bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of a Lombard response in migrating humpback whales (Megaptera novaeangliae)
Authors: Dunlop Rebecca A. , Cato Douglas H. , Noad Michael J.
Published: July 2014

The Lombard reflex is an increase in the subject's vocal levels in response to increased noise levels. This functions to maintain an adequate signal-to-noise ratio at the position of the receiver when noise levels vary. While it has been demonstrated in a small number of mammals and birds including some whales, it has not yet been shown to occur in one of the most vocal species of baleen whale, the humpback whale (Megaptera novaeangliae). Humpback whales were simultaneously visually and acoustically tracked (using an array of calibrated hydrophone buoys) as they migrated southward. Source levels of social vocalizations were estimated from measured received levels and a site-specific empirical sound propagation model developed. In total, 226 social vocalizations from 16 passing groups of whales were selected for final analysis. Noise levels were predominantly wind-dependent (from sea surface motion) and ranged from 81 to 108 dB re 1 μPa in the 36 Hz–2.8 kHz band. Vocalization source levels increased by 0.9 dB for every 1 dB increase in wind-dependent background noise levels, with source levels (at 1 m) being maintained ∼60 dB above the noise level.
Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics
Authors: Iversen Lars , Tu Hsiung-Lin , Lin Wan-Chen , Christensen Sune M. , Abel Steven M. , Iwig Jeff , Wu Hung-Jen , Gureasko Jodi , Rhodes Christopher , Petit Rebecca S. , Hansen Scott D. , Thill Peter , Yu Cheng-Han , Stamou Dimitrios , Chakraborty Arup K. , Kuriyan John , Groves Jay T.
Published: July 2014

Outliers dominate signaling at cell membrane

SOS enzymes act at cell membranes to activate Ras, a regulatory protein often overactive in cancer cells. Iversen
et al.
devised a system where they could observe the activity of individual enzymes at work. The single SOS molecules occupied stable states that varied greatly in their catalytic activity. Regulation appeared to occur by altering the time spent in active states. The overall activity of SOS was determined by just a few molecules that achieved the highest catalytic activity. The methods described should allow further detailed kinetic analysis of this and other signaling events that occur at the cell membrane — properties that it is not possible to discern from bulk biochemical measurements.

Science
, this issue p.
50
Just think: The challenges of the disengaged mind
Authors: Wilson Timothy D. , Reinhard David A. , Westgate Erin C. , Gilbert Daniel T. , Ellerbeck Nicole , Hahn Cheryl , Brown Casey L. , Shaked Adi
Published: July 2014

Don't leave me alone with my thoughts

Nowadays, we enjoy any number of inexpensive and readily accessible stimuli, be they books, videos, or social media. We need never be alone, with no one to talk to and nothing to do. Wilson
et al.
explored the state of being alone with one's thoughts and found that it appears to be an unpleasant experience. In fact, many of the people studied, particularly the men, chose to give themselves a mild electric shock rather than be deprived of external sensory stimuli.

Science
, this issue p.
75
A terrestrial planet in a ~1-AU orbit around one member of a ∼15-AU binary
Authors: Gould A. , Udalski A. , Shin I.-G. , Porritt I. , Skowron J. , Han C. , Yee J. C. , Kozłowski S. , Choi J.-Y. , Poleski R. , Wyrzykowski Ł. , Ulaczyk K. , Pietrukowicz P. , Mróz P. , Szymański M. K. , Kubiak M. , Soszyński I. , Pietrzyński G. , Gaudi B. S. , Christie G. W. , Drummond J. , McCormick J. , Natusch T. , Ngan H. , Tan T.-G. , Albrow M. , DePoy D. L. , Hwang K.-H. , Jung Y. K. , Lee C.-U. , Park H. , Pogge R.W. , Abe F. , Bennett D. P. , Bond I. A. , Botzler C. S. , Freeman M. , Fukui A. , Fukunaga D. , Itow Y. , Koshimoto N. , Larsen P. , Ling C. H. , Masuda K. , Matsubara Y. , Muraki Y. , Namba S. , Ohnishi K. , Philpott L. , Rattenbury N. J. , Saito To. , Sullivan D. J. , Sumi T. , Suzuki D. , Tristram P. J. , Tsurumi N. , Wada K. , Yamai N. , Yock P. C. M. , Yonehara A. , Shvartzvald Y. , Maoz D. , Kaspi S. , Friedmann M.
Published: July 2014

