected as dried blood spot (DBS) specimens from all participants for quality assurance. HIV infection results were delivered according to the national algorithm. According to the national HIV algorithm, 512 (30.5%) samples were reactive, 1,151 (68.7%) were nonreactive, and 13 (0.8%) were discordant. All discordant cases were retested with an enzyme immunoassay followed by Western blotting, and five (38.5%) were confirmed as HIV positive. The OraQuick OF test showed 518 (30.9%) reactive samples and 1,158 (69.1%) nonreactive samples, with a sensitivity and specificity of 99.8% and 99.8%, respectively. The Chembio DPP OF test showed 519 (31.0%) reactive samples and 1,157 (69.0%) nonreactive samples with a sensitivity and specificity of 100% and 99.8%, respectively. The participants perceived blood testing (49.9%) to be more accurate than OF testing (46.8%). The OF tests showed high performance for the diagnosis of HIV infection when examined individually and in an algorithm, compared with results according to the national testing algorithm.
Evaluation of a Commercially Developed Semiautomated PCR–Surface-Enhanced Raman Scattering Assay for Diagnosis of Invasive Fungal Disease
Authors: White P. Lewis , Hibbitts Samantha J. , Perry Michael D. , Green Julie , Stirling Emma , Woodford Luke , McNay Graeme , Stevenson Ross , Barnes Rosemary A.
Published: July 2014

Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of real-time PCR. Using this approach, the RenDx Fungiplex assay was developed to detect
Candida
and
Aspergillus
. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European
Aspergillus
PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of
Candida
and
Aspergillus
was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction–PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (
P
< 0.0001). The sensitivity of the assay was 82.8% and was similar for both
Candida
(80.0%) and
Aspergillus
(85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (≥2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility.
Performance of Two Resin-Containing Blood Culture Media in Detection of Bloodstream Infections and in Direct Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) Broth Assays for Isolate Identification: Clinical Comparison of the BacT/Alert Plus and Bactec Plus Systems
Authors: Fiori Barbara , D'Inzeo Tiziana , Di Florio Viviana , De Maio Flavio , De Angelis Giulia , Giaquinto Alessia , Campana Lara , Tanzarella Eloisa , Tumbarello Mario , Antonelli Massimo , Sanguinetti Maurizio , Spanu Teresa
Published: July 2014

We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P= 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P= 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P= 0.003), Gram-positive cocci (P= 0.013), andEscherichia coli(P= 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P= 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h;P= 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h;P< 0.001). Similarly high percentages of microorganisms in BacT/Alert and Bactec cultures (93.8% and 93.3%, respectively) were identified by direct matrix-assisted laser desorption ionization–time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings.
Clinical Significance of Low-Positive Histoplasma Urine Antigen Results
Authors: Theel Elitza S. , Ramanan Poornima
Published: July 2014

Histoplasma
urine antigen (UAg) detection is an important biomarker for histoplasmosis. The clinical significance of low-positive (<0.6 ng/ml) UAg results was evaluated in 25 patients without evidence of prior
Histoplasma
infection. UAg results from 12/25 (48%) patients were considered falsely positive, suggesting that low-positive UAg values should be interpreted cautiously.
Histone Deacetylase 6 Inhibits Influenza A Virus Release by Downregulating the Trafficking of Viral Components to the Plasma Membrane via Its Substrate, Acetylated Microtubules
Authors: Husain Matloob , Cheung Chen-Yi
Published: July 2014

Mammalian cells produce many proteins, such as IFITM3, ISG15, MxA, and viperin, that inhibit influenza A virus (IAV) infection. Here, we show that a new class of host protein, histone deacetylase 6 (HDAC6), inhibits IAV infection. We found that HDAC6-overexpressing cells release about 3-fold less IAV progeny, whereas HDAC6-depleted cells release about 6-fold more IAV progeny. The deacetylase activity of HDAC6 played a role in its anti-IAV function as tubacin, a specific small-molecule inhibitor of HDAC6, increased the release of IAV progeny in a dose-dependent manner. Further, as visualized by electron microscopy, tubacin-treated cells showed an increase in IAV budding at the plasma membrane, the site of IAV assembly. Tubacin is a domain-specific inhibitor and binds to one of the two HDAC6 catalytic domains possessing tubulin deacetylase activity. This indicated the potential involvement of acetylated microtubules in the trafficking of viral components to the plasma membrane. Indeed, as quantified by flow cytometry, there was about a 2.0- to 2.5-fold increase and about a 2.0-fold decrease in the amount of viral envelope protein hemagglutinin present on the plasma membrane of tubacin-treated/HDAC6-depleted and HDAC6-overexpressing cells, respectively. In addition, the viral ribonucleoprotein complex was colocalized with acetylated microtubule filaments, and viral nucleoprotein coimmunoprecipitated with acetylated tubulin. Together, our findings indicate that HDAC6 is an anti-IAV host factor and exerts its anti-IAV function by negatively regulating the trafficking of viral components to the host cell plasma membrane via its substrate, acetylated microtubules.

