bluuebunny/crossref_metadata_2025 huggingface dataset:
The dynamic AMPA receptor extracellular region: a platform for synaptic protein interactions
Authors: García‐Nafría J. , Herguedas B. , Watson J. F. , Greger I. H.
Published: February 2016

AMPA receptors (AMPARs) are glutamate‐gated cation channels that mediate fast excitatory neurotransmission and synaptic plasticity. Structures of GluA2 homotetramers in distinct functional states, together with simulations, emphasise the loose architecture of the AMPAR extracellular region (ECR). The ECR encompasses ∼80% of the receptor, and consists of the membrane‐distal N‐terminal domain (NTD) and ligand‐binding domain (LBD), which is fused to the ion channel domain. Minimal contacts within and between layers, together with flexible peptide linkers connecting these three domains give rise to an organisation capable of dynamic rearrangements. This building plan is uniquely suited to engage interaction partners in the crowded environment of synapses, permitting the formation of new binding sites and the loss of existing ones. ECR motions are thereby expected to impact signalling as well as synaptic anchorage and may thereby influence AMPAR clustering during synaptic plasticity.

image
Airway tree reconstruction in expiration chest CT scans facilitated by information transfer from corresponding inspiration scans
Authors: Bauer Christian , Eberlein Michael , Beichel Reinhard R.
Published: February 2016

Purpose:

Analysis and comparison of airways imaged in pairs of inspiration and expiration lung CT scans provides important information for quantitative assessment of lung diseases like chronic obstructive pulmonary disease. However, airway tree reconstruction in expiration CT scans is a challenging problem. Typically, only a low number of airway branches are found in expiration scans, compared to inspiration scans. To detect more airways in expiration CT scans, the authors introduce a novel airway reconstruction approach and assess its performance.

Methods:

The method requires a pair of inspiration and expiration CT scans and utilizes information from the inspiration scan to facilitate reconstructing the airway tree in the expiration lung CT scan. First, an initial airway tree (high confidence) and airway candidates (limited confidence) are reconstructed in the expiration scan by utilizing a 3D graph‐based reconstruction method. Then, the 3D airway tree is reconstructed in the inspiration scan. Second, correspondences between expiration and inspiration tree structures are identified by utilizing a novel hierarchical tree matching algorithm that utilizes a local CT image‐based similarity criterion. Third, the tree information from the inspiration airway tree is used to select expiration candidates, resulting in the final expiration tree structure. The approach was evaluated on a diverse set of 40 scan pairs and compared to the baseline method, which utilizes only the expiration CT scan.

Results:

The proposed method produced a significant (p < 0.05) increase in airway tree length by 13.35 cm, on average, which represents an 11.21% increase relative to the baseline result using only the expiration CT scan. A detailed analysis of all additionally identified airways resulted in a true and false positive rate of 94.8% and 5.2%, respectively. The true positive rate was found to be significantly higher than the false positive rate (p < 0.05).

Conclusions:

The proposed method allowed increasing the number of found airways in expiration scans significantly. In addition, the algorithm establishes correspondence between inspiration and expiration airway trees, which can facilitate label transfer between airway trees and quantitative assessment of change in airways. The approach can be adapted to facilitate airway reconstruction in several longitudinal lung CT scans by means of mutual information transfer.
Going nuts: Measuring free-fall acceleration by analyzing the sound of falling metal pieces
Authors: Kuhn Jochen , Vogt Patrik , Theilmann Florian
Published: February 2016

