bluuebunny/crossref_metadata_2025 huggingface dataset:
The Emergence of Flood Insurance in Canada: Navigating Institutional Uncertainty
Authors: Thistlethwaite Jason
Published: August 2016

Flood insurance has remained unavailable in Canada based on an assessment that it lacks economic viability. In response to Canada's costliest flood event to date in 2013, the Canadian insurance industry has started to develop a framework to expand existing property insurance to cover flood damage. Research on flood insurance has overlooked why and how insurance systems transition to expand insurance coverage without evidence of economic viability. This article will address this gap through a case study on the emergence of flood insurance in Canada, and the approach to its expansion. Between 2013 and 2016, insurance industry officials representing over 60% of premiums collected in Canada were interviewed. These interviews revealed that flood insurance is being expanded in response to institutional pressure, specifically external stakeholder expectations that the insurance industry will adopt a stronger role in managing flood risk through coverage of flood damage. Further evidence of this finding is explored by assessing the emergence of a unique flood insurance model that involves a risk‐adjusted and optional product along with an expansion of government policy supporting flood risk mitigation. This approach attempts to balance industry concerns about economic viability with institutional pressure to reduce flood risk through insurance. This analysis builds on existing research by providing the first scholarly analysis of flood insurance in Canada, important “empirical” teeth to existing conceptual analysis on the availability of flood insurance, and the influence of institutional factors on risk analysis within the insurance sector.
Advancing Risk Analysis for Nanoscale Materials: Report from an International Workshop on the Role of Alternative Testing Strategies for Advancement
Authors: Shatkin J. A. , Ong Kimberly J. , Beaudrie Christian , Clippinger Amy J. , Hendren Christine Ogilvie , Haber Lynne T. , Hill Myriam , Holden Patricia , Kennedy Alan J. , Kim Baram , MacDonell Margaret , Powers Christina M. , Sharma Monita , Sheremeta Lorraine , Stone Vicki , Sultan Yasir , Turley Audrey , White Ronald H.
Published: August 2016

The Society for Risk Analysis (SRA) has a history of bringing thought leadership to topics of emerging risk. In September 2014, the SRA Emerging Nanoscale Materials Specialty Group convened an international workshop to examine the use of alternative testing strategies (ATS) for manufactured nanomaterials (NM) from a risk analysis perspective. Experts in NM environmental health and safety, human health, ecotoxicology, regulatory compliance, risk analysis, and ATS evaluated and discussed the state of the science for in vitro and other alternatives to traditional toxicology testing for NM. Based on this review, experts recommended immediate and near‐term actions that would advance ATS use in NM risk assessment. Three focal areas—human health, ecological health, and exposure considerations—shaped deliberations about information needs, priorities, and the next steps required to increase confidence in and use of ATS in NM risk assessment. The deliberations revealed that ATS are now being used for screening, and that, in the near term, ATS could be developed for use in read‐across or categorization decision making within certain regulatory frameworks. Participants recognized that leadership is required from within the scientific community to address basic challenges, including standardizing materials, protocols, techniques and reporting, and designing experiments relevant to real‐world conditions, as well as coordination and sharing of large‐scale collaborations and data. Experts agreed that it will be critical to include experimental parameters that can support the development of adverse outcome pathways. Numerous other insightful ideas for investment in ATS emerged throughout the discussions and are further highlighted in this article.
Governmentality and the conduct of water: China's South–North Water Transfer Project
Authors: Rogers Sarah , Barnett Jon , Webber Michael , Finlayson Brian , Wang Mark
Published: August 2016

Governmentality is a way of thinking about dispersed practices of governing, including attempts to render space governable. China's South–North Water Transfer (SNWT) project, the world's largest interbasin water transfer project, is a programme of government that attempts to render the distribution of water across space more governable and administrable. This article analyses English and Chinese academic, media and government documents through a governmentality lens. It aims to examine the SNWT project's machinery, mentality and spatiality, including its narrative, its constitution of objects and subjects in space, its multiple techniques of government, and its physical and administrative assemblages. In decentring the problem of the state in relation to the SNWT project we can learn much about both the politics of water and the nature of Chinese governmentalities. This article shows how the SNWT naturalises water scarcity, normalises the pre‐eminence of North China, sustains engineering over regulatory solutions and reconfigures hydrosocial relations, while also outlining the limits to and endemic conflicts within this vast programme of government.
Fiscal Rules and Twin Deficits: The Link between Fiscal and External Balances
Authors: Badinger Harald , Fichet de Clairfontaine Aurélien , Reuter Wolf Heinrich
Published: August 2016