IMPORTANCE
Host cells produce many proteins that have the natural ability to restrict influenza virus infection. Here, we discovered that another host protein, histone deacetylase 6 (HDAC6), inhibits influenza virus infection. We demonstrate that HDAC6 exerts its anti-influenza virus function by negatively regulating the trafficking of viral components to the site of influenza virus assembly via its substrate, acetylated microtubules. HDAC6 is a multisubstrate enzyme and regulates multiple cellular pathways, including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of a wide range of diseases. Influenza virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza virus strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza virus potential of endogenous HDAC6 to inhibit influenza virus infection.
Inhibition of Antiviral Innate Immunity by Birnavirus VP3 Protein via Blockage of Viral Double-Stranded RNA Binding to the Host Cytoplasmic RNA Detector MDA5
Authors: Ye Chengjin , Jia Lu , Sun Yanting , Hu Boli , Wang Lun , Lu Xingmeng , Zhou Jiyong
Published: July 2014

Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-β) in DF-1 cells pretreated with IFN-α/β. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-β expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA
in vitro
and
in vivo
, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-β expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5.

IMPORTANCE
MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-β did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-β expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA
in vitro
and
in vivo
. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.
A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact
Authors: Harmache Abdallah
Published: July 2014

Marek's disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication
in vitro
and
in vivo
.
Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein
Authors: Kaever Thomas , Meng Xiangzhi , Matho Michael H. , Schlossman Andrew , Li Sheng , Sela-Culang Inbal , Ofran Yanay , Buller Mark , Crump Ryan W. , Parker Scott , Frazier April , Crotty Shane , Zajonc Dirk M. , Peters Bjoern , Xiang Yan
Published: July 2014

Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies.

IMPORTANCE
Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.
Amino Acid Substitutions in PB1 of Avian Influenza Viruses Influence Pathogenicity and Transmissibility in Chickens
Authors: Suzuki Yasushi , Uchida Yuko , Tanikawa Taichiro , Maeda Naohiro , Takemae Nobuhiro , Saito Takehiko
Published: July 2014

Amino acid substitutions were introduced into avian influenza virus PB1 in order to characterize the interaction between polymerase activity and pathogenicity. Previously, we used recombinant viruses containing the hemagglutinin (HA) and neuraminidase (NA) genes from the highly pathogenic avian influenza virus (HPAIV) H5N1 strain and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken and wild-bird hosts (LP and WB, respectively) to demonstrate that the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in chickens is regulated by the PB1 gene (Y. Uchida et al., J. Virol. 86:2686–2695, 2012, doi:http://dx.doi.org/10.1128/JVI.06374-11). In the present study, we introduced a C38Y substitution into WB PB1 and demonstrated that this substitution increased both polymerase activity in DF-1 cells in vitro and the pathogenicity of the recombinant viruses in chickens. The V14A substitution in LP PB1 reduced polymerase activity but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increased polymerase activity correlates directly with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis.

IMPORTANCEWe identified 2 novel amino acid substitutions in the avian influenza virus PB1 gene that affect the characteristics of highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype, such as viral replication and polymerase activityin vitroand pathogenicity and transmissibly in chickens. An amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens and was associated with changes in polymerase activityin vitro. A substitution at residue 14 reduced polymerase activityin vitro, while its effects on pathogenicity and transmissibility depended on the constellation of internal genes.
A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level
Authors: Bergua María , Zwart Mark P. , El-Mohtar Choaa , Shilts Turksen , Elena Santiago F. , Folimonova Svetlana Y.
Published: July 2014

Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels—the cellular and the whole-organism levels—by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases.

IMPORTANCE
Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.
Herpes Simplex Virus gE/gI Extracellular Domains Promote Axonal Transport and Spread from Neurons to Epithelial Cells
Authors: Howard Paul W. , Wright Catherine C. , Howard Tiffani , Johnson David C.
Published: July 2014

Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: (i) anterograde axonal transport of virus particles from neuron bodies to axon tips and (ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. The HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport, and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (∼400-residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants, gE-277 and gE-348, contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers, and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport; there were markedly reduced numbers of viral capsids and glycoproteins compared with wild-type HSV. The defects in axonal transport were manifest in neuronal cell bodies, involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild-type HSV. In contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite numbers of virus particles similar to those for HSV gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains function in both processes.