Galilei presented the kinematics of a one-dimensional accelerated motion with ease and in terms of elegant geometry. Moreover, he believed, “Philosophy [i.e. physics] is written in this grand book—I mean the universe—which stands continually open to our gaze, but it cannot be understood unless one first learns to comprehend the language and interpret the characters in which it is written. It is written in the language of mathematics, and its characters are triangles, circles, and other geometrical figures, without which it is humanly impossible to understand a single word of it.” In classroom practice, however, it can be difficult to reveal this mathematical heart of nature; free fall and other accelerated motions often get obscured by friction or other sources of errors. In this paper, we introduce a method of analyzing free-fall motion indirectly by evaluating the noise of freely falling metal pieces. The method connects a deeper understanding of the mathematical structure of accelerated motion with the possibility to derive a numerical value for the free-fall acceleration g.
Seeded Amplification of Chronic Wasting Disease Prions in Nasal Brushings and Recto-anal Mucosa-Associated Lymphoid Tissues from Elk by Real-Time Quaking-Induced Conversion
Authors: Haley Nicholas J. , Siepker Chris , Hoon-Hanks Laura L. , Mitchell Gordon , Walter W. David , Manca Matteo , Monello Ryan J. , Powers Jenny G. , Wild Margaret A. , Hoover Edward A. , Caughey Byron , Richt Jürgen A.
Published: February 2016

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, was first documented nearly 50 years ago in Colorado and Wyoming and has since been detected across North America and the Republic of Korea. The expansion of this disease makes the development of sensitive diagnostic assays and antemortem sampling techniques crucial for the mitigation of its spread; this is especially true in cases of relocation/reintroduction or prevalence studies of large or protected herds, where depopulation may be contraindicated. This study evaluated the sensitivity of the real-time quaking-induced conversion (RT-QuIC) assay of recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens and nasal brushings collected antemortem. These findings were compared to results of immunohistochemistry (IHC) analysis of ante- and postmortem samples. RAMALT samples were collected from populations of farmed and free-ranging Rocky Mountain elk (
Cervus elaphus nelsoni
;
n
= 323), and nasal brush samples were collected from a subpopulation of these animals (
n
= 205). We hypothesized that the sensitivity of RT-QuIC would be comparable to that of IHC analysis of RAMALT and would correspond to that of IHC analysis of postmortem tissues. We found RAMALT sensitivity (77.3%) to be highly correlative between RT-QuIC and IHC analysis. Sensitivity was lower when testing nasal brushings (34%), though both RAMALT and nasal brush test sensitivities were dependent on both the
PRNP
genotype and disease progression determined by the obex score. These data suggest that RT-QuIC, like IHC analysis, is a relatively sensitive assay for detection of CWD prions in RAMALT biopsy specimens and, with further investigation, has potential for large-scale and rapid automated testing of antemortem samples for CWD.
Multicenter Assessment of Gram Stain Error Rates
Authors: Samuel Linoj P. , Balada-Llasat Joan-Miquel , Harrington Amanda , Cavagnolo Robert
Published: February 2016

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories.
Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains
Authors: Kanda Teru , Furuse Yuki , Oshitani Hitoshi , Kiyono Tohru
Published: February 2016

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death.

IMPORTANCE
Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases.
Hepatitis E Virus (HEV) Genotype 3 Infection of Human Liver Chimeric Mice as a Model for Chronic HEV Infection
Authors: van de Garde Martijn D. B. , Pas Suzan D. , van der Net Guido , de Man Robert A. , Osterhaus Albert D. M. E. , Haagmans Bart L. , Boonstra Andre , Vanwolleghem Thomas
Published: February 2016

Genotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities of
in vivo
HEV studies in a human liver chimeric mouse model (uPA
+/+
Nod-SCID-IL2Rγ
−/−
) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. These
in vitro
HEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 days
in vitro
. In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV both
in vitro
and
in vivo
. Persistent HEV gt3 infections in chimeric mice show preferential viral shedding toward mouse bile and feces, paralleling the course of infection in humans.