This paper investigates the relationship between countries' fiscal balances and current accounts with an emphasis on the role of fiscal rules. The direct effect of fiscal policy on the current account via aggregate (import) demand is potentially amplified by indirect effects, materialising through interest rate effects and intergenerational transfers that reduce savings. On the other hand, the implied positive relation between fiscal and external balances is potentially attenuated by offsetting changes in savings through Ricardian equivalence considerations. We expect this attenuation effect to be stronger in countries with more stringent fiscal rules and test this hypothesis using a panel of 73 countries over the period 1985–2012. As with previous studies, we find a positive effect of fiscal balances on the current account, supporting the twin deficit hypothesis. However, the effect of fiscal balances on the current account depends on the stringency of fiscal (budget balance or debt) rules in place; it is reduced by one‐third on average and virtually eliminated for countries with the most stringent fiscal rules.
THE ATOMIC PRIESTHOOD AND NUCLEAR WASTE MANAGEMENT: RELIGION, SCI‐FI LITERATURE, AND THE END OF OUR CIVILIZATION
Authors: Musch Sebastian
Published: August 2016

This article discusses the idea of an “Atomic Priesthood,” a religious caste that would preserve and transmit the knowledge of nuclear waste management for future generations. In 1981, the US Department of Energy commissioned a “Human Interference Task Force” (HITF) that would examine the possibilities of how to maintain the security of nuclear waste storage sites for 10,000 years, a period during which our civilization would likely perish, but the dangerous nature of nuclear waste would persist. One option that was discussed was the establishment of an “atomic priesthood,” an idea that science fiction writers like Isaac Asimov and Arsen Darney had already toyed with. Reading the HITF report alongside sci‐fi novels, my article will shed light on the question of how the sheer force of nuclear power (and the longevity of nuclear waste) lends itself to religious interpretations and how the idea of the atomic priesthood is connected with the utopian/dystopian aspects of nuclear power.
THE FAULT IN US: ETHICS, INFINITY, AND CELESTIAL BODIES
Authors: Schaefer Donovan O.
Published: August 2016

Catherine Keller's Cloud of the Impossible knits together process theology and relational ontology with quantum mechanics. In quantum physics, she finds a new resource for undoing the architecture of classical metaphysics and its location of autonomous human subjects as the primary gears of ethical agency. Keller swarms theology with the quantum perspective, focusing in particular on the phenomenon of quantum entanglement, by which quantum particles are found to remain influential over each other long after they have been physically separated—what Albert Einstein and his collaborators recklessly dismissed as “spooky action at a distance.” This spooky action, Keller suggests, reroutes process thought—classically concerned with flux—to a new concern with intransigence—particularly the intransigence of the ethical relationship. Attending to the ethical urgency of the Other, she leaves process theology in a position of susceptibility to the moral imperative posed by the marginalized, the victimized, and the oppressed. This essay argues that although the ontological work of Keller's book productively integrates quantum physics into process theology, the ethical dimension of relationality is left cold in the quantum field. This is because, contra the ethical framework of contemporary deconstruction, which, following Emmanuel Levinas, sees ethical relationships as emerging out of a dynamic of infinite distance, moral connection has nothing to do with the remote reaches of the quantum scale or the macro‐scale limits of space—nothing to do with “infinity” at all. Ethics emerges out of a much messier landscape—the evolved dynamic of fleshy, finite, material bodies. Rather than seeing ethical labor as a matter of physics, my contention (and here I think I am arguing with, rather than against Keller) is that interdisciplinary undertakings like Cloud of the Impossible are ethical disciplinary practices, re‐acquainting us with the non‐sovereignty of the self in order to open up new habits of relating rather than spotlighting ethical imperatives.
Carotid sinus denervation ameliorates renovascular hypertension in adult Wistar rats
Authors: Pijacka Wioletta , McBryde Fiona D. , Marvar Paul J. , Lincevicius Gisele S. , Abdala Ana P. L. , Woodward Lavinia , Li Dan , Paterson David J. , Paton Julian F. R.
Published: August 2016

Key points

Peripheral chemoreflex sensitization is a feature of renovascular hypertension.

Carotid sinus nerve denervation (CSD) has recently been shown to relieve hypertension and reduce sympathetic activity in other rat models of hypertension.

We show that CSD in renovascular hypertension halts further increases in blood pressure.

Possible mechanisms include improvements in baroreceptor reflex sensitivity and renal function, restoration of cardiac calcium signalling towards control levels, and reduced neural inflammation.

