Assembly of Recombinant Israeli Acute Paralysis virus capsids

The dicistrovirus Israeli Acute Paralysis Virus (IAPV) has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer

Multiplatform Analysis of 12 Cancer Types Reveals Molecular Classification within and across Tissues of Origin

Recent genomic analyses of pathologically-defined tumor types identify “within-a-tissue” disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head & neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multi-platform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All datasets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies

The IAPV genome and origin of sequences used for IAPV fragment expression.

<p><b>A</b>. Genome structure of IAPV dicistrovirus with the two open reading frames and their constituent mature proteins shown. IRES – internal ribosome entry site, IGR – intergenic region. <b>B</b>. The fragments targeted for expression in <i>E.coli</i> and their encoded products. The precise endpoints used were: VP2, VTH→MQC; VP2-4, VTH→FGW; VP3, SKP→ELQ; VP1, INI→ISR. Expression screening for the proteins predicted from the fragments shown in B. <b>C</b>- left. Western blot with anti-His antibody. <b>C</b>-right. Purified VP2 used for the generation of a rabbit serum. Numbers to the left of the blot are protein molecular mass markers and are in kilodaltons.</p

Analysis of empty IAPV capsids.

<p><b>A</b>. Sucrose gradient analysis of Sf9 cell extracts infected with recombinant baculovirus ORF2-5TFS-3C. Each fraction represents a 5% step from 30% to 60% sucrose w/v and is blotted with the IAPV anti VP2 serum. <b>B</b>. EM analysis of peak fractions from the sucrose gradient shown in A. Empty capsids are indicated. A damaged particle is labelled D. Baculovirus nucleocapsids are labelled B. The bar is 100 nm.</p

Comparison between Subsequent Irradiation and Co-Irradiation into SIMP Steel

A modern Chinese ferritic/martensitic steel SIMP, is a new perspective nuclear structural material for the spallation target in accelerator driven sub-critical system. In this work, aimed at exploring the radiation resistance properties of this material, we investigate the differences between simultaneous Fe and He ions irradiation and He implantation of SIMP steel pre-irradiated by Fe self-ions. The irradiations were performed at 300 °C. The radiation-induced hardening was evaluated by nano-indentation, while the lattice disorder was investigated by transmission electron microscopy. Clear differences were found in the material microstructure after the two kinds of the ion irradiation performed. Helium cavities were observed in the co-irradiated SIMP steel, but not the case of He implantation with Fe pre-irradiation. In the same time, the size and density of Frank loops were different in the two different irradiation conditions. The reason for the different observed lattice disorders is discussed

Development of an IAPV capture ELISA.

<p><b>A</b>. Pepscan of 4 monoclonal antibodies to IAPV VP2 on an overlapping peptide library. Filled bars IAPVMAb8; stipple bars IAPVMAb12; striped bars IAPVMAb17; open bars IAPVMAb 27. The relationship between peptide identity and VP2 structure is shown. <b>B</b>. Twin site capture ELISA using capture MAbs 8 (triangles) and 27 (diamonds) as capture layer and probing with HRP-labelled IAPV MAb12. The test sample was a lysate of Sf9 cell infected with the baculovirus expressing ORF-2-5TFS-3C.</p

Design and test of baculovirus expression cassettes.

<p><b>A</b>. Cartoon representations of the various genetic constructs used to assess IAPV antigen expression in infected Sf9 cells. The sequences used are identified. FS- the HIV-1 frameshift site, the polypyrimidine tract of which is indicated. 3C-pro – the IAPV 3C like protease. <b>B</b>. Western blots of Sf9 cells infected for 2 days with recombinant baculoviruses constructed with the cassettes shown in A. In B the upper panel was blotted with rabbit anti-VP2 serum, the middle panel with anti-gp64 and the lower panel is the relative VP2:gp64 level. Numbers to the left of the blots are protein molecular mass markers (M) and are in kilodaltons. The expected position of the blotted antigens is indicated.</p

Receptor Binding Profiles of Avian Influenza Virus Hemagglutinin Subtypes on Human Cells as a Predictor of Pandemic Potential ▿ ‖

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed