bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Analysis of Air Jets for Sorting Applications
Authors: Ferreira Thiago , Sesmat Sylvie , Bideaux Eric , Sixdenier Fabien
Published: October 2014

Pulsed air jets are used in the industry to eject objects in sorting operation and understand the jet establishment and its spatial characteristics is important to optimize the application. This paper presents a first experimental analysis of jets issued from a high speed solenoid valve in terms of pressure, temperature, and velocity. Results will be first shown for steady state flows at different pressure conditions inducing subsonic or supersonic air jets and compared to the literature. For a subsonic jet, the results confirm the topography proposed in the literature. For the supersonic jets, a subsonic topography was identified after the supersonic zone. These supersonic jets have a constrained diameter which is appreciated in order to perform sorting with precision. Then, first unstationary experimental results will be presented and commented. This first measurement of the jet development is encouraging, since it was possible to identify the different delays linked to the propagation time from the valve outlet to the measurement point on opening and closing.
Energy Efficient Hydraulic Rotary Drive: Analysis and Comparison of Two Different Displacement Controlled Solutions
Authors: Padovani D. , Ivantysynova M.
Published: October 2014

This paper discusses, analyzes, and compares two different displacement controlled solutions for a rotary drive of a mobile machine. In particular, the first solution is a hydrostatic transmission based on a closed circuit structure while the second solution employs secondary controlled hydraulic drives. In detail, these alternatives are evaluated in the context of a machine with a huge mass that performs repetitive working cycles with frequent stops and goes that require a precise positioning. Consequently, the ability to recover energy via regenerative breaking becomes a key aspect in order to improve the global efficiency of the machine. Therefore, the paper will present a dynamic system model that was used to compare the two alternatives.
The Impact of the Surface Shape of the Piston on Power Losses
Authors: Wondergem Ashley M. , Ivantysynova Monika
Published: October 2014

Axial piston machines are widely used in industry thus new cost-effective and highly efficient designs are needed. One way to increase efficiency and decrease cost is by altering the geometry along with the configuration of the piston/cylinder interface influencing the fluid film generation and in turn the energy dissipation and load carrying capacity while still having a design that is cost effective and easy to manufacture. This paper presents a study on a reduction of energy dissipation between the piston and cylinder over a wide range of operating conditions at both full and partial displacements based on the surface shape of the piston along with the minimum clearance. First, it is necessary to measure a base design and then compare those results to simulations in order to verify the simulation results. Once a baseline is established, various piston surface shapes and minimum clearances are then also simulated and compared back to the simulated baseline. Not only is energy dissipation important to compare, but also the minimum gap height over one revolution. The minimum gap height is in direct correlation to friction loss and wear. Therefore, this paper also includes an understanding of how the gap height affects the total losses thus leading to the importance of finding a relative clearance that satisfies a median between torque losses and leakage along with the importance of reducing the occurrence of critical gap heights to reduce the need for wear in in the machine.
Technical Cleanliness of Assembled Fluid Power Components
Authors: Elo Lauri , Pekkonen Juuso , Rinkinen Jari
Published: October 2014

The demands on the cleanliness of fluid power systems increase continuously. The KompuNW project, which was launched in 2011, aimed to find out the technical cleanliness of fluid power components in Finland, and the technical cleanliness was measured with commercial cleanliness cabinets. These cabinets apply well for hydraulic components where a rinsing pen should reach the test component surfaces that need to be controlled. In some cases the flow rate or the pressure produced by the cleanliness cabinets is insufficient for detaching particles from the surfaces of hollow components e.g. hydraulic cylinders or oil reservoirs. For this reason the functional test bench, designed and constructed in IHA, uses low viscosity hydraulic oil to extract contaminants from the assembled fluid power components. The extracted particles can then be analysed in the test bench with a microscope or an inline particle counter.

This paper has several aims. First the backgrounds of fluid power components are described. Then this paper will present the differences in the definitions and codes of oil cleanliness (ISO 4406:1999, SAE AS4059E:2005) and component cleanliness (ISO 16232:2007). In addition, the differences between the test bench and the commercial cleanliness cabinets are presented. Standards ISO 16232:2007-5 and ISO 18413:2002 were applied in the design of the test bench. The main points of these standards are introduced in this paper, too.

