bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Iron Cross-Section in Thermal Region on Neutron Transport in VVER-1000 Mock-Up in LR-0 Reactor
Authors: Schulc Martin , Košťál Michal , Harutyunyan Davit , Švadlenková Marie , Rypar Vojtěch , Milčák Ján , Kolros Antonín
Published: September 2016

The iron cross-section in thermal regions influences the thermal neutron flux prediction in steel structural components of reactors and also in regions adjoining them. The thermal neutron flux level is proportional to pin power density in fuel. This quantity is an important criterion reflected in limits and conditions of reactor operation. The new power density evaluation shows notable, well distinguishable discrepancy between calculations realized using the CENDL-3.1 nuclear data library and experimentally determined pin power density in boundary rows of pins. All experiments were carried out in a water–water energetic reactor (VVER-1000) transport mock-up placed in the LR-0 reactor.
Discrimination of frequency modulated sweeps by mice
Authors: Screven Laurel A. , Dent Micheal L.
Published: September 2016

Mice often produce ultrasonic vocalizations (USVs) that sweep upwards in frequency from around 60 to around 80 kHz and downwards in frequency from 80 to 60 kHz. Whether or not these USVs are used for communication purposes is still unknown. Here, mice were trained and tested using operant conditioning procedures and positive reinforcement to discriminate between synthetic upsweeps and downsweeps. The stimuli varied in bandwidth, duration, and direction of sweep. The mice performed significantly worse when discriminating between background and test stimuli when the stimuli all occupied the same bandwidths. Further, the mice's discrimination performance became much worse for stimuli that had durations similar to those natural vocalizations of the mice. Sweeps composed of different frequency ranges and longer durations had improved discrimination. These results collected using artificial stimuli created to mimic natural USVs indicate that the bandwidth of the vocalizations may be much more important for communication than the frequency contours of the vocalizations.
Tree growth acceleration and expansion of alpine forests: The synergistic effect of atmospheric and edaphic change
Authors: Silva Lucas C. R. , Sun Geng , Zhu-Barker Xia , Liang Qianlong , Wu Ning , Horwath William R.
Published: September 2016

Soil-plant-atmosphere interactions regulate the impact of climate on forest ecosystems.
Loss-of-function variants influence the human serum metabolome
Authors: Yu Bing , Li Alexander H. , Metcalf Ginger A. , Muzny Donna M. , Morrison Alanna C. , White Simon , Mosley Thomas H. , Gibbs Richard A. , Boerwinkle Eric
Published: September 2016

Loss-of-function variants, which often lead to greatly truncated protein product, influence human metabolite levels.
Genetically engineered crops and pesticide use in U.S. maize and soybeans
Authors: Perry Edward D. , Ciliberto Federico , Hennessy David A. , Moschini GianCarlo
Published: September 2016

The impact on pesticide use of genetically engineered maize and soybean varieties has changed over time.
NASA to launch asteroid-sampling mission
Authors: Voosen Paul
Published: September 2016

OSIRIS-REx plans to return more than 60 grams of dust from carbon-rich Bennu.
London's biomedical behemoth opens its doors
Authors: Stokstad Erik
Published: September 2016

The £650 million Francis Crick Institute aims to foster collaboration—but Brexit casts a shadow.
A painstaking overhaul for cardiac safety testing
Authors: Servick Kelly
Published: September 2016

New methods may better predict arrhythmia risk in vitro.
Second chapter
Authors: Couzin-Frankel Jennifer
Published: September 2016

T cell therapy has successfully targeted blood cancers. A bigger challenge is to make it work on solid tumors.
Ligand-accelerated enantioselective methylene C(sp
3
)–H bond activation
Authors: Chen Gang , Gong Wei , Zhuang Zhe , Andrä Michal S. , Chen Yan-Qiao , Hong Xin , Yang Yun-Fang , Liu Tao , Houk K. N. , Yu Jin-Quan
Published: September 2016