IMPORTANCE
Hepatitis E virus (HEV) genotype 3 infections are emerging in Western countries and are of great concern for immunosuppressed patients at risk for developing chronic HEV infection. Lack of adequate model systems for chronic HEV infection hampers studies on HEV infectivity and transmission and antiviral drugs. We compared the
in vivo
infectivity of clinical samples from chronic HEV patients in human liver chimeric mice to an
in vitro
virus culture system. Efficient
in vivo
HEV infection is observed after inoculation with feces- and liver-derived HEV but not with HEV RNA-containing plasma or cell culture supernatant. HEV in chimeric mice is preferentially shed toward bile and feces, mimicking the HEV infection course in humans. The observed
in vivo
infectivity differences may be relevant for the epidemiology of HEV in humans. This novel small-animal model therefore offers new avenues to unravel HEV's pathobiology.
Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation
Authors: Zhang Kuan , Brownlie Robert , Snider Marlene , van Drunen Littel-van den Hurk Sylvia
Published: February 2016

VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status.

IMPORTANCE
In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation.
A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4
+
T Cells
Authors: Offersen Rasmus , Nissen Sara Konstantin , Rasmussen Thomas A. , Østergaard Lars , Denton Paul W. , Søgaard Ole Schmeltz , Tolstrup Martin
Published: February 2016

Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a “shock-and-kill” approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703
ex vivo
exhibited increased secretion of interferon alpha (IFN-α) (
P
= 0.005) and CXCL10 (
P
= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56
dim
CD16
+
NK cells (
P
= 0.001) as well as higher proportions of CD107a-positive (
P
= 0.002) and IFN-γ-producing (
P
= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4
+
and CD8
+
T cells. Furthermore, CD4
+
T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (
P
= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4
+
T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (
P
= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4
+
T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials.

IMPORTANCE
We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel “dumbbell-shape” structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4
+
T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4
+
T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.
SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication
Authors: Su Chan-I , Tseng Chung-Hsin , Yu Chia-Yi , Lai Michael M. C.
Published: February 2016

Small ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular gene
Ubc9
, which encodes the sole E2-conjugating enzyme required for SUMOylation. By
in vivo
and
in vitro
SUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication.

IMPORTANCE
SUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.
Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity
Authors: Li Lian-Feng , Yu Jiahui , Li Yongfeng , Wang Jinghan , Li Su , Zhang Lingkai , Xia Shui-Li , Yang Qian , Wang Xiao , Yu Shaoxiong , Luo Yuzi , Sun Yuan , Zhu Yan , Munir Muhammad , Qiu Hua-Ji
Published: February 2016

Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione
S
-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.

IMPORTANCE
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.
The Attenuation Phenotype of a Ribavirin-Resistant Porcine Reproductive and Respiratory Syndrome Virus Is Maintained during Sequential Passages in Pigs
Authors: Khatun Amina , Shabir Nadeem , Seo Byoung-Joo , Kim Bum-Seok , Yoon Kyoung-Jin , Kim Won-Il
Published: February 2016

In a previous study, ribavirin-resistant porcine reproductive and respiratory syndrome virus (PRRSV) mutants (RVRp13 and RVRp22) were selected, and their resistance against random mutation was shown in cultured cells. In the present study, these ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability during three pig-to-pig passages in comparison with modified live virus (MLV) (Ingelvac PRRS MLV). Pigs challenged with RVRp22 had significantly lower (
P
< 0.05) viral loads in sera and tissues than pigs challenged with MLV or RVRp13 at the first passage, and the attenuated replication of RVRp22 was maintained until the third passage. Viral loads in sera and tissues dramatically increased in pigs challenged with MLV or RVRp13 during the second passage. Consistently, all five sequences associated with the attenuation of virulent PRRSV in RVRp13 and MLV quickly reverted to wild-type sequences during the passages, but two attenuation sequences were maintained in RVRp22 even after the third passage. In addition, RVRp22 showed a significantly lower (
P
< 0.001) mutation frequency in nsp2, which is one of the most variable regions in the PRRSV genome, than MLV. Nine unique mutations were found in open reading frames (ORFs) 1a, 2, and 6 in the RVRp22 genome based on full-length sequence comparisons with RVRp13, VR2332 (the parental virus of RVRp13 and RVRp22), and MLV. Based on these results, it was concluded that RVRp22 showed attenuated replication in pigs; further, because of the high genetic stability of RVRp22, its attenuated phenotype was stable even after three sequential passages in pigs.

