VP2 mediates the release of the feline calicivirus RNA genome by puncturing the endosome membrane of infected cells.

Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.

Application of a Gas-Kinetic BGK Scheme in Thermal Protection System Analysis for Hypersonic Vehicles.

One major problem in the development of hypersonic vehicles is severe aerodynamic heating; thus, the implementation of a thermal protection system is required. A numerical investigation on the reduction of aerodynamic heating using different thermal protection systems is conducted using a novel gas-kinetic BGK scheme. This method adopts a different solution strategy from the conventional computational fluid dynamics technique, and has shown a lot of benefits in the simulation of hypersonic flows. To be specific, it is established based on solving the Boltzmann equation, and the obtained gas distribution function is used to reconstruct the macroscopic solution of the flow field. Within the finite volume framework, the present BGK scheme is specially designed for the evaluation of numerical fluxes across the cell interface. Two typical thermal protection systems are investigated by using spikes and opposing jets, separately. Both their effectiveness and mechanisms to protect the body surface from heating are analyzed. The predicted distributions of pressure and heat flux, and the unique flow characteristics brought by spikes of different shapes or opposing jets of different total pressure ratios all verify the reliability and accuracy of the BGK scheme in the thermal protection system analysis.

A highly sensitive europium nanoparticle-based immunoassay for detection of influenza A/B virus antigen in clinical specimens.

We report the development of a novel europium nanoparticle-based immunoassay (ENIA) for rapid detection of influenza A and influenza B viruses. The ENIA demonstrated sensitivities of 90.7% (147/162) for influenza A viruses and 81.80% (9/11) for influenza B viruses compared to those for an in-house reverse transcription (RT)-PCR assay in testing of influenza-positive clinical samples.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Electrocatalytic properties of Au@Pt nanoparticles: effects of Pt shell packing density and Au core size.

A detailed study of electrocatalytic properties of Au@Pt nanoparticles (NPs) as functions of Pt shell packing density and Au core size in terms of CO/methanol oxidation and oxygen reduction reactions is reported here. While most samples studied showed inferior catalytic activities to those of the commercial Pt black that fall reasonably well in a d-band-center up-shift (i.e., stronger surface bonding) regime, the steepest activity recovery trend as manifested by the smallest Au-core samples suggests a plausible transition to a d-band-center down-shift (i.e., weaker surface bonding) regime as the Au core becomes smaller.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

A coverage-dependent study of pt spontaneously deposited onto Au and Ru surfaces: direct experimental evidence of the ensemble effect for methanol electro-oxidation on Pt.

Direct experimental evidence that can be unambiguously attributed to the need of an ensemble of a minimum number of neighboring Pt atoms for methanol electro-oxidation has been observed for the first time. This was realized by a Pt coverage-dependent investigation of methanol and CO electro-oxidation on Pt sites generated via spontaneous deposition onto both Au and Ru surfaces. CO stripping voltammograms also show clear evidence of a substantially strengthened CO-Pt bonding for submonolayer Pt deposited on the Au substrate over a range of ca. 0.22 to 0.77, which is in qualitative agreement with the theoretical prediction based on the Hammer-Nørskov d-band center model. However, the degree of the bond strengthening depends on the Pt coverage, being stronger for lower coverage. Additionally, evidence of an Ostwald ripening process for Pt islands formation has also been observed.

Development of a microchip Europium nanoparticle immunoassay for sensitive point-of-care HIV detection.

Rapid, sensitive and specific diagnostic assays play an indispensable role in determination of HIV infection stages and evaluation of efficacy of antiretroviral therapy. Recently, our laboratory developed a sensitive Europium nanoparticle-based microtiter-plate immunoassay capable of detecting target analytes at subpicogram per milliliter levels without the use of catalytic enzymes and signal amplification processes. Encouraged by its sensitivity and simplicity, we continued to miniaturize this assay to a microchip platform for the purpose of converting the benchtop assay technique to a point-of-care test. It was found that detection capability of the microchip platform could be readily improved using Europium nanoparticle probes. We were able to routinely detect 5 pg/mL (4.6 attomoles) of HIV-1 p24 antigen at a signal-to-blank ratio of 1.5, a sensitivity level reasonably close to that of microtiter-plate Europium nanoparticle assay. Meanwhile, use of the microchip platform effectively reduced sample/reagent consumption 4.5 fold and shortened total assay time 2 fold in comparison with microtiter plate assays. Complex matrix substance in plasma negatively affected the microchip assays and the effects could be minimized by diluting the samples before loading. With further improvements in sensitivity, reproducibility, usability, assay process simplification, and incorporation of portable time-resolved fluorescence reader, Europium nanoparticle immunoassay technology could be adapted to meet the challenges of point-of-care diagnosis of HIV or other health-threatening pathogens at bedside or in resource-limited settings.