Our data suggest that the peripheral chemoreflex may be a viable therapeutic target for renovascular hypertension.

The peripheral chemoreflex is known to be hyper‐responsive in both spontaneously hypertensive (SHR) and Goldblatt hypertensive (two kidney one clip; 2K1C) rats. We have previously shown that carotid sinus nerve denervation (CSD) reduces arterial blood pressure (ABP) in SHR. In the present study, we show that CSD ameliorates 2K1C hypertension and reveal the potential underlying mechanisms. Adult Wistar rats were instrumented to record ABP via telemetry, and then underwent CSD (n = 9) or sham CSD (n = 9) 5 weeks after renal artery clipping, in comparison with normal Wistar rats (n = 5). After 21 days, renal function was assessed, and tissue was collected to assess sympathetic postganglionic intracellular calcium transients ([Ca2+]i) and immune cell infiltrates. Hypertensive 2K1C rats showed a profound elevation in ABP (Wistar: 98 ± 4 mmHg vs. 2K1C: 147 ± 8 mmHg; P < 0.001), coupled with impairments in renal function and baroreflex sensitivity, increased neuroinflammatory markers and enhanced [Ca2+]I in stellate neurons (P < 0.05). CSD reduced ABP in 2K1C+CSD rats and prevented the further progressive increase in ABP seen in 2K1C+sham CSD rats, with a between‐group difference of 14 ± 2 mmHg by week 3 (P < 0.01), which was accompanied by improvements in both baroreflex control and spectral indicators of cardiac sympatho‐vagal balance. Furthermore, CSD improved protein and albuminuria, decreased [Ca2+]i evoked responses from stellate neurons, and also reduced indicators of brainstem inflammation. In summary, CSD in 2K1C rats reduces the hypertensive burden and improves renal function. This may be mediated by improvements in autonomic balance, functional remodelling of post‐ganglionic neurons and reduced inflammation. Our results suggest that the peripheral chemoreflex may be considered as a potential therapeutic target for controlling renovascular hypertension.
Biomimetic coatings enhance tribocorrosion behavior and cell responses of commercially pure titanium surfaces
Authors: Marques Isabella da Silva Vieira , Alfaro Maria Fernanda , Saito Miki Taketomi , da Cruz Nilson Cristino , Takoudis Christos , Landers Richard , Mesquita Marcelo Ferraz , Nociti Junior Francisco Humberto , Mathew Mathew T. , Sukotjo Cortino , Barão Valentim Adelino Ricardo
Published: August 2016

Biofunctionalized surfaces for implants are currently receiving much attention in the health care sector. Our aims were (1) to create bioactive Ti-coatings doped with Ca, P, Si, and Ag produced by microarc oxidation (MAO) to improve the surface properties of biomedical implants, (2) to investigate the TiO2 layer stability under wear and corrosion, and (3) to evaluate human mesenchymal stem cells (hMSCs) responses cultured on the modified surfaces. Tribocorrosion and cell experiments were performed following the MAO treatment. Samples were divided as a function of different Ca/P concentrations and treatment duration. Higher Ca concentration produced larger porous and harder coatings compared to the untreated group (p < 0.001), due to the presence of rutile structure. Free potentials experiments showed lower drops (−0.6 V) and higher coating lifetime during sliding for higher Ca concentration, whereas lower concentrations presented similar drops (−0.8 V) compared to an untreated group wherein the drop occurred immediately after the sliding started. MAO-treated surfaces improved the matrix formation and osteogenic gene expression levels of hMSCs. Higher Ca/P ratios and the addition of Ag nanoparticles into the oxide layer presented better surface properties, tribocorrosive behavior, and cell responses. MAO is a promising technique to enhance the biological, chemical, and mechanical properties of dental implant surfaces.
Design, fabrication, and characterization of polymer-based cantilever probes for atomic force microscopes
Authors: Yu Fangzhou , Liu Jiangbo , Zhang Xiao , Lin Ai-Lian , Khan Nabeela , Pan Yanbiao , Gao Nan , Zou Qingze , Jeon Jaeseok
Published: August 2016