Impolite planet ignores host's partner

Many known exoplanets (planets outside our own solar system) are hosted by binary systems that contain two stars. These planets normally circle around both of their stars. Using microlensing data taken with a worldwide network of telescopes, Gould
et al.
found a planet twice the mass of Earth that circles just one of a pair of stars. The same approach has the potential to uncover other similar star systems and help to illuminate some of the mysteries of planet formation.

Science
, this issue p.
46
Climate change and wind intensification in coastal upwelling ecosystems
Authors: Sydeman W. J. , García-Reyes M. , Schoeman D. S. , Rykaczewski R. R. , Thompson S. A. , Black B. A. , Bograd S. J.
Published: July 2014

Strong winds, upwelling, and teeming shores

Climate warming has produced stronger winds along some coasts, a result of growing differences in temperature and pressure between land and sea. These winds cause cold nutrient-rich seawater to rise to the surface, affecting climate and fueling marine productivity. Sydeman
et al.
examined data from the five major world regions where upwelling is occurring. Particularly in the California, Humboldt, and Benguela upwelling systems, winds have become stronger over the past 60 years. These regions represent up to a fifth of wild marine fish catches and are hot spots of biodiversity.

Science
, this issue p.
77
Emergent complex states in bilayer graphene
Authors: LeRoy Brian J. , Yankowitz Matthew
Published: July 2014

Experiments probe a wealth of exotic electronic behavior.

[Also see Reports by

Kou
et al.

,

Lee
et al.

, and

Maher
et al.

]
A challenge to pseudoscience
Authors: Bohannon John
Published: July 2014

Egyptian expat questions claims that devices noninvasively detect viruses in blood and treat infected people.
IPCC lessons from Berlin
Authors: Wible Brad
Published: July 2014

Did the “Summary for Policymakers” become a summary by policy-makers?
Development of a Rapid Diagnostic Kit That Uses an Immunochromatographic Device To Detect Antibodies in Human Sparganosis
Authors: Yamasaki Hiroshi , Nakamura Takeshi , Intapan Pewpan M. , Maleewong Wanchai , Morishima Yasuyuki , Sugiyama Hiromu , Matsuoka Hiroyuki , Kobayashi Kaoru , Takayama Katsuyoshi , Kobayashi Yukuharu
Published: July 2014

A diagnostic kit using an immunochromatographic device was developed to replace the time-consuming immunodiagnostic methods for human sparganosis. The kit was found to be faster and easier to use than an enzyme-linked immunosorbent assay (ELISA) and showed higher sensitivity and specificity. It will be useful for the laboratory diagnosis of hospitalized cases of sparganosis.
Post Hoc
Analysis of a Randomized Double-Blind Trial of the Correlation of Functional and Binding Antibody Responses Elicited by 13-Valent and 7-Valent Pneumococcal Conjugate Vaccines and Association with Nasopharyngeal Colonization
Authors: Juergens Christine , Patterson Scott , Trammel James , Greenberg David , Givon-Lavi Noga , Cooper David , Gurtman Alejandra , Gruber William C. , Scott Daniel A. , Dagan Ron
Published: July 2014

