Q.F. Fang

Characterization of infectious particles of grass carp reovirus by treatment with proteases

Proteolytic cleavages play an important role in reovirus infection during entry into cells. The effects of protease digestion on the morphology, infectivity and polypeptide composition of grass carp reovirus (GCRV) were investigated. Following treatment with chymotrypsin, the different subviral particles of GCRV were isolated using density gradient centrifugation and examined by electron microscope (EM). Analysis of protein components revealed that the viral outer capsid was composed of VP5 and VP7. Of particular note, VP5 was found to primarily exist within virions as cleaved fragments, which was consistent with observations for its analogue μ1/μ1C, generated by autolysis of μ1 at the μ1N/μ1C junction for mammalian orthoreoviruses (MRVs). Meanwhile, both trypsin- and chymotrypsin-treated GCRV particles appeared to have an enhanced infectivity. Moreover, the corresponding assays between infectivity and protein component indicated that the enhancement of infectivity was correlated to the complete digestion of the outer capsid protein VP7 and partial cleavage of VP5. Overall, the results presented in this paper provided strong evidence that the proteins VP5 and VP7 of GCRV play an indispensable role in viral infection.

Magnetic and charge ordering in nanosized manganites

Perovskite manganites exhibit a wide range of functional properties, such as colossal magneto-resistance, magnetocaloric effect, multiferroic property, and some interesting physical phenomena including spin, charge, and orbital ordering. Recent advances in science and technology associated with perovskite oxides have resulted in the feature sizes of microelectronic devices down-scaling into nanoscale dimensions. The nanoscale perovskite manganites display novel magnetic and electronic properties that are different from their bulk and film counterparts. Understanding the size effects of perovskite manganites at the nanoscale is of importance not only for the fundamental scientific research but also for developing next generation of electronic and magnetic nanodevices. In this paper, the current understanding and the fundamental issues related to the size effects on the magnetic properties and charge ordering in manganites are reviewed, which covers lattice structure, magnetic and electronic properties in both ferromagnetic and antiferromagnetic based manganites. In addition to review the literatures, this article identifies the promising avenues for the future research in this area.

Characterization of the nonstructural protein NS80 of grass carp reovirus

Nonstructural proteins of members of the family Reoviridae are believed to play significant roles in the virus replication cycle. Phylogenetic analyses indicate that the nonstructural protein NS80 of grass carp reovirus, encoded by a gene of Segment 4 (S4), is a primary determinant that is related to the formation of viroplasmic inclusion bodies (VIB), where viral replication and assembly are thought to occur. To understand the role of the NS80 protein in viral replication, an initial investigation of NS80 gene expression in both infected and transfected cells was conducted. Transmission electron microscopy results indicate that replication and assembly of GCRV occur within VIB-like structures in the perinuclear region of the cell cytoplasm. Furthermore, expression of the S4 gene in infected cells was detected with an NS80-specific antibody by western blot and immunofluorescence. Moreover, globular VIB-like structures were observed when expressing GFP-derived full-length NS80 (pEGFP-C1/NS80) and recombinants containing the C-terminal conserved region (pEGFP-C1/NS80335–724) in transfected Vero. No such structures were detected in cells transfected with an N-terminal recombinant (pEGFP-C1/NS801–334), suggesting that the NS80 C-terminal conserved region may be involved in the formation of inclusion structures. These data provide a foundation for further functional studies of NS80 related to viral inclusion formation in viral replication.

Manufacture, microstructure and mechanical properties of Mo-W-N nanostructured hard films

Mo1−xWxNy (x = 0–0.67) hard films were fabricated on wafers of silicon and high speed steel by dc magnetron sputtering technique. The effect of tungsten concentration on the phase composition, microstructure, surface morphology, hardness, adhesion, and corrosion resistance of the films was studied by X-ray diffraction, scanning electron microscopy, nano-indentation, and scratch test. It was found that if the W concentration (x) in the film is in the range of 0–0.52, the films exhibit fcc (Mo,W)Ny single phase where larger W atoms substituted Mo atoms in fcc MoNy. At higher x values (x > 0.52) the films exhibit a two-phase structure consisting of fcc (Mo,W)Ny and pure bcc tungsten phase. The hardness of the Mo1−xWxNy films increases at first with increasing x, and then decreases after passing a maximum. The maximum hardness of 47 GPa is obtained at x = 0.37 corresponding to an adhesion strength of 60 N. The Mo–W–N coated high speed steel has a lower corrosion current density and higher corrosion potential than the bare high speed steel substrates.

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid, which was named as pR/GCRV-VP7, was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover, the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein) genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5 days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

Internal friction in Al bicrystals with ⟨111⟩ tilt and twist grain boundaries

Internal friction measurements were performed on various ⟨111⟩ tilt and twist grain boundaries in high-purity Al bicrystals. The temperature dependence of the grain boundary internal friction peak was determined, and the activation parameters of grain boundary relaxation were obtained. These parameters were found to change in a wide range depending on boundary geometry. The activation enthalpy of boundary relaxation and the pre-exponential factor of the relaxation time are related according to the compensation effect. The results are discussed in terms of the model of correlated relaxations. Bicrystals with vicinal Σ3 boundaries were observed to behave like single crystals, i.e. an internal friction peak did not appear. This evidences that both coherent and incoherent (60° ⟨111⟩ tilt) twins possess a high mechanical resistance.

First-principles calculations of transition metal solute interactions with hydrogen in tungsten

We have performed systematic first-principles calculations to predict the interaction between transition metal (TM) solutes and hydrogen in the interstitial site as well as the vacancy in tungsten. We showed that the site preference of the hydrogen atom is significantly influenced by the solute atoms, which can be traced to the charge density perturbation in the vicinity of the solute atom. The solute-H interactions are mostly attractive except for Re, which can be well understood in terms of the competition between the chemical and elastic interactions. The chemical interaction dominates the solute-H interaction for the TM solutes with a large atomic volume and small electronegativity compared to tungsten, while the elastic interaction is primarily responsible for the solute-H interaction for the TM solutes with a small atomic volume and large electronegativity relative to tungsten. The presence of a hydrogen atom near the solute atom has a negative effect on the binding of other hydrogen atoms. The large positive binding energies among the solute, vacancy and hydrogen suggest that they would easily form a defect cluster in tungsten, where the solute-vacancy and vacancy-H interaction contribute greatly while the solute-H interaction contributes a little. Our result provides a sound theoretical explanation for recent experimental phenomena of hydrogen retention in the tungsten alloy and further recommends a suitable W–Re–Ta ternary alloy for possible plasma-facing materials (PFMs) including the consideration of the hydrogen retention.

High-resolution 3D Structures Reveal the Biological Functions of Reoviruses

Viruses in the family Reoviridae are non-enveloped particles comprising a segmented double-stranded RNA genome surrounded by a two-layered or multi-layered icosahedral protein capsid. These viruses are classified into two sub-families based on their particle structural organization. Recent studies have focused on high-resolution three-dimensional structures of reovirus particles by using cryo-electron microscopy (cryo-EM) to approach the resolutions seen in X-ray crystallographic structures. The results of cryo-EM image reconstructions allow tracing of most of the protein side chains, and thus permit integration of structural and functional information into a coherent mechanism for reovirus assembly and entry.

Molecular Characterization of Nonstructural Protein NS38 of Grass Carp Reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 °C rather than 37 °C. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.