Micromachined polymer-based cantilever probes have been proposed for atomic force microscopes (AFMs) in order to enable noninvasive, rapid high-resolution topography imaging and mechanical measurements of live biological samples. Polymer-based probes developed to date still consist of a rather stiff cantilever with a relatively high spring constant prone to causing deformation and/or distortion of biological sample surfaces during scanning and a rather blunt tip that limits high-resolution topography imaging. This paper reports the design, fabrication, and characterization of soft polymer-based AFM probes. The proposed probe design and fabrication process utilize an acrylic polymer as the structural material, sequential depositions of low and high viscosity acrylic polymers, and a properly patterned optical reflection coating in order to achieve low effective spring constants (less than 0.01 N/m), relatively small tip radii (as small as 40 nm), and relatively low strain gradient (around 10−4/μm). The fabricated polymer AFM probes are calibrated and characterized through experiments and used to obtain AFM height and deflection images of a live mammalian cell in liquid. Experimental results show that the above design specifications have been successfully achieved.
Short delays and low pulse amplitudes produce widespread activation in the target-distance processing area of auditory cortex of the mustached bat
Authors: Macías Silvio , Hechavarría Julio C.
Published: August 2016

While approaching an object, echolocating bats decrease the amplitude of their vocalizations. This behavior is known as “echo-level compensation.” Here, the activation pattern of the cortical FM-FM (frequency modulated) area of the mustached bat is assessed by using acoustic stimuli that correspond to sonar signals and their echoes emitted during echo-level compensation behavior. Activation maps were calculated from the delay response areas of 86 cortical neurons, and these maps were used to explore the topography of cortical activation during echolocation and its relation to the bats' cortical “chronotopy.” Chronotopy predicts short echo-delays to be represented by rostral auditory cortex neurons while caudal neurons represent long echo-delays. The results show that a chronotopic activation of the cortex is evident only at loud pulse amplitudes [80–90 dB sound pressure level (SPL)]. In response to fainter pulse levels (60–70 dB SPL), as those produced as the animals zoom-in on targets, chronotopic activation of the cortex becomes less clear because units throughout the FM-FM area start firing, especially in response to short echo-delays. The fact that cortical activity is more widespread in response to combinations of short echo-delays and faint pulse amplitudes could represent an adaptation that enhances cortical activity in the late stages of echo-level compensation.
Absolute pitch in Costa Rica: Distribution of pitch identification ability and implications for its genetic basis
Authors: Chavarria-Soley Gabriela
Published: August 2016

Absolute pitch is the unusual ability to recognize a pitch without an external reference. The current view is that both environmental and genetic factors are involved in the acquisition of the trait. In the present study, 127 adult musicians were subjected to a musical tone identification test. Subjects were university music students and volunteers who responded to a newspaper article. The test consisted of the identification of 40 piano and 40 pure tones. Subjects were classified in three categories according to their pitch naming ability: absolute pitch (AP), high accuracy of tone identification (HA), and non-absolute pitch (non-AP). Both the percentage of correct responses and the mean absolute deviation showed a statistically significant variation between categories. A very clear pattern of higher accuracy for white than for black key notes was observed for the HA and the non-AP groups. Meanwhile, the AP group had an almost perfect pitch naming accuracy for both kinds of tones. Each category presented a very different pattern of deviation around the correct response. The age at the beginning of musical training did not differ between categories. The distribution of pitch identification ability in this study suggests a complex inheritance of the trait.
Controlled deformation of vesicles by flexible structured media
Authors: Zhang Rui , Zhou Ye , Martínez-González José A. , Hernández-Ortiz Juan P. , Abbott Nicholas L. , de Pablo Juan J.
Published: August 2016

Vesicles immersed in a liquid crystal can adopt either spindle- or pancake-like shapes through control of anchoring conditions.
Can captive breeding save Mexico's vaquita?
Authors: Goldfarb Ben
Published: August 2016

Scientists mull a risky strategy to save an imperiled porpoise
Particle no-show at LHC prompts anxiety
Authors: Cho Adrian
Published: August 2016

Accelerator enters prime discovery phase, but the clock is ticking
Scientists split on Oregon old-growth forest plan
Authors: Cornwall Warren
Published: August 2016

The northern spotted owl is again the center of controversy
From bark to bedside
Authors: Grimm David
Published: August 2016

Clinical trials in pets are ramping up, but will they benefit human health?
Eye lens radiocarbon reveals centuries of longevity in the Greenland shark (
Somniosus microcephalus
)
Authors: Nielsen Julius , Hedeholm Rasmus B. , Heinemeier Jan , Bushnell Peter G. , Christiansen Jørgen S. , Olsen Jesper , Ramsey Christopher Bronk , Brill Richard W. , Simon Malene , Steffensen Kirstine F. , Steffensen John F.
Published: August 2016

Deep living for centuries

We tend to think of vertebrates as living about as long as we do, give or take 50 to 100 years. Marine species are likely to be very long-lived, but determining their age is particularly difficult. Nielsen
et al.
used the pulse of carbon-14 produced by nuclear tests in the 1950s—specifically, its incorporation into the eye during development—to determine the age of Greenland sharks. This species is large yet slow-growing. The oldest of the animals that they sampled had lived for nearly 400 years, and they conclude that the species reaches maturity at about 150 years of age.