In a randomized double-blind trial in healthy Israeli infants in Israel who received the 13-valent or 7-valent pneumococcal conjugate vaccine (PCV13 or PCV7, respectively) at 2, 4, 6, and 12 months, PCV13 significantly reduced nasopharyngeal (NP) colonization of serotypes 1, 6A, 7F, 19A, cross-reacting 6C, and the common PCV7 serotype 19F, from ages 7 to 24 months. No differences were observed between the vaccine groups for serotype 3 or for the remaining common PCV7 serotypes. For serotype 5, too few events were observed to draw an inference. Generally consistent with these findings, PCV13 elicited significantly higher enzyme-linked immunosorbent assay (ELISA) IgG-binding antibody responses than did PCV7 for the additional PCV13 serotypes 1, 3, 5, 6A, 7F, 19A, and for the common serotype 19F, with similar or lower responses for the remaining common serotypes. To further assess immunogenicity and colonization, we conducted a
post hoc
analysis of PCV13 functional antibody responses measured by opsonophagocytic activity (OPA) assays in a randomly selected subset of subjects. The pattern of functional antibody OPA responses elicited by PCV13 relative to PCV7 was similar to that of the ELISA anticapsular IgG-binding antibody responses described above. In addition, the OPA responses generally correlated positively with IgG responses for all 13 serotypes among the PCV13 recipients and for all 7 common serotypes and the additional serotype 6A but not for 19A or the other serotypes unique to PCV13 among the PCV7 recipients. This
post hoc
analysis supports an association between serum OPA functional and IgG-binding antibody levels, allowing for a transfer of inferred associations between IgG responses and NP colonization to OPA responses.
Immune Responses in Macaques to a Prototype Recombinant Adenovirus Live Oral Human Papillomavirus 16 Vaccine
Authors: Berg Michael G. , Adams Robert J. , Gambhira Ratish , Siracusa Mark C. , Scott Alan L. , Roden Richard B. S. , Ketner Gary
Published: July 2014

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.
Immunogenicity of the Trivalent Inactivated Influenza Vaccine in Young Children Less than 4 Years of Age, with a Focus on Age and Baseline Antibodies
Authors: Mugitani Ayumi , Ito Kazuya , Irie Shin , Eto Takashi , Ishibashi Motoki , Ohfuji Satoko , Fukushima Wakaba , Maeda Akiko , Hirota Yoshio
Published: July 2014

In this study, we assessed the effects of the prevaccination titer and age on the immunogenicity of a low dose of influenza vaccine in children less than 4 years of age. A total of 259 children received two vaccine doses (0.1 ml for 0-year-olds and 0.2 ml for children 1 year of age or older) 4 weeks apart during the 2005/2006 season. The hemagglutination inhibition antibody titers were measured before vaccination and 4 weeks after the first and second doses. The geometric mean titer, mean fold rise, seroresponse proportion (≥4-fold rise in titer), and seroprotection proportion (titer ≥1:40) were calculated for the prevaccination titer and age categories. A multivariate logistic regression analysis was performed using the seroresponse and seroprotection proportions as dependent variables and the prevaccination titer and age as explanatory variables. As for the seroresponse against the H1 antigen after the first dose, the adjusted odds ratios of the prevaccination titers (versus <1:10) were 2.2 (95% confidence interval, 0.8 to 5.8) at 1:10 to 1:20 and 0.14 (0.04 to 0.49) at ≥1:40. The corresponding figures for ages were 0.03 (0.01 to 0.07) for the 0-year-olds and 0.17 (0.08 to 0.34) for the 1-year-olds compared with the 2- to 3-year-olds (Ptrend< 0.001). Similar results were also obtained for the H3 and B strains. Significantly elevated odds ratios for seroprotection were observed with greater prevaccination titers and older ages for all strains. The prevaccination titer and age were independently associated with the antibody response in young children. The immune response was weaker in the younger children and those without preexisting immunity.
dl-2-Hydroxyisocaproic Acid Attenuates Inflammatory Responses in a Murine Candida albicans Biofilm Model
Authors: Nieminen M. T. , Hernandez M. , Novak-Frazer L. , Kuula H. , Ramage G. , Bowyer P. , Warn P. , Sorsa T. , Rautemaa R.
Published: July 2014