Effective differentiation of prochiral carbon–hydrogen (C–H) bonds on a single methylene carbon via asymmetric metal insertion remains a challenge. Here, we report the discovery of chiral acetyl-protected aminoethyl quinoline ligands that enable asymmetric palladium insertion into prochiral C–H bonds on a single methylene carbon center. We apply these palladium complexes to catalytic enantioselective functionalization of β-methylene C–H bonds in aliphatic amides. Using bidentate ligands to accelerate C–H activation of otherwise unreactive monodentate substrates is crucial for outcompeting the background reaction driven by substrate-directed cyclopalladation, thereby avoiding erosion of enantioselectivity. The potential of ligand acceleration in C–H activation is also demonstrated by enantioselective β-C–H arylation of simple carboxylic acids without installing directing groups.
A highly active and stable IrO

x

/SrIrO
3
catalyst for the oxygen evolution reaction
Authors: Seitz Linsey C. , Dickens Colin F. , Nishio Kazunori , Hikita Yasuyuki , Montoya Joseph , Doyle Andrew , Kirk Charlotte , Vojvodic Aleksandra , Hwang Harold Y. , Norskov Jens K. , Jaramillo Thomas F.
Published: September 2016

Oxygen electrochemistry plays a key role in renewable energy technologies such as fuel cells and electrolyzers, but the slow kinetics of the oxygen evolution reaction (OER) limit the performance and commercialization of such devices. Here we report an iridium oxide/strontium iridium oxide (IrO

x

/SrIrO
3
) catalyst formed during electrochemical testing by strontium leaching from surface layers of thin films of SrIrO
3
. This catalyst has demonstrated specific activity at 10 milliamps per square centimeter of oxide catalyst (OER current normalized to catalyst surface area), with only 270 to 290 millivolts of overpotential for 30 hours of continuous testing in acidic electrolyte. Density functional theory calculations suggest the formation of highly active surface layers during strontium leaching with IrO
3
or anatase IrO
2
motifs. The IrO

x

/SrIrO
3
catalyst outperforms known IrO

x

and ruthenium oxide (RuO

x

) systems, the only other OER catalysts that have reasonable activity in acidic electrolyte.
Radiative human body cooling by nanoporous polyethylene textile
Authors: Hsu Po-Chun , Song Alex Y. , Catrysse Peter B. , Liu Chong , Peng Yucan , Xie Jin , Fan Shanhui , Cui Yi
Published: September 2016

Thermal management through personal heating and cooling is a strategy by which to expand indoor temperature setpoint range for large energy saving. We show that nanoporous polyethylene (nanoPE) is transparent to mid-infrared human body radiation but opaque to visible light because of the pore size distribution (50 to 1000 nanometers). We processed the material to develop a textile that promotes effective radiative cooling while still having sufficient air permeability, water-wicking rate, and mechanical strength for wearability. We developed a device to simulate skin temperature that shows temperatures 2.7° and 2.0°C lower when covered with nanoPE cloth and with processed nanoPE cloth, respectively, than when covered with cotton. Our processed nanoPE is an effective and scalable textile for personal thermal management.
Plant development regulated by cytokinin sinks
Authors: Zürcher Evelyne , Liu Jingchun , di Donato Martin , Geisler Markus , Müller Bruno
Published: September 2016

Morphogenetic signals control the patterning of multicellular organisms. Cytokinins are mobile signals that are perceived by subsets of plant cells. We found that the responses to cytokinin signaling during
Arabidopsis
development are constrained by the transporter PURINE PERMEASE 14 (PUP14). In our experiments, the expression of
PUP14
was inversely correlated to the cytokinin signaling readout. Loss of
PUP14
function allowed ectopic cytokinin signaling accompanied by aberrant morphogenesis in embryos, roots, and the shoot apical meristem. PUP14 protein localized to the plasma membrane and imported bioactive cytokinins, thus depleting apoplastic cytokinin pools and inhibiting perception by plasma membrane–localized cytokinin sensors to create a sink for active ligands. We propose that the spatiotemporal cytokinin sink patterns established by PUP14 determine the cytokinin signaling landscape that shapes the morphogenesis of land plants.
Effects of a warming Arctic
Authors: Shepherd Theodore G.
Published: September 2016