Science
, this issue p.
702
Laser spectroscopy of muonic deuterium
Authors: Pohl Randolf , Nez François , Fernandes Luis M. P. , Amaro Fernando D. , Biraben François , Cardoso João M. R. , Covita Daniel S. , Dax Andreas , Dhawan Satish , Diepold Marc , Giesen Adolf , Gouvea Andrea L. , Graf Thomas , Hänsch Theodor W. , Indelicato Paul , Julien Lucile , Knowles Paul , Kottmann Franz , Le Bigot Eric-Olivier , Liu Yi-Wei , Lopes José A. M. , Ludhova Livia , Monteiro Cristina M. B. , Mulhauser Françoise , Nebel Tobias , Rabinowitz Paul , dos Santos Joaquim M. F. , Schaller Lukas A. , Schuhmann Karsten , Schwob Catherine , Taqqu David , Veloso João F. C. A. , Antognini Aldo ,   The CREMA Collaboration
Published: August 2016

The deuteron is too small, too

The radius of the proton has remained a point of debate ever since the spectroscopy of muonic hydrogen indicated a large discrepancy from the previously accepted value. Pohl
et al.
add an important clue for solving this so-called proton radius puzzle. They determined the charge radius of the deuteron, a nucleus consisting of a proton and a neutron, from the transition frequencies in muonic deuterium. Mirroring the proton radius puzzle, the radius of the deuteron was several standard deviations smaller than the value inferred from previous spectroscopic measurements of electronic deuterium. This independent discrepancy points to experimental or theoretical error or even to physics beyond the standard model.

Science
, this issue p.
669
Atmospheric photochemistry at a fatty acid–coated air-water interface
Authors: Rossignol Stéphanie , Tinel Liselotte , Bianco Angelica , Passananti Monica , Brigante Marcello , Donaldson D. James , George Christian
Published: August 2016

Active fatty acid layers

Saturated fatty acids are considered to be inert, but they can be surprisingly reactive when present as a coating at an air-water interface. Rossignol
et al.
show that nonanoic acid is photochemically active when it is present as a monolayer on a water surface (see the Perspective by Vaida). Fatty acids are ubiquitous in the environment, and their photochemical processing could have a substantial impact on local ozone and particle formation.

Science
, this issue p.
699
; see also p.
650
In real life
The Four-Dimensional Human Ways of Being in the Digital World
Laurence Scott
Norton, 2016. 272 pp.
Authors: Golbeck Jennifer
Published: August 2016

An essayist offers an intimate glimpse into the digital technologies that are redefining the human experience
Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle
Authors: Surtees Rebecca , Dowall Stuart D. , Shaw Amelia , Armstrong Stuart , Hewson Roger , Carroll Miles W. , Mankouri Jamel , Edwards Thomas A. , Hiscox Julian A. , Barr John N.
Published: August 2016

The
Nairovirus
genus of the
Bunyaviridae
family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease.

IMPORTANCE
Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance.
B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies
Authors: Patrusheva Irina , Perelygina Ludmila , Torshin Ivan , LeCher Julia , Hilliard Julia
Published: August 2016

B virus (
Macacine herpesvirus 1
) can cause deadly zoonotic disease in humans. Molecular mechanisms of B virus cell entry are poorly understood for both macaques and humans. Here we investigated the abilities of clinical B virus isolates to use entry receptors of herpes simplex viruses (HSV). We showed that resistant B78H1 cells became susceptible to B virus clinical strains upon expression of either human nectin-2 or nectin-1. Antibody against glycoprotein D (gD) protected these nectin-bearing cells from B virus infection, and a gD-negative recombinant B virus failed to enter these cells, indicating that the nectin-mediated B virus entry depends on gD. We observed that the infectivity of B virus isolates with a single amino acid substitution (D122N) in the IgV-core of the gD ectodomain was impaired on nectin-1-bearing cells. Computational homology-based modeling of the B virus gD–nectin-1 complex revealed conformational differences between the structures of the gD-122N and gD-122D variants that affected the gD–nectin-1 protein-protein interface and binding affinity. Unlike HSV, B virus clinical strains were unable to use herpesvirus entry mediator (HVEM) as a receptor, regardless of conservation of the gD amino acid residues essential for HSV-1 entry via HVEM. Based on the model of the B virus gD-HVEM interface, we predict that residues R7, R11, and G15 are largely responsible for the inability of B virus to utilize HVEM for entry. The ability of B virus to enter cells of a human host by using a combination of receptors distinct from those for HSV-1 or HSV-2 suggests a possible mechanism of enhanced neuropathogenicity associated with zoonotic infections.