Chronic biofilm infections are often accompanied by a chronic inflammatory response, leading to impaired healing and increased, irreversible damage to host tissues. Biofilm formation is a major virulence factor forCandida albicansand a challenge for treatment. Most current antifungals have proved ineffective in eradicating infections attributed to biofilms. The biofilm structure protectsCandidaspecies against antifungals and provides a way for them to evade host immune systems. This leads to a very distinct inflammatory response compared to that seen in planktonic infections. Previously, we showed the superior efficacy ofdl-2-hydroxyisocaproic acid (HICA) against various bacteria and fungi. However, the immunomodulatory properties of HICA have not been studied. Our aim was to investigate the potential anti-inflammatory response to HICAin vivo. We hypothesized that HICA reduces the levels of immune mediators and attenuates the inflammatory response. In a murine model, a robust biofilm was formed for 5 days in a diffusion chamber implanted underneath mouse skin. The biofilm was treated for 12 h with HICA, while caspofungin and phosphate-buffered saline (PBS) were used as controls. The pathophysiology and immunoexpression in the tissues surrounding the chamber were determined by immunohistochemistry. Histopathological examination showed an attenuated inflammatory response together with reduced expression of matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO) compared to those of chambers containing caspofungin and PBS. Interestingly, the expression of developmental endothelial locus 1 (Del-1), an antagonist of neutrophil extravasation, increased after treatment with HICA. Considering its anti-inflammatory and antimicrobial activity, HICA may have enormous therapeutic potential in the treatment of chronic biofilm infections and inflammation, such as those seen with chronic wounds.
Prospective Cohort Study of the Safety of an Influenza A(H1N1) Vaccine in Pregnant Chinese Women
Authors: Ma Fubao , Zhang Longhua , Jiang Renjie , Zhang Jinlin , Wang Huaqing , Gao Xiaozhi , Li Xiuhong , Liu Yuanbao
Published: July 2014

To monitor and evaluate the safety of the influenza A(H1N1) vaccine in pregnant women and its influence on the fetus and neonate, we performed a prospective study in which 122 pregnant Chinese women who received the influenza A(H1N1) vaccine and 104 pregnant women who did not receive any vaccine (serving as controls) were observed. The results indicated that the seroconversion rate in the vaccinated group was 90.4% (95% confidence interval [CI], 82.6% to 95.5%). The rate of adverse events following immunization in the pregnant women who received the influenza A(H1N1) vaccine was 3.3%. The spontaneous abortion rates in the vaccinated group and the unvaccinated group were 0.8% and 1.9%, respectively (exact probability test,P= 0.470), the prolonged-pregnancy rates were 8.2% and 4.8%, respectively (χ2= 1.041,P= 0.308), the low-birth-weight rates were 1.6% and 0.95%, respectively (exact probability test,P= 1.000), and the spontaneous-labor rates were 70.5% and 75%, respectively (χ2= 0.573,P= 0.449). All newborns who have an Apgar score of ≥7 are considered healthy; Apgar scores of ≥9 were observed in 38.5% and 57.7% of newborns in the vaccinated group and the unvaccinated group, respectively (χ2= 8.274,P= 0.004). From these results, we conclude that the influenza A(H1N1) vaccine is safe for pregnant women and has no observed adverse effects on fetal growth. (This study has been registered at ClinicalTrials.gov under registration no. NCT01842997.)
Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Authors: Yoshikawa Tomoki , Fukushi Shuetsu , Tani Hideki , Fukuma Aiko , Taniguchi Satoshi , Toda Shoichi , Shimazu Yukie , Yano Koji , Morimitsu Toshiharu , Ando Katsuyuki , Yoshikawa Akira , Kan Miki , Kato Nobuyuki , Motoya Takumi , Kuzuguchi Tsuyoshi , Nishino Yasuhiro , Osako Hideo , Yumisashi Takahiro , Kida Kouji , Suzuki Fumie , Takimoto Hirokazu , Kitamoto Hiroaki , Maeda Ken , Takahashi Toru , Yamagishi Takuya , Oishi Kazunori , Morikawa Shigeru , Saijo Masayuki , Shimojima Masayuki
Published: July 2014

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.
Same-Day Subtyping of Campylobacter jejuni and C. coli Isolates by Use of Multiplex Ligation-Dependent Probe Amplification–Binary Typing
Authors: Cornelius Angela J. , Vandenberg Olivier , Robson Beth , Gilpin Brent J. , Brandt Stephanie M. , Scholes Paula , Martiny Delphine , Carter Philip E. , van Vught Paul , Schouten Jan , On Stephen L. W.
Published: July 2014