It remains unclear to what extent the warming Arctic affects climate and weather extremes at lower latitudes
The great debate
Fact and Fiction in Global Energy Policy 15 Contentious Questions
Benjamin K. Sovacool, Marilyn A. Brown, and Scott V. Valentine
Johns Hopkins University Press, 2016. 387 pp.
Authors: Wong-Parodi Gabrielle
Published: September 2016

Competing perspectives face off in a quest to find common ground on controversial energy issues
Remembering 9/11
Who Owns the Dead? The Science and Politics of Death at Ground Zero
Jay D. Aronson
Harvard University Press, 2016. 326 pp.
Authors: Black Sue
Published: September 2016

A historian probes the bitter conflicts that accompanied America's most costly forensic investigation
Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study
Authors: Riddle Mark S. , Kaminski Robert W. , Di Paolo Claudio , Porter Chad K. , Gutierrez Ramiro L. , Clarkson Kristen A. , Weerts Hailey E. , Duplessis Christopher , Castellano Amy , Alaimo Cristina , Paolino Kristopher , Gormley Robert , Gambillara Fonck Veronica
Published: September 2016

Several candidate vaccines against
Shigella
spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered
Shigella
vaccine development over the past several decades. Bioconjugation technology, exploited here for an
Shigella flexneri
2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel
S. flexneri
2a bioconjugate vaccine made of the polysaccharide component of the
S. flexneri
2a O-antigen, conjugated to the exotoxin protein A of
Pseudomonas aeruginosa
(EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 μg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant
S. flexneri
2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against
S. flexneri
2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to
S. flexneri
2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.)
Impact of Delays between Clinical and Laboratory Standards Institute and Food and Drug Administration Revisions of Interpretive Criteria for Carbapenem-Resistant Enterobacteriaceae
Authors: Bartsch Sarah M. , Huang Susan S. , Wong Kim F. , Slayton Rachel B. , McKinnell James A. , Sahm Daniel F. , Kazmierczak Krystyna , Mueller Leslie E. , Jernigan John A. , Lee Bruce Y.
Published: September 2016

Delays often occur between CLSI and FDA revisions of antimicrobial interpretive criteria. Using our Regional Healthcare Ecosystem Analyst (RHEA) simulation model, we found that the 32-month delay in changing carbapenem-resistant
Enterobacteriaceae
(CRE) breakpoints might have resulted in 1,821 additional carriers in Orange County, CA, an outcome that could have been avoided by identifying CRE and initiating contact precautions. Policy makers should aim to minimize the delay in the adoption of new breakpoints for antimicrobials against emerging pathogens when containment of spread is paramount; delays of <1.5 years are ideal.
Alternative Molecular Methods for Improved Detection of Meningococcal Carriage and Measurement of Bacterial Density
Authors: Manigart Olivier , Okeakpu Jacinta , Odutola Aderonke , Jarju Sheikh , Foster-Nyarko Ebenezer , Diallo Kanny , Roca Anna , Kampmann Beate , D'Alessandro Umberto , Sow Samba , Antonio Martin , Maiden Martin J. , Borrow Ray , Stuart James M. , Trotter Caroline L. , Greenwood Brian M.
Published: September 2016

Conventional methods for detecting pharyngeal carriage of
Neisseria meningitidis
are complex. There is a need for simpler methods with improved performance. We have investigated two alternative approaches. Three pharyngeal swabs were collected from 999 pupils aged 10 to 18 years in The Gambia. Carriage of
N. meningitidis
was investigated by using three different methods: (i) plating on Thayer-Martin selective medium and testing by conventional microbiological methods followed by PCR testing; (ii) seeding in Todd-Hewitt broth (THB) and, after culture overnight, testing by PCR; and (iii) compression of the swab on filter paper and, after DNA concentration, testing by PCR. PCR after culture in THB was more than twice as sensitive as conventional methods in detecting
N. meningitidis
(13.2% versus 5.7%;
P
< 0.0001). PCR after DNA extraction from filter paper had a sensitivity similar to that of conventional methods (4.9% versus 5.7%;
P
= 0.33). Capsular genogroups detected by broth culture were genogroups W (21 isolates), B (12 isolates), Y (8 isolates), E (3 isolates), and X (2 isolates), and 68 meningococci had the capsule-null intergenic region. The distributions of genogroups and of capsule-null organisms were similar with each of the three methods. The carriage density in samples extracted from filter paper ranged from 1 to 25,000 DNA copies. PCR of broth cultures grown overnight doubled the yield of
N. meningitidis
carriage isolates compared with conventional methods. This approach could improve the efficiency of carriage studies. Collection on filter paper followed by quantitative PCR could be useful for density measurement and for carriage studies in areas with limited resources.
Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics
Authors: Scherer Julian , Yaffe Zachary A. , Vershinin Michael , Enquist Lynn W.
Published: September 2016

Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins.

IMPORTANCE
Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.
Residues K465 and G467 within the Cytoplasmic Domain of GP2 Play a Critical Role in the Persistence of Lymphocytic Choriomeningitis Virus in Mice
Authors: Iwasaki Masaharu , Ng Cherie T. , Cubitt Beatrice , de la Torre Juan C.
Published: September 2016

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose serious public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. We have documented that a recombinant LCMV containing the glycoprotein (GPC) gene of LASV within the backbone of the immunosuppressive clone 13 (Cl-13) variant of the Armstrong strain of LCMV (rCl-13/LASV-GPC) exhibited Cl-13-like growth properties in cultured cells, but in contrast to Cl-13, rCl-13/LASV-GPC was unable to establish persistence in immunocompetent adult mice, which prevented its use for some
in vivo
experiments. Recently, V459K and K461G mutations within the GP2 cytoplasmic domain (CD) of rCl-13/LASV-GPC were shown to increase rCl-13/LASV-GPC infectivity in mice. Here, we generated rCl-13(GPC/VGKS) by introducing the corresponding revertant mutations K465V and G467K within GP2 of rCl-13 and we show that rCl-13(GPC/VGKS) was unable to persist in mice. K465V and G467K mutations did not affect GPC processing, virus RNA replication, or gene expression. In addition, rCl-13(GPC/VGKS) grew to high titers in cultured cell lines and in immunodeficient mice. Further analysis revealed that rCl-13(GPC/VGKS) infected fewer splenic plasmacytoid dendritic cells than rCl-13, yet the two viruses induced similar type I interferon responses in mice. Our findings have identified novel viral determinants of Cl-13 persistence and also revealed that virus GPC-host interactions yet to be elucidated critically contribute to Cl-13 persistence.

IMPORTANCE
The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), provides investigators with a superb experimental model system to investigate virus-host interactions. The Armstrong strain (ARM) of LCMV causes an acute infection, whereas its derivative, clone 13 (Cl-13), causes a persistent infection. Mutations F260L and K1079Q within GP1 and L polymerase, respectively, have been shown to play critical roles in Cl-13's ability to persist in mice. However, there is an overall lack of knowledge about other viral determinants required for Cl-13's persistence. Here, we report that mutations K465V and G467K within the cytoplasmic domain of Cl-13 GP2 resulted in a virus, rCl-13(GPC/VGKS), that failed to persist in mice despite exhibiting Cl-13 wild-type-like fitness in cultured cells and immunocompromised mice. This finding has uncovered novel viral determinants of viral persistence, and a detailed characterization of rCl-13(GPC/VGKS) can provide novel insights into the mechanisms underlying persistent viral infection.
Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site
Authors: Wibmer Constantinos Kurt , Gorman Jason , Anthony Colin S. , Mkhize Nonhlanhla N. , Druz Aliaksandr , York Talita , Schmidt Stephen D. , Labuschagne Phillip , Louder Mark K. , Bailer Robert T. , Abdool Karim Salim S. , Mascola John R. , Williamson Carolyn , Moore Penny L. , Kwong Peter D. , Morris Lynn
Published: September 2016

All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.