IMPORTANCE
PRRSV is a rapidly evolving RNA virus. MLV vaccines are widely used to control PRRS; however, there have been serious concerns regarding the use of MLV as a vaccine virus due to the rapid reversion to virulence during replication in pigs. As previously reported, ribavirin is an effective antiviral drug against many RNA viruses. Ribavirin-resistant mutants reemerged by escaping lethal mutagenesis when the treatment concentration was sublethal, and those mutants were genetically more stable than parental viruses. In a previous study, two ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22) were selected, and their higher genetic stability was shown
in vitro
. Consequently, in the present study, both of the ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability
in vivo
. RVRp22 was found to exhibit higher genetic and phenotypic stability than MLV, and nine unique mutations were identified in the RVRp22 genome based on a full-length sequence comparison with the RVRp13, VR2332, and MLV genomes.
Viral Persistence Induces Antibody Inflation without Altering Antibody Avidity
Authors: Welten Suzanne P. M. , Redeker Anke , Toes René E. M. , Arens Ramon
Published: February 2016

Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4
+
T follicular helper cells (T
FH
) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses.

IMPORTANCE
Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4
+
T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.
Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line
Authors: Shabman Reed S. , Shrivastava Susmita , Tsibane Tshidi , Attie Oliver , Jayaprakash Anitha , Mire Chad E. , Dilley Kari E. , Puri Vinita , Stockwell Timothy B. , Geisbert Thomas W. , Sachidanandam Ravi , Basler Christopher F.
Published: February 2016

Bats are of significant interest as reservoirs for zoonotic viral pathogens; however, tools to dissect bat-virus interactions are limited in availability. This study serendipitously identified, in an established bat cell line, a fully replication-competent gammaherpesvirus; determined the complete genome sequence of the virus; and generated a viral transcript map. This virus can replicate in select human and nonhuman primate cell lines. However, analyses of viral sequences support a bat origin for this virus; we therefore refer to the virus as bat gammaherpesvirus 8 (BGHV8). The viral genome contains unique open reading frames that likely encode modulators of bat innate and adaptive immune signaling pathways and expresses viral miRNAs. The virus and its gene products should provide a unique tool to dissect both bat and gammaherpesvirus biology.
Fighting Ebola in Sierra Leone: a view from the frontline
Authors: Owens Stephen , Leyland Thomas
Published: February 2016

The largest and most complex Ebola epidemic in history is believed to have started with the infection of a 2-year-old boy in South-eastern Guinea in late 2013. Within a year, thousands of children and their families had contracted the virus, many had died and many more were orphaned. We reflect on our experiences of volunteering at the Kerry Town Ebola Treatment Centre in Sierra Leone between January and February 2015, where we were deployed to care for just a few of these children as part of the Save The Children team.
Muscle injury is the principal injury type and hamstring muscle injury is the first injury diagnosis during top-level international athletics championships between 2007 and 2015
Authors: Edouard Pascal , Branco Pedro , Alonso Juan-Manuel
Published: February 2016

Background

During top-level international athletics championships, muscle injuries are frequent.

Objective

To analyse the incidence and characteristics of muscle injuries and hamstring muscle injuries (hamstring injuries) occurring during top-level international athletics championships.

Methods

During 16 international championships held between 2007 and 2015, national medical team and local organising committee physicians reported daily all injuries on a standardised injury report form. Only muscle injuries (muscle tears and muscle cramps) and hamstring injuries have been analysed.

Results

40.9% of all recorded injuries (n=720) were muscle injuries, with 57.5% of them resulting in time loss. The overall incidence of muscle injuries was higher in male athletes than female athletes (51.9±6.0 vs 30.3±5.0 injuries per 1000 registered athletes, respectively; RR=1.71; 95% CI 1.45 to 2.01). Muscle injuries mainly affected the thigh (52.9%) and lower leg (20.1%), and were mostly caused by overuse with sudden onset (38.2%) and non-contact trauma (24.6%). Muscle injury risk varied according to the event groups. Hamstring injuries represented 17.1% of all injuries, with a higher risk in male compared to female athletes (22.4±3.4 vs 11.5±2.6 injuries per 1000 registered athletes, respectively; RR=1.94; 95% CI 1.42 to 2.66).