Campylobacteriosis is the most commonly reported form of human bacterial gastroenteritis in the world. Sound identification of infectious sources requires subtyping, but the most widely used methods have turnaround times measured in days and require specialist equipment and skills. A multiplex ligation-dependent probe amplification-binary typing (MBiT) assay was developed for subtyping
Campylobacter jejuni
and
Campylobacter coli
. It was tested on 245 isolates, including recent isolates from Belgium and New Zealand, and compared to multilocus sequence typing (MLST). When used in an outbreak setting, MBiT identified the predominant genotype and possible additional cases days before pulsed-field gel electrophoresis (PFGE) results were available. MBiT was more discriminatory than MLST and, being a single assay with results produced within 6 h, was more rapid and cost-effective than both MLST and PFGE. In addition, MBiT requires only basic molecular biology equipment and skills.
Relevance of a Full-Length Genomic RNA Standard and a Thermal-Shock Step for Optimal Hepatitis Delta Virus Quantification
Authors: Homs Maria , Giersch Katja , Blasi Maria , Lütgehetmann Marc , Buti Maria , Esteban Rafael , Dandri Maura , Rodriguez-Frias Francisco
Published: July 2014

Hepatitis D virus (HDV) is a defective RNA virus that requires the surface antigens of hepatitis B virus (HBV) (HBsAg) for viral assembly and replication. Several commercial and in-house techniques have been described for HDV RNA quantification, but the methodologies differ widely, making a comparison of the results between studies difficult. In this study, a full-length genomic RNA standard was developed and used for HDV quantification by two different real-time PCR approaches (fluorescence resonance energy transfer [FRET] and TaqMan probes). Three experiments were performed. First, the stability of the standard was determined by analyzing the effect of thawing and freezing. Second, because of the strong internal base pairing of the HDV genome, which leads to a rod-like structure, the effect of intense thermal shock (95°C for 10 min and immediate cooling to −80°C) was tested to confirm the importance of this treatment in the reverse transcription step. Lastly, to investigate the differences between the DNA and RNA standards, the two types were quantified in parallel with the following results: the full-length genomic RNA standard was stable and reliably mimicked the behavior of HDV-RNA-positive samples, thermal shock enhanced the sensitivity of HDV RNA quantification, and the DNA standard underquantified the HDV RNA standard. These findings indicate the importance of using complete full-length genomic RNA and a strong thermal-shock step for optimal HDV RNA quantification.
Evaluation of Alere i Influenza A&B for Rapid Detection of Influenza Viruses A and B
Authors: Nie Shuping , Roth Richard B. , Stiles Jeffrey , Mikhlina Albina , Lu Xuedong , Tang Yi-Wei , Babady N. Esther
Published: July 2014

Rapid and accurate diagnosis of influenza is important for infection control, as well as for patient management. Alere i Influenza A&B is an isothermal nucleic acid amplification-based integrated system for detection and differentiation of influenza virus A and influenza virus B. The performance of the Alere i Influenza A&B was screened using frozen nasopharyngeal-swab specimens collected in viral transport medium (VTM) that were originally tested fresh with the FilmArray Respiratory Panel (RP) assay during the 2012–2013 influenza outbreak. In total, 360 VTM specimens were selected for Alere i Influenza A&B testing: 40 influenza virus A H1N1-2009 (influenza virus A-1), 40 influenza virus A H3N2 (influenza virus A-3), 37 influenza virus A “equivocal” or “no subtype detected” (influenza virus A-u), 41 influenza virus B, and 202 influenza virus-negative specimens, as initially determined by the FilmArray RP assay. The Alere assay showed sensitivities of 87.2%, 92.5%, 25.0%, and 97.4% for influenza virus A-1, influenza virus A-3, influenza virus A-u, and influenza virus B, respectively, after discordant resolution by Prodesse ProFLU+ PCR. The specificities were 100% for both influenza virus A and influenza virus B. In general, the Alere i Influenza A&B provided good sensitivity, although the assay did show poorer sensitivity with samples determined to have low influenza virus A titers by Prodesse ProFlu+ PCR (a mean real-time PCR threshold cycle [C