Finally the extraction procedures of a hydraulic cylinder and oil reservoir are shown with examples of the extraction curves of these components.
Output Feedback Control of Electro-Hydraulic Cylinder Drives Using the Twisting Algorithm
Authors: Schmidt Lasse , Andersen Torben O. , Pedersen Henrik C.
Published: October 2014

This paper discusses the utilization of the so-called twisting algorithm when applied in output feedback position control schemes for electro-hydraulic cylinder drives. The twisting controller was the first second order sliding controller ever introduced, and can structure-wise be considered a straight forward extension of the simplest first order sliding controller, that is, a relay controller. Such a controller may be implemented without the knowledge of system time constants etc., as opposed to the surface based first order sliding controllers which has been presented in numerous contributions in literature. This paper considers the twisting algorithm when applied directly for output feedback control, and with the design based on a reduced order model representation of an arbitrary valve driven hydraulic cylinder drive. The consequence of implementing such a controller with the well-known saturation-, or boundary layer method is discussed, and the control operation inside- and out-side the boundary layer region is considered. Furthermore, the global stability of such a controller is discussed, with emphasis on possible local instability modes. Results demonstrate that the proposed output feedback controller may be successfully applied to hydraulic valve driven cylinder drives, with performance being on the level with a conventional surface based first order sliding mode controller.
Study on Dynamics of Air Pressure Reducing Valve With Focus on the Noise Attenuation Problem
Authors: Badykova Lilia , Stadnick Dmitry , Afanasev Kirill , Igolkin Alexander , Sverbilov Victor
Published: October 2014

The noise in natural gas distribution systems is mainly caused by pressure reduction in shut-off and control valves. The aim of present research is to analyze dynamics of an air pressure reducing valve with a muffler installed at its output. An increase of output impedance can lead to a loss in stability and control accuracy. Therefore, the valve simulation model in software package MATLAB/Simulink is developed in order to study the muffler’s impact on static and dynamic performances of the pressure reducing valve. As a result of performed modeling and simulation the influence of the output throttle resistance on the system main operating parameters is obtained. The output data can be used to optimize sizing of the muffler for the air pressure reducing valve.
Reconstruction of Hydrostatic Drive and Control System Dedicated for Small Mobile Platform
Authors: Guzowski Artur , Sobczyk Andrzej
Published: October 2014

The paper presents modification of drive and control system for specially developed small size multipurpose mobile platform which has been built based on existing vehicle undercarriage. For such redesigned object new concept of hydrostatic drive and control system is proposed. The control system consists of selected hydraulic components and controllers which were chosen from Sauer-Danfoss PLUS+1 package which is the system dedicated for mobile hydraulic control systems application. The hydraulic system has been equipped with extra valves, enabling to obtain of assumed functions.
Scanning probes in nanostructure fabrication
Authors: Kaestner Marcus , Ivanov Tzvetan , Schuh Andreas , Ahmad Ahmad , Angelov Tihomir , Krivoshapkina Yana , Budden Matthias , Hofer Manuel , Lenk Steve , Zoellner Jens-Peter , Rangelow Ivo W. , Reum Alexander , Guliyev Elshad , Holz Mathias , Nikolov Nikolay
Published: October 2014

Scanning probes have enabled modern nanoscience and are still the backbone of today's nanotechnology. Within the technological development of AFM systems, the cantilever evolved from a simple passive deflection element to a complex microelectromechanical system through integration of functional groups, such as piezoresistive detection sensors and bimaterial based actuators. Herein, the authors show actual trends and developments of miniaturization efforts of both types of cantilevers, passive and active. The results go toward the reduction of dimensions. For example, the authors have fabricated passive cantilever with a width of 4 μm, a length of 6 μm and thickness of 50–100 nm, showing one order of magnitude lower noise levels. By using active cantilevers, direct patterning on calixarene is demonstrated employing a direct, development-less phenomena triggered by tip emitted low energy (<50 eV) electrons. The scanning probes are not only applied for lithography, but also for imaging and probing of the surface before and immediately after scanning probe patterning. In summary, piezoresistive probes are comparable to passive probes using optical read-out. They are able to routinely obtain atomic step resolution at a low thermal noise floor. The active cantilever technology offers a compact, integrated system suited for integration into a table-top scanning probe nanolithography tool.
Atomic layer deposition TiO2–Al2O3 stack: An improved gate dielectric on Ga-polar GaN metal oxide semiconductor capacitors
Authors: Wei Daming , Edgar James H. , Briggs Dayrl P. , Retterer Scott T. , Srijanto Bernadeta , Hensley Dale K. , Meyer Harry M.
Published: October 2014