Conclusions

During international athletics championships, muscle injury is the principal type of injury, and among those, the hamstring is the most commonly affected, with a two times higher risk in male than female athletes. Athletes in explosive power events, male athletes and older male athletes, in specific were more at risk of muscle injuries and hamstring injuries. Injury prevention strategies should be sex-specific.
Clinical outcomes in 995 unselected real-world patients treated with an ultrathin biodegradable polymer-coated sirolimus-eluting stent: 12-month results from the FLEX Registry
Authors: Lemos Pedro A , Chandwani Prakash , Saxena Sudheer , Ramachandran Padma Kumar , Abhyankar Atul , Campos Carlos M , Marchini Julio Flavio , Galon Micheli Zanotti , Verma Puneet , Sandhu Manjinder Singh , Parikh Nikhil , Bhupali Ashok , Jain Sharad , Prajapati Jayesh
Published: February 2016

Objectives

To evaluate, in the FLEX Registry, clinical outcomes of an ultrathin (60 µm) biodegradable polymer-coated Supraflex sirolimus-eluting stent (SES) for the treatment of coronary artery disease. Additionally, to determine the vascular response to the Supraflex SES through optical coherence tomography (OCT) analysis.

Setting

Multicentre, single-arm, all-comers, observational registry of patients who were treated with the Supraflex SES, between July 2013 and May 2014, at nine different centres in India.

Participants

995 patients (1242 lesions) who were treated with the Supraflex SES, between July 2013 and May 2014, at nine different centres in India. A total of 47 participants underwent OCT analysis at 6 months’ follow-up.

Interventions

Percutaneous coronary intervention with Supraflex SES,

Primary and secondary outcome measures

The primary endpoint—the rate of major adverse cardiac events (defined as a composite of cardiac death, myocardial infarction (MI), target lesion revascularisation (TLR))—was analysed during 12 months.

Results

At 12 months, the primary endpoint occurred in 36 (3.7%) of 980 patients, consisting of 18 (1.8%) cardiac deaths, 16 (1.6%) MI, 7 (0.7%) TLR and 2 (0.2%) cases of non-target lesion target vessel revascularization. In a subset of 47 patients, 1227 cross-sections (9309 struts) were analysed at 6 months by OCT. Overall, a high percentage of struts was covered (98.1%), with a mean neointimal thickness of 0.13±0.06 µm.

Conclusions

The FLEX Registry evaluated clinical outcomes in real-world and more complex cohorts and thus provides evidence that the Supraflex SEX can be used safely and routinely in a broader percutaneous coronary intervention population. Also, the Supraflex SES showed high percentage of stent strut coverage and good stent apposition during OCT follow-up.
ASPECTS discrepancies between CT and MR imaging: analysis and implications for triage protocols in acute ischemic stroke
Authors: Hui Ferdinand K , Obuchowski Nancy A , John Seby , Toth Gabor , Katzan Irene , Wisco Dolora , Cheng-Ching Esteban , Uchino Ken , Man Shu-Mei , Hussain Shazam
Published: February 2016

Background

Optimal imaging triage for intervention for large vessel occlusions remains unclear. MR-based imaging provides ischemic core volumes at the cost of increased imaging time. CT Alberta Stroke Program Early CT Score (ASPECTS) estimates are faster, but may be less sensitive.

Objective

To assesses the rate at which MRI changed management in comparison with CT imaging alone.

Methods

Retrospective analysis of patients with acute ischemic stroke undergoing imaging triage for endovascular therapy was performed between 2008 and 2013. Univariate and multivariate analyses were performed. Multivariate logistic regression was used to evaluate the effect of time on disagreement in MRI and CT ASPECTS scores.