T

] value of 31.9 ± 2.0), which included the majority of the samples called influenza virus A “equivocal” or “no subtype detected” by a single BioFire FilmArray RP test. The integrated, rapid, and simple characteristics of the Alere i Influenza A&B assay make it a potential candidate for point-of-care testing, with a test turnaround time of less than 15 min.
How Viruses Hijack the ERAD Tuning Machinery
Authors: Noack Julia , Bernasconi Riccardo , Molinari Maurizio
Published: July 2014

An essential step during the intracellular life cycle of many positive-strand RNA viruses is the rearrangement of host cell membranes to generate membrane-bound replication platforms. For example,
Nidovirales
and
Flaviviridae
subvert the membrane of the endoplasmic reticulum (ER) for their replication. However, the absence of conventional ER and secretory pathway markers in virus-induced ER-derived membranes has for a long time hampered a thorough understanding of their biogenesis. Recent reports highlight the analogies between mouse hepatitis virus-, equine arteritis virus-, and Japanese encephalitis virus-induced replication platforms and ER-associated degradation (ERAD) tuning vesicles (or EDEMosomes) that display nonlipidated LC3 at their cytosolic face and segregate the ERAD factors EDEM1, OS-9, and SEL1L from the ER lumen. In this Gem, we briefly summarize the current knowledge on ERAD tuning pathways and how they might be hijacked for viral genome replication. As ERAD tuning components, such as SEL1L and nonlipidated LC3, appear to contribute to viral infection, these cellular pathways represent novel candidate drug targets to combat positive-strand RNA viruses.
The Immunodominant Influenza A Virus M158–66Cytotoxic T Lymphocyte Epitope Exhibits Degenerate Class I Major Histocompatibility Complex Restriction in Humans
Authors: Choo Joanna A. L. , Liu Jingxian , Toh Xinyu , Grotenbreg Gijsbert M. , Ren Ee Chee
Published: July 2014

Cytotoxic T lymphocytes recognizing conserved peptide epitopes are crucial for protection against influenza A virus (IAV) infection. The CD8 T cell response against the M158–66(GILGFVFTL) matrix protein epitope is immunodominant when restricted by HLA-A*02, a major histocompatibility complex (MHC) molecule expressed by approximately half of the human population. Here we report that the GILGFVFTL peptide is restricted by multiple HLA-C*08 alleles as well. We observed that M158–66was able to elicit cytotoxic T lymphocyte (CTL) responses in both HLA-A*02- and HLA-C*08-positive individuals and that GILGFVFTL-specific CTLs in individuals expressing both restriction elements were distinct and not cross-reactive. The crystal structure of GILGFVFTL–HLA-C*08:01 was solved at 1.84 Å, and comparison with the known GILGFVFTL–HLA-A*02:01 structure revealed that the antigen bound both complexes in near-identical conformations, accommodated by binding pockets shaped from shared as well as unique residues. This discovery of degenerate peptide presentation by both HLA-A and HLA-C allelic variants eliciting unique CTL responses to IAV infection contributes fundamental knowledge with important implications for vaccine development strategies.

IMPORTANCEThe presentation of influenza A virus peptides to elicit immunity is thought to be narrowly restricted, with a single peptide presented by a specific HLA molecule. In this study, we show that the same influenza A virus peptide can be more broadly presented by both HLA-A and HLA-C molecules. This discovery may help to explain the differences in immunity to influenza A virus between individuals and populations and may also aid in the design of vaccines.
Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
Authors: Csibra Eszter , Brierley Ian , Irigoyen Nerea
Published: July 2014

Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV),
gag
and
pol
are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.