IMPORTANCE
An essential phase of the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is the capacity to reactivate from latency and then spread from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to axon tips followed by (ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important for identifying novel targets to reduce alphaherpesvirus disease.
Structural Insights into the Human Metapneumovirus Glycoprotein Ectodomain
Authors: Leyrat Cedric , Paesen Guido C. , Charleston James , Renner Max , Grimes Jonathan M.
Published: July 2014

Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.
Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication
Authors: Han Song-Hee , Kim Seong-Jun , Kim Eun-Jung , Kim Tae-Eun , Moon Jae-Su , Kim Geon-Woo , Lee Seung-Hoon , Cho Kun , Yoo Jong Shin , Son Woo Sung , Rhee Jin-Kyu , Han Seung Hyun , Oh Jong-Won
Published: July 2014

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues
in vitro
and
in vivo
. Using
in vitro
kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for
de novo
RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.

IMPORTANCE
While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the Δ1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.
Propionibacterium
-Produced Coproporphyrin III Induces Staphylococcus aureus Aggregation and Biofilm Formation
Authors: Wollenberg Michael S. , Claesen Jan , Escapa Isabel F. , Aldridge Kelly L. , Fischbach Michael A. , Lemon Katherine P.
Published: July 2014

The majority of bacteria detected in the nostril microbiota of most healthy adults belong to three genera:
Propionibacterium
,
Corynebacterium
, and
Staphylococcus
. Among these staphylococci is the medically important bacterium
Staphylococcus aureus
. Almost nothing is known about interspecies interactions among bacteria in the nostrils. We observed that crude extracts of cell-free conditioned medium from
Propionibacterium
spp. induce
S. aureus
aggregation in culture. Bioassay-guided fractionation implicated coproporphyrin III (CIII), the most abundant extracellular porphyrin produced by human-associated
Propionibacterium
spp., as a cause of
S. aureus
aggregation. This aggregation response depended on the CIII dose and occurred during early stationary-phase growth, and a low pH (~4 to 6) was necessary but was not sufficient for its induction. Additionally, CIII induced plasma-independent
S. aureus
biofilm development on an abiotic surface in multiple
S. aureus
strains. In strain UAMS-1, CIII stimulation of biofilm depended on
sarA
, a key biofilm regulator. This study is one of the first demonstrations of a small-molecule-mediated interaction among medically relevant members of the nostril microbiota and the first description of a role for CIII in bacterial interspecies interactions. Our results indicate that CIII may be an important mediator of
S. aureus
aggregation and/or biofilm formation in the nostril or other sites inhabited by
Propionibacterium
spp. and
S. aureus
.

IMPORTANCE
Very little is known about interspecies interactions among the bacteria that inhabit the adult nostril, including
Staphylococcus aureus
, a potential pathogen that colonizes about a quarter of adults. We demonstrated that coproporphyrin III (CIII), a diffusible small molecule excreted by nostril- and skin-associated
Propionibacterium
spp., induces
S. aureus
aggregation in a manner dependent on dose, growth phase, and pH. CIII also induces
S. aureus
to form a plasma-independent surface-attached biofilm. This report is the first description of a role for CIII in bacterial interspecies interactions at any human body site and a novel demonstration that nostril microbiota physiology is influenced by small-molecule-mediated interactions.
Detection of the Middle East Respiratory Syndrome Coronavirus Genome in an Air Sample Originating from a Camel Barn Owned by an Infected Patient
Authors: Azhar Esam I. , Hashem Anwar M. , El-Kafrawy Sherif A. , Sohrab Sayed Sartaj , Aburizaiza Asad S. , Farraj Suha A. , Hassan Ahmed M. , Al-Saeed Muneera S. , Jamjoom Ghazi A. , Madani Tariq A.
Published: July 2014

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel betacoronavirus that has been circulating in the Arabian Peninsula since 2012 and causing severe respiratory infections in humans. While bats were suggested to be involved in human MERS-CoV infections, a direct link between bats and MERS-CoV is uncertain. On the other hand, serological and virological data suggest dromedary camels as the potential animal reservoirs of MERS-CoV. Recently, we isolated MERS-CoV from a camel and its infected owner and provided evidence for the direct transmission of MERS-CoV from the infected camel to the patient. Here, we extend this work and show that identical MERS-CoV RNA fragments were detected in an air sample collected from the same barn that sheltered the infected camel in our previous study. These data indicate that the virus was circulating in this farm concurrently with its detection in the camel and in the patient, which warrants further investigations for the possible airborne transmission of MERS-CoV.

IMPORTANCE
This work clearly highlights the importance of continuous surveillance and infection control measures to control the global public threat of MERS-CoV. While current MERS-CoV transmission appears to be limited, we advise minimal contact with camels, especially for immunocompromised individuals, and the use of appropriate health, safety, and infection prevention and control measures when dealing with infected patients. Also, detailed clinical histories of any MERS-CoV cases with epidemiological and laboratory investigations carried out for any animal exposure must be considered to identify any animal source.
Health information technology interventions enhance care completion, engagement in HIV care and treatment, and viral suppression among HIV-infected patients in publicly funded settings
Authors: Shade Starley B , Steward Wayne T , Koester Kimberly A , Chakravarty Deepalika , Myers Janet J
Published: July 2014

Background The National HIV/AIDS Strategy (NHAS) emphasizes the use of technology to facilitate coordination of comprehensive care for people with HIV. We examined the effect of six health information technology (HIT) interventions in a Ryan White-funded Special Projects of National Significance (SPNS) on care completion services, engagement in HIV care, and viral suppression.