Results

A total of 241 patients underwent both diffusion-weighted imaging (DWI) and CT. Six patients with DWI ASPECTS ≥6 and CT ASPECTS <6 were omitted, leaving 235 patients. For 47 patients, disagreement between the two modalities resulted in different treatment recommendations. The estimated probability of disagreement was 20.0% (95% CI 15.4% to 25.6%). In a multivariate logistic regression, CT ASPECTS >7 (p=0.004) and admission National Institutes of Health Stroke Scale (NIHSS) score <16 (p=0.008) were simultaneously significant predictors of agreement in ASPECTS. The time between modalities was a marginally significant predictor (p=0.080).

Conclusions

The study suggests that patients with NIHSS scores at admission of <16 and patients with CT ASPECTS >7 have a higher likelihood of agreement between CT and DWI based on an ASPECTS cut-off value of 6. Additional MRI for triage in patients with NIHSS at admission of >16, and ASPECTS of 6 or 7 may be more likely to change management. Unsurprisingly, patients with low CT ASPECTS had good correlation with MRI ASPECTS.
Time to redefine success? TICI 3 versus TICI 2b recanalization in middle cerebral artery occlusion treated with thrombectomy
Authors: Kleine Justus F , Wunderlich Silke , Zimmer Claus , Kaesmacher Johannes
Published: February 2016

Background

The Thrombolysis in Cerebral Infarction (TICI) scale is the most widely applied scoring system to grade technical results of recanalizing therapies in acute ischemic stroke (AIS). TICI 2b and TICI 3 are conventionally subsumed as ‘successful recanalization’. Previous studies reported conflicting results for the clinical relevance of achieving complete (TICI 3) versus ‘almost’ complete reperfusion (TICI 2b).

Objective

To examine if neurologic outcome differs significantly between TICI 2b and TICI 3 in patients with AIS with middle cerebral artery (MCA) occlusion treated ‘successfully’ with mechanical thrombectomy (MTE).

Methods

Retrospective analysis of prospectively collected data from 352 consecutive patients with isolated MCA occlusion subjected to MTE between January 2007 and July 2015.

Results

262 of the 277 successfully treated patients had adequate follow-up and were included. Patients (n=119) in the TICI 3 group had a lower National Institutes of Health Stroke Scale score at discharge (NIHSS-DIS; median 5 vs 7, p=0.005), and showed higher rates of strong neurologic improvement (ΔNIHSS≥8 or NIHSS-DIS≤1, 68.4% vs 37.1%, p=0.002) and favorable NIHSS outcome (NIHSS-DIS≤5, 49.2% vs 31.9%, p=0.005). Hospital stays were shorter in the TICI 3 group (median 10 vs 12 days, p=0.014). After adjusting for relevant baseline and treatment parameters, TICI 3 was independently associated with strong neurologic improvement (OR=4.3, 95% CI 2.2 to 8.3, p<0.001) and favorable NIHSS outcome (OR=3.0, 95% CI 1.5 to 6.3, p=0.003).

Conclusions

Neurologic outcome is substantially better in TICI 3 than TICI 2b patients, and hospital stays are shorter. Endovascular strategies that consequently strive to achieve TICI 3 may be warranted and cost-effective, and should be examined by future research. TICI 3 rates should be included as a safety measure in studies evaluating MTE devices and techniques.
Passion for poultry
Authors: Garton William
Published: February 2016

William Garton's interest in poultry began when he was a boy. Despite trying many aspects of veterinary medicine as a student, it was poultry‐specific work that he enjoyed most. As a poultry intern with the Minster Veterinary Practice he wrote a monthly blog for Vet Record Careers, and he is now associate poultry director for the practice's north‐west branches
Gentiopicroside and sweroside from Veratrilla baillonii Franch. induce phosphorylation of Akt and suppress Pck1 expression in hepatoma cells
Authors: Huang Xian-Ju , Li Jun , Mei Zhi-Yi , Chen Guoxun
Published: February 2016