IMPORTANCE
Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.
Recent H3N2 Influenza Virus Clinical Isolates Rapidly Acquire Hemagglutinin or Neuraminidase Mutations When Propagated for Antigenic Analyses
Authors: Chambers Benjamin S. , Li Yang , Hodinka Richard L. , Hensley Scott E.
Published: July 2014

Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated nine primary H3N2 viral isolates from respiratory secretions of children. Upon propagation
in vitro
, five of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses.
Ectopic DNMT3L Triggers Assembly of a Repressive Complex for Retroviral Silencing in Somatic Cells
Authors: Kao Tzu-Hao , Liao Hung-Fu , Wolf Daniel , Tai Kang-Yu , Chuang Ching-Yu , Lee Hsuan-Shu , Kuo Hung-Chih , Hata Kenichiro , Zhang Xing , Cheng Xiaodong , Goff Stephen P. , Ooi Steen K. T. , Bestor Timothy H. , Lin Shau-Ping
Published: July 2014

Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransferase 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1β and KAP1) in ES cells and orchestrates retroviral silencing activity with TRIM28 through mechanisms including, but not limited to,
de novo
DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney murine leukemia virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1γ), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late-passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly integrated retroviruses.

IMPORTANCE
Almost half of the mammalian genome is composed of endogenous retroviruses and other retrotransposable elements that threaten genomic integrity. These elements are usually subject to epigenetic silencing. We discovered that two epigenetic regulators that lack enzymatic activity, DNA methyltransferase 3-like (DNMT3L) and tripartite motif-containing protein 28 (TRIM28), collaborate with each other to impose retroviral silencing. In addition to modulating
de novo
DNA methylation, we found that by interacting with TRIM28, DNMT3L can attract various enzymes to form a DNMT3L-induced repressive complex to remove active marks and add repressive marks to histone proteins. Collectively, these results reveal a novel and pivotal function of DNMT3L in shaping the chromatin modifications necessary for retroviral and retrotransposon silencing.
The Rhesus Rhadinovirus CD200 Homologue Affects Immune Responses and Viral Loads during
In Vivo
Infection
Authors: Estep Ryan D. , Rawlings Stephanie D. , Li Helen , Manoharan Minsha , Blaine Elizabeth T. , O'Connor Megan A. , Messaoudi Ilhem , Axthelm Michael K. , Wong Scott W.
Published: July 2014

Rhesus macaque rhadinovirus (RRV) is a gammaherpesvirus of rhesus macaque (RM) monkeys that is closely related to human herpesvirus 8 (HHV-8)/Kaposi's Sarcoma-associated herpesvirus (KSHV), and it is capable of inducing diseases in simian immunodeficiency virus (SIV)-infected RM that are similar to those seen in humans coinfected with HIV and HHV-8. Both HHV-8 and RRV encode viral CD200 (vCD200) molecules that are homologues of cellular CD200, a membrane glycoprotein that regulates immune responses and helps maintain immune homeostasis via interactions with the CD200 receptor (CD200R). Though the functions of RRV and HHV-8 vCD200 molecules have been examined
in vitro
, the precise roles that these viral proteins play during
in vivo
infection remain unknown. Thus, to address the contributions of RRV vCD200 to immune regulation and disease
in vivo
, we generated a form of RRV that lacked expression of vCD200 for use in infection studies in RM. Our data indicated that RRV vCD200 expression limits immune responses against RRV at early times postinfection and also impacts viral loads, but it does not appear to have significant effects on disease development. Further, examination of the distribution pattern of CD200R in RM indicated that this receptor is expressed on a majority of cells in peripheral blood mononuclear cells, including B and T cells, suggesting potentially wider regulatory capabilities for both vCD200 and CD200 that are not strictly limited to myeloid lineage cells. In addition, we also demonstrate that RRV infection affects CD200R expression levels
in vivo
, although vCD200 expression does not play a role in this phenomenon.

IMPORTANCE
Cellular CD200 and its receptor, CD200R, compose a pathway that is important in regulating immune responses and is known to play a role in a variety of human diseases. A number of pathogens have been found to modulate the CD200-CD200R pathway during infection, including human herpesvirus 8 (HHV-8), the causative agent of Kaposi's sarcoma and B cell neoplasms in AIDS patients, and a closely related primate virus, rhesus macaque rhadinovirus (RRV), which infects and induces disease in rhesus macaque monkeys. HHV-8 and RRV encode homologues of CD200, termed vCD200, which are thought to play a role in preventing immune responses against these viruses. However, neither molecule has been studied in an
in vivo
model of infection to address their actual contributions to immunoregulation and disease. Here we report findings from our studies in which we analyzed the properties of a mutant form of RRV that lacks vCD200 expression in infected rhesus macaques.
The P4-P2′ Amino Acids Surrounding Human Norovirus Polyprotein Cleavage Sites Define the Core Sequence Regulating Self-Processing Order
Authors: May Jared , Viswanathan Prasanth , Ng Kenneth K.-S. , Medvedev Alexei , Korba Brent
Published: July 2014