IMPORTANCE
The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site.
Multiple Roles of the Cytoplasmic Domain of Herpes Simplex Virus 1 Envelope Glycoprotein D in Infected Cells
Authors: Arii Jun , Shindo Keiko , Koyanagi Naoto , Kato Akihisa , Kawaguchi Yasushi
Published: September 2016

Herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) plays an essential role in viral entry. The functional regions of gD responsible for viral entry have been mapped to its extracellular domain, whereas the gD cytoplasmic domain plays no obvious role in viral entry. Thus far, the role(s) of the gD cytoplasmic domain in HSV-1 replication has remained to be elucidated. In this study, we show that ectopic expression of gD induces microvillus-like tubular structures at the plasma membrane which resemble the reported projection structures of the plasma membrane induced in HSV-1-infected cells. Mutations in the arginine cluster (residues 365 to 367) in the gD cytoplasmic domain greatly reduced gD-induced plasma membrane remodeling. In agreement with this, the mutations in the arginine cluster in the gD cytoplasmic domain reduced the number of microvillus-like tubular structures at the plasma membrane in HSV-1-infected cells. In addition, the mutations produced an accumulation of unenveloped nucleocapsids in the cytoplasm and reduced viral replication and cell-cell spread. These results suggest that the arginine cluster in the gD cytoplasmic domain is required for the efficient induction of plasma membrane projections and viral final envelopment, and these functions of the gD domain may lead to efficient viral replication and cell-cell spread.

IMPORTANCE
The cytoplasmic domain of HSV-1 gD, an envelope glycoprotein essential for viral entry, was reported to promote viral replication and cell-cell spread, but the role(s) of the domain during HSV-1 infection has remained unknown. In this study, we clarify two functions of the arginine cluster in the HSV-1 gD cytoplasmic domain, both of which require host cell membrane remodeling, i.e., the formation of microvillus-like projections at the plasma membrane and viral final envelopment in HSV-1-infected cells. We also show that the gD arginine cluster is required for efficient HSV-1 replication and cell-cell spread. This is the first report clarifying not only the functions of the gD cytoplasmic domain but also identifying the gD arginine cluster to be the HSV-1 factor responsible for the induction of plasma membrane projections in HSV-1-infected cells. Our results elucidate some of the functions of this multifunctional envelope glycoprotein during HSV-1 infection.
Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid
Authors: Severin Chelsea , Terrell James R. , Zengel James R. , Cox Robert , Plemper Richard K. , He Biao , Luo Ming
Published: September 2016

In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the α-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA.

IMPORTANCE
Mumps virus (MuV) infection may cause serious diseases, including hearing loss, orchitis, oophoritis, mastitis, and pancreatitis. MuV is a negative-strand RNA virus, similar to rabies virus or Ebola virus, that has a unique mechanism of viral RNA synthesis. They all make their own RNA-dependent RNA polymerase (RdRp). The viral RdRp uses the genomic RNA inside the viral nucleocapsid as the template to synthesize viral RNAs. Since the template RNA is always sequestered in the nucleocapsid, the viral RdRp must find a way to open it up in order to gain access to the covered template. Our work reported here shows that a helix structural element in the MuV nucleocapsid protein becomes open when the sequestered RNA is released. The amino acids related to this helix are required for RdRp to synthesize viral RNA. We propose that the viral RdRp pulls this helix open to release the genomic RNA.
Visualizing Herpesvirus Procapsids in Living Cells
Authors: Maier Oana , Sollars Patricia J. , Pickard Gary E. , Smith Gregory A.
Published: September 2016

A complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components is present on the initial capsid assembly, the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded green fluorescent protein (GFP) fused in frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kinetics
in vitro
and approximated wild-type virulence
in vivo
. The fusion also proved to be well tolerated in herpes simplex virus. Viruses encoding GFP-VP24, along with a traditional capsid reporter fusion (pUL25/mCherry), demonstrated that GFP-VP24 was a reliable capsid marker and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection and monitoring of capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation.

IMPORTANCE
The family
Herpesviridae
consists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share structurally related icosahedral capsids that encase the double-stranded DNA (dsDNA) viral genome. The dynamics of capsid assembly and maturation have been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection.
Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis
Authors: Harrison Kate , Haga Ismar R. , Pechenick Jowers Tali , Jasim Seema , Cintrat Jean-Christophe , Gillet Daniel , Schmitt-John Thomas , Digard Paul , Beard Philippa M.
Published: September 2016

Poxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growth
in vitro
and reduced weight loss and clinical signs in an
in vivo
murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.