The use of phytochemicals and herbal medicines has accompanied human history. Advances in modern biomedical sciences have allowed us to investigate the functional mechanisms of herbal medicines and phytochemicals. Veratrilla baillonii Franch. has long been used as a medicinal herb in southwestern China. Here, we analyzed the effects of an ethanol extract from V. baillonii (VBFE) on the expression levels of the cytosolic form of the phosphoenolpyruvate carboxykinase gene (Pck1) mRNA and components of the insulin signalling cascade in HL1C hepatoma cells. Compared with the insulin control, VBFE treatment inhibited the expression of Pck1 mRNA in a dose-dependent manner. This was associated with the phosphorylation of Akt and Erk1/2 in a time-dependent manner. Further analysis of the purified components of VBFE indicated that gentiopicroside and sweroside from VBFE, alone and in combination, suppressed Pck1 expression and induced Akt and Erk1/2 phosphorylation. In conclusion, gentiopicroside and sweroside suppress Pck1 expression and induce phosphorylation of components in the insulin signalling cascade. This is the first study to demonstrate that gentiopicroside and sweroside show insulin-mimicking effects on the regulation of Pck1 expression. Further studies are warranted to explore the potential of gentiopicroside and sweroside in the control of blood glucose in animals.
Carbon isotope (δ13Ccarb) and facies variability at the Wenlock–Ludlow boundary (Silurian) of the Midland Platform, UK
Authors: Blain John A. , Ray David C. , Wheeley James R.
Published: February 2016

The Wenlock–Ludlow series boundary (Silurian) has been recognized as a time of pronounced sea-level rise and the end of a globally recognized Late Homerian Stage (Mulde) positive carbon isotope excursion (CIE). However, the precise timing and synchronicity of the end of the excursion with respect to the Wenlock–Ludlow boundary is debated. Within the type Wenlock and Ludlow areas (UK), high-resolution δ13Ccarb isotope data are presented across the Wenlock–Ludlow boundary, and within a range of carbonate platform settings. Correlation between sections and depositional settings has been based upon the characteristics of high-order sea-level fluctuations (parasequences). Comparisons between parasequence-bounded δ13Ccarb values reveal clear spatial variations, with lighter values recorded from more distal settings and heavier values from shallower settings. Temporal variations in the δ13Ccarb values are also documented and appear to reflect local variations in carbonate provenance and productivity in response to sea-level rise. While δ13Ccarb values converge in all sections towards the Wenlock–Ludlow boundary, the apparent end of the Mulde CIE appears diachronous and is progressively older within more distal settings.
The effects of grey seal predation and commercial fishing on the recovery of a depleted cod stock
Authors: Cook Robin M. , Trijoulet Vanessa
Published: February 2016

Cod (Gadus morhua) are preyed upon by grey seals (Halichoerus grypus), and there is debate over the impact this has had on the decline of stocks and their prospects for recovery. We analysed a depleted stock to the West of Scotland and show that seal predation rate is consistent with a type II functional response. Forward projections of a model including the functional response under varying levels of fishing and seal population size suggest that stock recovery is possible under current conditions, but there is a modest probability that the stock will decline further in both the short and long term. The potential recovery is fragile and sensitive to relatively small increases in either fishing or seal predation. Forward projection models that exclude the functional response estimate a lower probability of stock decline and may underestimate the risk to the stock. At low stock sizes and high fishing mortality rates, functional response models project slower recovery but the opposite is true at low fishing mortality.
Formation of ground and excited hydrogen atoms in proton–rubidium inelastic scattering
Authors: Elkilany S.A.
Published: February 2016

Inelastic collisions of protons with rubidium atoms are treated for the first time within the framework of the three channel coupled static, and frozen core approximations. The method is used for calculating partial and total cross sections with the assumption that only three channels (elastic; non-excited hydrogen, 1s-state; and excited hydrogen, 2s-state) are open. We have used the Lipmann–Schwinger equation and the Green’s functions iterative numerical method technique to solve the derived coupled integro-differential equations to obtain the computer code. The present results for total hydrogen formation cross sections are in agreement with results of other available ones in a wide range of incident energy.