A volcano curve: optimizing methanol electro-oxidation on Pt-decorated Ru nanoparticles.

Controlled Pt adlayers were deposited on commercial Ru nanoparticles (NPs) using an industrially scalable one-pot ethylene glycol (EG) reduction based method and were characterized by X-ray diffraction (XRD), electrochemical (EC) CO stripping voltammetry, inductively-coupled plasma optical emission spectrometry (ICP-OES), X-ray photoemission spectroscopy (XPS), and transmission electron microscopy (TEM). Compared with the previously used "spontaneous deposition", the wet chemistry-based EG method is less technically demanding, i.e. no need to handle high-temperature hydrogen reduction, offers a better control of the Pt packing density (PD), enables the formation of stable, segregated Pt surface adlayers for optimal tuning and use of Pt, and effectively prevents NPs sintering. Two batches of a total of 11 (8 vs. 3) samples with different values of Pt PD ranging from 0.05 to 0.93 were prepared, with a time interval of more than 18 months between the sytheses of the two batches of samples, and an excellent reproducibility of results was observed. All samples were investigated in terms of methanol (MeOH) electro-oxidation (EO) by cyclic voltammetry (CV) and chronoamperometry (CA). Although the peak current of CV increased as the Pt content increased, the long-term steady-state MeOH electro-oxidation current density of the Pt-decorated Ru NPs measured by CA showed a volcano curve as a function of the Pt PD, with the maximum appearing at the PD of 0.31. The optimal peak activity was approximately 150% higher than that of the industrial benchmark PtRu (1 : 1) alloy NPs and could deliver the same performance at half the electrode material cost. Fundamentally, such a volcano curve in the reaction current is the result of two competing processes of the EO of MeOH: the triple dehydrogenation of MeOH that prefers more Pt ensemble sites, and the elimination of poisonous CO that is enhanced by more adjacent Ru/Pt sites via the so-called bifunctional mechanism and also by possible electronic effects at low Pt coverages.

Spatially resolved 195Pt NMR of carbon-supported PtRu electrocatalysts: local electronic properties, elemental composition, and catalytic activity.

While bimetallic nanoparticles (NPs) offer greater tunability of their catalytic performance than their monometallic counterparts, their detailed mechanistic characterizations are still of a great challenging prospect, particularly at nanoscale. In this paper, we describe a unique (195)Pt nuclear magnetic resonance (NMR) based in situ technique that in principle enables us to access local elemental composition and electronic information across the dimension of the Pt-based NPs with decent spatial resolution. When combined with electrochemical analysis, it opens a way to correlate the local elemental composition and electronic properties with the catalytic activity of the bimetallic NPs. Specifically, from the (195)Pt NMR analysis we concluded that (1) for the PtRu/carbon nanofibers, Ru segregates at the surface while Pt does so inside the NPs; (2) alloying Ru substantially reduces the E(F) local density of states (LDOS) at the Pt atoms; (3) the larger variation in s-like E(F) LDOS at the surface region of the PtRu/graphite nanofibers may imply a higher diversity of catalytic sites available for reactions, therefore explains the observed higher reactivity in methanol electro-oxidation.

A comparative in situ 195Pt electrochemical-NMR investigation of PtRu nanoparticles supported on diverse carbon nanomaterials.

This paper reports a detailed in situ 195Pt electrochemical-nuclear magnetic resonance (EC-NMR) study of PtRu nanoparticles (NPs) that had a nominal atomic ratio of Pt : Ru = 1 : 1 and were supported on carbon nanocoils and carbon black (Vulcan XC-72) respectively. The particle sizes of the two samples were determined by X-ray diffraction using the Sherrer equation: 3.6 nm for the former and 3.2 nm for the latter, which were further corroborated by transmission electron microscope measurements. By taking advantage of a unique correlation between the spectral frequency of the 195Pt NMR resonance and the radial atomic position in a particle, qualitatively- and spatially-resolved local Pt atomic fractions in the particles were deduced by using a Ruderman-Kittel-Kasuya-Yosida (RKKY) J-coupling-based method as a function of different electrode potentials. The results indicated that both samples had Pt-enriched cores and Pt-deprived surfaces and, most importantly, the local Pt concentration varied as the electrochemical environment changed. The spatially-resolved Fermi level local densities of states (E(f)-LDOS), which are a measure of the electronic frontier orbitals in metals, were deduced across the NMR spectrum and correlated with the EC activity in methanol electro-oxidation. The results were also compared to those obtained previously from Pt/Ru NPs supported respectively on carbon and graphite nanofibers.