IMPORTANCE
Cellular retrograde transport pathways traffic cargo from endosomes to the
trans
-Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small-molecule inhibitors reduced the replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the wrapping step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of antipoxvirus drugs.
Sindbis Virus Can Exploit a Host Antiviral Protein To Evade Immune Surveillance
Authors: Wang Xinlu , Li Melody M. H. , Zhao Jing , Li Shenglan , MacDonald Margaret R. , Rice Charles M. , Gao Xiang , Gao Guangxia
Published: September 2016

Viral infection induces production of type I interferons (IFNs), which stimulate the expression of a variety of antiviral factors to inhibit viral replication. To establish effective infection, viruses need to develop strategies to evade the immune responses. A neurovirulent Sindbis virus strain with neuroinvasive properties (SVNI) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc finger antiviral protein (ZAP), a host factor that specifically inhibits the replication of certain viruses by binding to the viral mRNAs, repressing the translation of target mRNA, and promoting the degradation of target mRNA. We report here that murine embryonic fibroblast cells from ZAP knockout mice supported more efficient SVNI replication than wild-type cells. SVNI infection of 10-day-old suckling mice led to reduced survival in the knockout mice. Unexpectedly, however, SVNI infection of 23-day-old weanling mice, whose immune system is more developed than that of the suckling mice, resulted in significantly improved survival in ZAP knockout mice. Further analyses revealed that in the weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of IFN production, which restricted viral spread to the central nervous system. Blocking IFN activity through the use of receptor-neutralizing antibodies rendered knockout mice more sensitive to SVNI infection than wild-type mice. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance.

IMPORTANCE
Sindbis virus, a prototypic member of the
Alphavirus
genus, has been used to study the pathogenesis of acute viral encephalitis in mice for many years. How the virus evades immune surveillance to establish effective infection is largely unknown. ZAP is a host antiviral factor that potently inhibits Sindbis virus replication in cell culture. We show here that infection of ZAP knockout suckling mice with an SVNI led to faster disease progression. However, SVNI infection of weanling mice led to slower disease progression in knockout mice. Further analyses revealed that in weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection and induced higher levels of interferon production, which restricted viral spread to the central nervous system. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance and allow enhanced neuroinvasion.
Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C
Authors: Zhang Zeli , Gu Qinyong , Jaguva Vasudevan Ananda Ayyappan , Jeyaraj Manimehalai , Schmidt Stanislaw , Zielonka Jörg , Perković Mario , Heckel Jens-Ove , Cichutek Klaus , Häussinger Dieter , Smits Sander H. J. , Münk Carsten
Published: September 2016

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F.

IMPORTANCE
The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.
Increased Protein Degradation Improves Influenza Virus Nucleoprotein-Specific CD8+T Cell ActivationIn Vitrobut Not in C57BL/6 Mice
Authors: Altenburg Arwen F. , van de Sandt Carolien E. , van Trierum Stella E. , De Gruyter Heidi L. M. , van Run Peter R. W. A. , Fouchier Ron A. M. , Roose Kenny , Saelens Xavier , Volz Asisa , Sutter Gerd , de Vries Rory D. , Rimmelzwaan Guus F.
Published: September 2016

Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in the case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long-lasting immunity are preferred. Because influenza virus-specific CD8+T cells are directed mainly against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8+T cells. To optimize the induction of CD8+T cell responses, we made several modifications to NP, aiming at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve the induction of CD8+T cell responses. We showed that NP with increased degradation rates improved CD8+T cell activationin vitroif the amount of antigen was limited or if CD8+T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and wild-type NP (NPwt), since NPwt already induced optimal CD8+T cell responses.