IMPORTANCE
B virus causes brainstem destruction in infected humans in the absence of timely diagnosis and intervention. Nectins are cell adhesion molecules that are widely expressed in human tissues, including neurons and neuronal synapses. Here we report that human nectin-2 is a target receptor for B virus entry, in addition to the reported receptor human nectin-1. Similar to a B virus lab strain, B virus clinical strains can effectively use both nectin-1 and nectin-2 as cellular receptors for entry into human cells, but unlike HSV-1 and HSV-2, none of the clinical strains uses an HVEM-mediated entry pathway. Ultimately, these differences between B virus and HSV-1 and -2 may provide insight into the neuropathogenicity of B virus during zoonotic infections.
Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins
Authors: Conde Jonas Nascimento , da Silva Emiliana Mandarano , Allonso Diego , Coelho Diego Rodrigues , Andrade Iamara da Silva , de Medeiros Luciano Neves , Menezes Joice Lima , Barbosa Angela Silva , Mohana-Borges Ronaldo
Published: August 2016

Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system.

IMPORTANCE
Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.
Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag
Authors: Wen Yi , Dick Robert A. , Feigenson Gerald W. , Vogt Volker M.
Published: August 2016

The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an
in vitro
liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes.

IMPORTANCE
Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.
Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates
Authors: Verkerke Hans P. , Williams James A. , Guttman Miklos , Simonich Cassandra A. , Liang Yu , Filipavicius Modestas , Hu Shiu-Lok , Overbaugh Julie , Lee Kelly K.
Published: August 2016

Soluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers.

IMPORTANCE
The production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.
Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice
Authors: Matundan Harry H. , Mott Kevin R. , Allen Sariah J. , Wang Shaohui , Bresee Catherine J. , Ghiasi Yasamin N. , Town Terrence , Wechsler Steven L. , Ghiasi Homayon
Published: August 2016

We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT
(−)
McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation.

IMPORTANCE
Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.
Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies
Authors: Khurana Surender , Chung Ka Yan , Coyle Elizabeth M. , Meijer Adam , Golding Hana
Published: August 2016

Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of low-pathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. An in-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole-genome gene fragment phage display libraries (GFPDLs) expressing peptides of 15 to 350 amino acids across the complete genome of the HPAI H7N7 A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of 15 H7N7-exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (<1:10) and those with HI titers of >1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface plasmon resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7-exposed individuals, providing the first evidence for
in vivo
expression of PA-X and its recognition by the immune system during human influenza A virus infection. This knowledge can help inform the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza virus.

IMPORTANCE
An outbreak of pathogenic H7N7 virus occurred in poultry farms in The Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethal infection. In this study, we investigated convalescent-phase sera from H7N7-exposed individuals by using a whole-genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7-exposure antibodies. PA-X is a recently identified influenza virus virulence protein generated by ribosomal frameshifting in segment 3 of influenza virus coding for PA. However, PA-X expression during influenza virus infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7-exposed individuals (but not in unexposed adults), providing the first evidence for
in vivo
expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza virus.
Graf1 Controls the Growth of Human Parainfluenza Virus Type 2 through Inactivation of RhoA Signaling
Authors: Ohta Keisuke , Goto Hideo , Matsumoto Yusuke , Yumine Natsuko , Tsurudome Masato , Nishio Machiko
Published: August 2016

Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation.

IMPORTANCE
Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causes RhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation.
ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle
Authors: Wood Jennifer J. , Boyne James R. , Paulus Christina , Jackson Brian R. , Nevels Michael M. , Whitehouse Adrian , Hughes David J.
Published: August 2016

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle—viral latency and the productive lytic cycle—and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.

IMPORTANCE
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi's sarcoma (KS). Herpesviruses are able to establish a latent infection, in which they escape immune detection by restricting viral gene expression. Importantly, however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we have identified a novel cellular protein (AT-rich interacting domain protein 3B [ARID3B]) that we show is able to temper lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle-dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.