This research focuses on the benefits and properties of TiO2–Al2O3 nanostack thin films deposited on Ga2O3/GaN by plasma-assisted atomic layer deposition (PA–ALD) for gate dielectric development. This combination of materials achieved a high dielectric constant, a low leakage current, and a low interface trap density. Correlations were sought between the films' structure, composition, and electrical properties. The gate dielectrics were approximately 15 nm thick and contained 5.1 nm TiO2, 7.1 nm Al2O3, and 2 nm Ga2O3 as determined by spectroscopic ellipsometry. The interface carbon concentration, as measured by x-ray photoelectron spectroscopy depth profile, was negligible for GaN pretreated by thermal oxidation in O2 for 30 min at 850 °C. The RMS roughness slightly increased after thermal oxidation and remained the same after ALD of the nanostack, as determined by atomic force microscopy. The dielectric constant of TiO2–Al2O3 on Ga2O3/GaN was increased to 12.5 compared to that of pure Al2O3 (8–9) on GaN. In addition, the nanostack's capacitance–voltage (C-V) hysteresis was small, with a total trap density of 8.74 × 1011 cm−2. The gate leakage current density (J = 2.81 × 10−8 A/cm2) was low at +1 V gate bias. These results demonstrate the promising potential of PA–ALD deposited TiO2/Al2O3 for serving as the gate dielectric on Ga2O3/GaN based MOS devices.
Electron-stimulated desorption from polished and vacuum fired 316LN stainless steel coated with Ti-Zr-Hf-V
Authors: Malyshev Oleg B. , Valizadeh Reza , Hogan Benjamin T. , Hannah Adrian N.
Published: October 2014

In this study, two identical 316LN stainless steel tubular samples, which had previously been polished and vacuum-fired and then used for the electron-stimulated desorption (ESD) experiments, were coated with Ti-Zr-Hf-V with different morphologies: columnar and dense. ESD measurement results after nonevaporable getter (NEG) activation to 150, 180, 250, and 350 °C indicated that the values for the ESD yields are significantly (2–20 times) lower than the data from our previous study with similar coatings on nonvacuum-fired samples. Based on these results, the lowest pressure and best long-term performance in particle accelerators will be achieved with a vacuum-fired vacuum chamber coated with dense Ti-Zr-Hf-V coating activated at 180 °C. This is likely due to the following facts: after NEG activation, the hydrogen concentration inside the NEG was lower than in the bulk stainless steel substrate; the NEG coating created a barrier for gas diffusion from the sample bulk to vacuum; the dense NEG coating performed better as a barrier than the columnar NEG coating.
Directionality of the ambient noise field in an Arctic, glacial bay
Authors: Deane Grant B. , Glowacki Oskar , Tegowski Jaroslaw , Moskalik Mateusz , Blondel Philippe
Published: October 2014

The directionality of ambient noise in an Arctic tidewater glacier bay was measured using two horizontally spaced, broadband hydrophones. Segments of noise were divided into two frequency bands and analyzed for arrival angle. These data show that different classes of source radiate noise in distinct spectral bands and are spatially diverse. A previously unidentified source, the interaction of surface gravity waves with underside of ice ledges at the periphery of icebergs, is described. The generation of noise by ice-wave interaction suggests that surface waves should be measured if ambient noise is to be used to monitor ice dynamics in glacial fjords.
Core Concepts: Understanding the Complexity of the Spinal Stabilizing Systems in Local and Global Injury Prevention and Treatment
Authors: Warren Lindsay , Baker Russell , Nasypany Alan , Seegmiller Jeffrey
Published: October 2014