Methods Interventions included use of surveillance data to identify out-of-care individuals, extending access to electronic health records to support service providers, use of electronic laboratory ordering and prescribing, and development of a patient portal. Data from a sample of electronic patient records from each site were analyzed to assess changes in utilization of comprehensive care (prevention screening, support service utilization), engagement in primary HIV medical care (receipt of services and use of antiretroviral therapy), and viral suppression. We used weighted generalized estimating equations to estimate outcomes while accounting for the unequal contribution of data and differences in the distribution of patient characteristics across sites and over time.

Results We observed statistically significant changes in the desired direction in comprehensive care utilization and engagement in primary care outcomes targeted by each site. Five of six sites experienced statistically significant increases in viral suppression.

Discussion These results provide additional support for the use of HIT as a valuable tool for achieving the NHAS goal of providing comprehensive care for all people living with HIV. HIT has the potential to increase utilization of services, improve health outcomes for people with HIV, and reduce community viral load and subsequent transmission of HIV.
British athletics muscle injury classification: a new grading system
Authors: Pollock Noel , James Steven L J , Lee Justin C , Chakraverty Robin
Published: July 2014

The commonly used muscle injury grading systems based on three grades of injury, representing minor, moderate and complete injuries to the muscle, are lacking in diagnostic accuracy and provide limited prognostic information to the clinician. In recent years, there have been a number of proposals for alternative grading systems. While there is recent evidence regarding the prognostic features of muscle injuries, this evidence has not often been incorporated into the grading proposals. The British Athletics Muscle Injury Classification proposes a new system, based on the available evidence, which should provide a sound diagnostic base for therapeutic decision-making and prognostication. Injuries are graded 0–4 based on MRI features, with Grades 1–4 including an additional suffix ‘a’, ‘b’ or ‘c’ if the injury is ‘myofascial’, ‘musculo-tendinous’ or ‘intratendinous’. Retrospective and prospective studies in elite track and field athletes are underway to validate the classification for use in hamstring muscle injury management. It is intended that this grading system can provide a suitable diagnostic framework for enhanced clinical decision-making in the management of muscle injuries and assist with future research to inform the development of improved prevention and management strategies.
A randomised controlled trial for the evaluation of risk for type 2 diabetes in hypertensive patients receiving thiazide diuretics: Diuretics In the Management of Essential hypertension (DIME) study
Authors: Ueda Shinichiro , Morimoto Takeshi , Ando Shin-ichi , Takishita Shu-ichi , Kawano Yuhei , Shimamoto Kazuaki , Ogihara Toshio , Saruta Takao ,   for the DIME Investigators
Published: July 2014

Objectives

Thiazide diuretics are one of the first choice antihypertensives but not optimally utilised because of concerns regarding their adverse effects on glucose metabolism. The Diuretics In the Management of Essential hypertension (DIME) study was designed, for the first time, to assess the risk for type 2 diabetes mellitus in patients with essential hypertension during antihypertensive treatment with low-dose thiazide diuretics compared to those not treated with diuretics.

Design

Multicentre, unblinded, pragmatic, randomised, controlled trial with blinded assessment of end points and intention-to-treat analysis that was started in 2004 and finished in 2012.

Setting

Hypertension clinics at 106 sites in Japan, including general practitioners’ offices and teaching hospitals.

Participants

Non-diabetic patients with essential hypertension.

Interventions

Antihypertensive treatment with low-dose thiazide diuretics at 12.5 mg/day of hydrochlorothiazide or equivalent (Diuretics group) or that without thiazide diuretics (No-diuretics group).

Main outcome

The primary outcome was new onset of type 2 diabetes diagnosed according to WHO criteria and the criteria of Japanese Society of Diabetes.

Results

1130 patients were allocated to Diuretics (n=544) or No-diuretics group (n=586). Complete end point information was collected for 1049 participants after a median follow-up of 4.4 years. Diabetes developed in 25 (4.6%) participants in the Diuretics group, as compared with 29 (4.9%) in the No-diuretics group (HR 0.93; 95% CI 0.55 to 1.58; p=0.800).

Conclusions

Antihypertensive treatment with thiazide diuretics at low doses may not be associated with an increased risk for new onset of type 2 diabetes. This result might suggest safety of use of low doses of thiazide diuretics.

Trial registration number

ClinicalTrials.govNCT00131846.