Noroviruses (NoV) are members of the family
Caliciviridae
. The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with “early” sites (NS1/2-3 and NS3-4) being cleaved rapidly and three “late” sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2′ (P4-P2′) surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in
in vitro
transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (
k
cat
and
k
cat

/K
m

), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease.

IMPORTANCE
Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated that the core sequences spanning positions P4-P2′ surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This article builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.
Identification of Conserved Residues in Hepatitis C Virus Envelope Glycoprotein E2 That Modulate Virus Dependence on CD81 and SRB1 Entry Factors
Authors: Lavie Muriel , Sarrazin Stéphane , Montserret Roland , Descamps Véronique , Baumert Thomas F. , Duverlie Gilles , Séron Karin , Penin François , Dubuisson Jean
Published: July 2014

In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies.

IMPORTANCE
Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.
The Phosphatidylinositol 3-Kinase (PI3K) Isoform Dependence of Tumor Formation Is Determined by the Genetic Mode of PI3K Pathway Activation Rather than by Tissue Type
Authors: Utermark Tamara , Schmit Fabienne , Lee Sang Hyun , Gao Xueliang , Schaffhausen Brian S. , Roberts Thomas M.
Published: July 2014

Previous work has shown that prostate cancer in a
Pten
-null murine model is dependent on the p110β isoform of phosphatidylinositol 3-kinase (PI3K), while breast cancer driven by either polyoma middle T antigen (MT) or HER2 is p110α dependent. Whether these differences in isoform dependence arise from tissue specificity or from the nature of the oncogenic signal activating the PI3K pathway is important, given increasing interest in using isoform-specific PI3K inhibitors in cancer therapy. To approach this question, we studied the PI3K isoform dependence of our recently constructed prostate cancer model driven by MT. Since MT activates a number of signaling pathways, we first confirmed that the MT-driven prostate cancer model was actually dependent on PI3K. A newly generated transgenic prostate line expressing an MT allele (Y315F) known to be defective for PI3K binding displayed a markedly reduced ability to drive tumor formation. We next selectively ablated expression of either p110α or p110β in mice in which wild-type MT was expressed in the prostate. We found that tumor formation driven by MT was significantly delayed by the loss of
p110α
expression, while ablation of
p110β
had no effect. Since the tumor formation driven by MT is p110α dependent in the prostate as well as in the mammary gland, our data suggest that PI3K isoform dependence is driven by the mode of PI3K pathway activation rather than by tissue type.

IMPORTANCE
Middle T antigen (MT), the oncogene of polyomavirus, can drive tumor formation in a variety of cell types and tissues. Interestingly, MT has no intrinsic enzymatic activity but instead functions by binding and activating cellular signaling proteins. One of the most important of these is the lipid kinase PI3K, which was first studied in MT immunoprecipitates. Ubiquitously expressed PI3K comes in two major isoforms: p110α and p110β. Previous work in animal models showed that p110α was the key isoform in breast tumors driven by oncogenes, including MT and HER2, while p110β was key in prostate tumors driven by
Pten
loss. We asked the simple question of whether a prostate tumor driven by MT depends on p110α, which would suggest that the mode of activation determines p110 isoform dependence, or p110β, which would suggest that tissue type determines isoform dependence. The clear answer is that MT depends on p110α in both the prostate and breast.
Comprehensive High-Throughput RNA Sequencing Analysis Reveals Contamination of Multiple Nasopharyngeal Carcinoma Cell Lines with HeLa Cell Genomes
Authors: Strong Michael J. , Baddoo Melody , Nanbo Asuka , Xu Miao , Puetter Adriane , Lin Zhen
Published: July 2014

In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18
+
NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication.

IMPORTANCE
Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.