The core is central to almost all extremity movements, especially in athletics. Running, jumping, kicking, and throwing are dependent on core function to create a stable base for movement. Poor core strength, endurance, stiffness, control, coordination, or a combination thereof can lead to decreased performance and increased risk of injury. Due to the core’s many complex elements, none of which are more or less important than the next, it is imperative that athletic trainers have a systematic and comprehensive plan for assessing and treating patients with stability or motor control dysfunctions of the entire spinal stabilizing system. The purpose of this clinical commentary is to outline the structural (anatomical) components of the core and their functions, establish the elements of core stability (functional), review these elements’ importance in decreasing the risk of injury, and discuss the application of this information in athletic training.
Large carnivores make savanna tree communities less thorny
Authors: Ford Adam T. , Goheen Jacob R. , Otieno Tobias O. , Bidner Laura , Isbell Lynne A. , Palmer Todd M. , Ward David , Woodroffe Rosie , Pringle Robert M.
Published: October 2014

Understanding how predation risk and plant defenses interactively shape plant distributions is a core challenge in ecology. By combining global positioning system telemetry of an abundant antelope (impala) and its main predators (leopards and wild dogs) with a series of manipulative field experiments, we showed that herbivores’ risk-avoidance behavior and plants’ antiherbivore defenses interact to determine tree distributions in an African savanna. Well-defended thorny
Acacia
trees (
A. etbaica
) were abundant in low-risk areas where impala aggregated but rare in high-risk areas that impala avoided. In contrast, poorly defended trees (
A. brevispica
) were more abundant in high- than in low-risk areas. Our results suggest that plants can persist in landscapes characterized by intense herbivory, either by defending themselves or by thriving in risky areas where carnivores hunt.
Loss-induced suppression and revival of lasing
Authors: Peng B. , Özdemir Ş. K. , Rotter S. , Yilmaz H. , Liertzer M. , Monifi F. , Bender C. M. , Nori F. , Yang L.
Published: October 2014

Controlling and reversing the effects of loss are major challenges in optical systems. For lasers, losses need to be overcome by a sufficient amount of gain to reach the lasing threshold. In this work, we show how to turn losses into gain by steering the parameters of a system to the vicinity of an exceptional point (EP), which occurs when the eigenvalues and the corresponding eigenstates of a system coalesce. In our system of coupled microresonators, EPs are manifested as the loss-induced suppression and revival of lasing. Below a critical value, adding loss annihilates an existing Raman laser. Beyond this critical threshold, lasing recovers despite the increasing loss, in stark contrast to what would be expected from conventional laser theory. Our results exemplify the counterintuitive features of EPs and present an innovative method for reversing the effect of loss.
Laboratory formation of a scaled protostellar jet by coaligned poloidal magnetic field
Authors: Albertazzi B. , Ciardi A. , Nakatsutsumi M. , Vinci T. , Béard J. , Bonito R. , Billette J. , Borghesi M. , Burkley Z. , Chen S. N. , Cowan T. E. , Herrmannsdörfer T. , Higginson D. P. , Kroll F. , Pikuz S. A. , Naughton K. , Romagnani L. , Riconda C. , Revet G. , Riquier R. , Schlenvoigt H.-P. , Skobelev I. Yu. , Faenov A.Ya. , Soloviev A. , Huarte-Espinosa M. , Frank A. , Portugall O. , Pépin H. , Fuchs J.
Published: October 2014

Although bipolar jets are seen emerging from a wide variety of astrophysical systems, the issue of their formation and morphology beyond their launching is still under study. Our scaled laboratory experiments, representative of young stellar object outflows, reveal that stable and narrow collimation of the entire flow can result from the presence of a poloidal magnetic field whose strength is consistent with observations. The laboratory plasma becomes focused with an interior cavity. This gives rise to a standing conical shock from which the jet emerges. Following simulations of the process at the full astrophysical scale, we conclude that it can also explain recently discovered x-ray emission features observed in low-density regions at the base of protostellar jets, such as the well-studied jet HH 154.
Nutrient computation for root architecture
Authors: Bisseling Ton , Scheres Ben
Published: October 2014