IMPORTANCEDue to the continuous antigenic drift of seasonal influenza viruses and the threat of a novel pandemic, there is a great need for the development of novel influenza vaccines that offer broadly protective immunity against multiple subtypes. CD8+T cells can provide immunity against multiple subtypes of influenza viruses by the recognition of relatively conserved internal antigens. In this study, we aimed at optimizing the CD8+T cell response to influenza A virus by making modifications to influenza A virus nucleoprotein (NP) expressed from the modified vaccinia virus Ankara (MVA) vaccine vector. These modifications resulted in increased antigen degradation, thereby producing elevated levels of peptides that can be presented on major histocompatibility complex (MHC) class I molecules to CD8+T cells. Although we were unable to increase the NP-specific immune response in the mouse strain used, this approach may have benefits for vaccine development using less-immunogenic proteins.
In situ observations of earthquake-driven fluid pulses within the Japan Trench plate boundary fault zone
Authors: Fulton Patrick M. , Brodsky Emily E.
Published: September 2016

Transient fluid flow within faults is suspected to be an important component of the earthquake cycle and subduction zone evolution. However, an understanding of the mechanisms and time scales involved has been limited due to a paucity of direct measurements. Here we report on in situ observations that appear to capture the thermal signature of earthquake-driven fluid pulses within the damage zone of the Japan Trench plate boundary fault. The data are from a sub-seafloor temperature observatory installed through the fault following the March 2011 Mw 9.0 Tohoku-oki earthquake as part of the Integrated Ocean Drilling Program’s Japan Trench Fast Drilling Project (JFAST). High-resolution temperature time series data reveal spatially correlated transients in response to earthquakes that are indicative of advection by transient fluid flow. We interpret the observed phenomenon as reflecting pressure redistribution in a fault zone and a potential mechanism for earthquake triggering and episodic heat and chemical transport.
Iron status of young children from immigrant families
Authors: Saunders Natasha Ruth , Parkin Patricia C , Birken Catherine S , Maguire Jonathon L , Borkhoff Cornelia M
Published: September 2016

Objectives

Children from immigrant families may be at risk for iron deficiency (ID) due to differences in pre-migration and post-migration exposures. Our objectives were to determine whether there is an association between family immigrant status and iron stores and to evaluate whether known dietary, environmental or biological determinants of low iron status influence this relationship.

Design

This was a cross-sectional study of healthy urban preschool children (12–72 months) recruited from seven primary care practices in Toronto. Laboratory assessment of serum ferritin and haemoglobin and standardised parent-completed surveys were completed between 2008 and 2013 during routine health maintenance visits. Multiple regression analyses were used to evaluate the association between family immigrant status and serum ferritin, ID (ferritin <14 μg/L) and iron deficiency anaemia (IDA) (ferritin <14 μg/L and haemoglobin ≤110 g/L).

Results

Of 2614 children included in the analysis, 47.6% had immigrant family status. The median serum ferritin was 30 μg/L and 10.4% of all children had ID and 1.9% had IDA. After adjusting for maternal ethnicity and education, age, sex, income quintile, cow's milk intake, breastfeeding duration and bottle use, there were no significant associations between immigrant status and ferritin, ID or IDA. Significant predictors of low iron status included age, sex, cow's milk intake and breastfeeding duration.

Conclusions

We found no association between family immigrant status and iron status after including clinically important covariates in the models. These data suggest immigrant children may not need enhanced screening for iron status or targeted interventions for iron supplementation.
Is physiological glucocorticoid replacement important in children?
Authors: Porter John , Blair Joanne , Ross Richard J
Published: September 2016

Cortisol has a distinct circadian rhythm with low concentrations at night, rising in the early hours of the morning, peaking on waking and declining over the day to low concentrations in the evening. Loss of this circadian rhythm, as seen in jetlag and shift work, is associated with fatigue in the short term and diabetes and obesity in the medium to long term. Patients with adrenal insufficiency on current glucocorticoid replacement with hydrocortisone have unphysiological cortisol concentrations being low on waking and high after each dose of hydrocortisone. Patients with adrenal insufficiency complain of fatigue, a poor quality of life and there is evidence of poor health outcomes including obesity potentially related to glucocorticoid replacement. New technologies are being developed that deliver more physiological glucocorticoid replacement including hydrocortisone by subcutaneous pump, Plenadren, a once-daily modified-release hydrocortisone and Chronocort, a delayed and sustained absorption hydrocortisone formulation that replicates the overnight profile of cortisol. In this review, we summarise the evidence regarding physiological glucocorticoid replacement with a focus on relevance to paediatrics.