Plants sense and respond to nutrients using a peptide signaling system
‘Nonadherence’: A bitter pill for drug trials
Authors: Servick Kelly
Published: October 2014

Drug developers seek new ways to ensure that subjects take their medicine.
Ebola vaccine trials raise ethical issues
Authors: Cohen Jon , Kupferschmidt Kai
Published: October 2014

Randomized studies may offer fastest answer.
Baboon watch
Authors: Pennisi Elizabeth
Published: October 2014

An epic baboon study shows how social interactions shape health and reproduction in all primates—including humans.
Part 1: Targeting cancer pathways: Tumor resistance
Authors: Yaffe Michael B. , Engelman Jeffrey , Deininger Michael W.
Published: October 2014

This webinar is the first in a series focusing on the pathways that allow for cancer development and progression, the emerging research in identifying and targeting these pathways, and the innovations in the development of cancer treatment options. Recent advances in our understanding of cancer have revealed that the disease cannot be understood through simple analysis of genetic mutations within cancerous cells. Instead, tumors should be considered complex tissues in which the cancer cells evolve and communicate with the surrounding cellular microenvironment to promote their own survival and dissemination. Although therapies against specific signaling proteins or pathways have been remarkably successful at treating certain cancers, the tumors frequently develop resistance, leading to even more aggressive forms of the disease.

This webinar will focus on how rewiring of signaling pathways in response to drug treatment contributes to resistance and how this knowledge can be leveraged to develop more effective treatment strategies.
Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor
Authors: Shi Jiong , Zhou Jing , Halambage Upul D. , Shah Vaibhav B. , Burse Mallori J. , Wu Hua , Blair Wade S. , Butler Scott L. , Aiken Christopher
Published: October 2014

The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes
in vitro
and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.

IMPORTANCE
The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.
Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases
Authors: Campagnola Grace , McDonald Seth , Beaucourt Stéphanie , Vignuzzi Marco , Peersen Olve B.
Published: October 2014

Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and
in vivo
fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on
in vitro
nucleotide discrimination, and these effects are strongly correlated with elongation rates and
in vivo
mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.

IMPORTANCE
Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on
in vitro
nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth
in vivo
. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.
The Putative Herpes Simplex Virus 1 Chaperone Protein UL32 Modulates Disulfide Bond Formation during Infection
Authors: Albright Brandon S. , Kosinski Athena , Szczepaniak Renata , Cook Elizabeth A. , Stow Nigel D. , Conway James F. , Weller Sandra K.
Published: October 2014

During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.

IMPORTANCE
Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
Variations in pH Sensitivity, Acid Stability, and Fusogenicity of Three Influenza Virus H3 Subtypes
Authors: Costello Deirdre A. , Whittaker Gary R. , Daniel Susan
Published: October 2014

Influenza A virus strains adapt to achieve successful entry into host species. Entry is mediated by the viral membrane protein hemagglutinin (HA), which triggers membrane fusion and genome release under acidic conditions in the endosome. In addition to changes in the receptor binding domain, the acid stability of HA has been linked to the successful transmission of virus between avian and human hosts. However, to fully understand the connection between changes in HA and host tropism, additional factors relevant to HA structure-function and membrane fusion are also likely to be important. Using single-particle-tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which provide detailed information regarding HA pH sensitivity and acid stability and the rate and extent of membrane fusion. This provides a comparative way to characterize and distinguish influenza virus fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn), and A/Brisbane/07/H3N2 (Brisbane). The rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while the fusion of the egg-adapted strains Udorn and X:31 is strongly dependent on pH (and is faster) as the pH decreases. All strains exhibit similar acid stability (the length of time that they remain fusogenic in an acidic environment) at higher pHs, but the egg-adapted strains become less acid stable at lower pHs. Thus, it appears that the laboratory-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH, with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain.

IMPORTANCE
The ability of influenza virus to release its genome under different acidic conditions has recently been linked to the transmission of influenza virus between different species. However, it is yet to be determined how acid-induced membrane fusion varies with virus strain and influences tropism. The results presented here are the results of an intra-H3-subtype study of acid stability and fusion kinetics. Using a single-particle-tracking (SPT) technique, we show here that the highest pH that initiates fusion is not necessarily the pH at which the kinetics of fusion is fastest and most abundant for a given strain. Strains exhibit different fusion behaviors, as evidenced by their unique kinetic trends; pH sensitivities, as evidenced by the differences when the first fusion events commence; and HA stabilities, as evidenced by the length of time that virions can persist in an acidic environment and still be fusion competent.
Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells
Authors: Gao Shijuan , Wu Jiaoxiang , Liu Ran-Yi , Li Jiandong , Song Liping , Teng Yan , Sheng Chunjie , Liu Dong , Yao Chen , Chen Huiming , Jiang Wei , Chen Shuai , Huang Wenlin
Published: October 2014

Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione
S
-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication.

IMPORTANCE
Although the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication.
Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles
Authors: Grover Jonathan R. , Veatch Sarah L. , Ono Akira
Published: October 2014

HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag–PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants.

IMPORTANCE
Nascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote the association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using superresolution localization microscopy suggest the presence of a novel molecular mechanism promoting the association of PSGL-1, CD43, and CD44 with assembling HIV-1 which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein.
An Interleukin-1 Beta-Encoding Retrovirus Exhibits Enhanced Replication
In Vivo
Authors: Browne Edward P.
Published: October 2014

Interleukin-1 beta (IL-1β) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1β secretion through the DNA-sensing molecule IFI16, the effect of IL-1β on the course of infection is unknown. To test whether IL-1β secretion affects retroviral replication
in vivo
, I constructed a novel murine leukemia virus strain (FMLV-IL-1β) that encodes the mature form of IL-1β. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1β exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1β-infected mice correlated with a large expansion of Gr-1
+
CD11b
+
myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1β infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1β was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1β secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1β represents a new model of proinflammatory retroviral infection.

IMPORTANCE
Interleukin-1 beta (IL-1β) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1β on retroviral replication
in vivo
, I constructed a novel mouse retrovirus strain (FMLV-IL-1β) that encodes IL-1β and promotes abundant IL-1β secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1β as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.
Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7
Authors: Rodrigues Louise , Freitas Natalia , Kallakury Bhaskar V. , Menne Stephan , Gudima Severin O.
Published: October 2014

The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection.

IMPORTANCE
This study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.
Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice
Authors: Bi Yuhai , Xie Qing , Zhang Shuang , Li Yun , Xiao Haixia , Jin Tao , Zheng Weinan , Li Jing , Jia Xiaojuan , Sun Lei , Liu Jinhua , Qin Chuan , Gao George F. , Liu Wenjun
Published: October 2014

The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated
in vitro
and
in vivo
. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans.

IMPORTANCE
To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus
in vitro
and
in vivo
. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.
Identification of a Neutralizing Epitope within Antigenic Domain 5 of Glycoprotein B of Human Cytomegalovirus
Authors: Wiegers Anna-Katharina , Sticht Heinrich , Winkler Thomas H. , Britt William J. , Mach Michael
Published: October 2014

Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infectedin utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus-neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus-neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a target site on gB, despite originating from different, healthy, HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND mutations in AD-5 were resistant to virus-neutralizing MAbs. Competition enzyme-linked immunosorbent assays (ELISAs) with human HCMV-convalescent-phase sera from unselected donors confirmed the conserved antibody response for the YNND epitope in HCMV-infected individuals and, because a significant fraction of the gB AD-5 response was directed against the YNND epitope, further argued that this epitope is a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed reactivity to AD-5 and neutralization activity toward gB mutant viruses that were similar to those of AD-5-specific MAbs. Taken together, our data indicate that the YNND epitope represents an important target for anti-gB antibody responses as well as for anti-AD-5 virus-neutralizing antibodies.

IMPORTANCEHCMV is a major global health concern, and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence an interesting molecule for intervention strategies, e.g., vaccination. Mapping the target structures of neutralizing antibodies induced by naturally occurring HCMV infection can aid in defining the properties required for a protective capacity of vaccine antigens. The data presented here extend our knowledge of neutralizing epitopes within gB to include AD-5. Collectively, our data will contribute to optimal vaccine design and development of antibody-based therapies.