Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)(1). Assembly of an infectious virion proceeds in two stages(2). In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation(3). However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-angstrom resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.

Investigation of the [−/A]8 and C1236T genetic variations within the human toll-like receptor 3 gene for association with multiple sclerosis

Multiple sclerosis (MS) is a serious cause of neurological disability among young adults. The clinical course remains difficult to predict, and the pathogenesis of the disease is still modestly understood. Autoimmunity is thought to be a key aspect of the disease, with autoreactive T cells thought to mediate central nervous system (CNS) inflammation to some extent. Toll-like receptors are known to mediate cellular recognition of pathogens by way of patterns of molecular presentation. Toll-like receptor 3 is coded by the gene TLR3 and is recognized as an important factor in virus recognition and is known to be involved in the expression of neuroprotective mediators. We set out to investigate two variations within the TLR3 gene, an 8 bp insertion-deletion \[-/A](8) and a single base-pair variation C1236T, in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We used capillary gel electrophoresis and TaqMan genotyping assay techniques to resolve genotypes for each marker, respectively. Our work found no significant difference between frequencies for TLR3 \[-/A](8) by genotype (chi(2)=1.03, p=0.60) or allele (chi(2)=1.09, p=0.30). Similarly, we found no evidence for the association of TLR3 C1236T by genotype (chi(2)=0.35, p=0.84) or allele frequency (chi(2)=0.31, p=0.58). This work reveals no evidence to suggest that these markers are associated with MS in the tested population. Although the role of TLR3 and the wider toll-like receptor family remain significant in neurological and CNS inflammatory disorders, our current work does not support a role for the two tested variants in this gene with regard to MS susceptibility.

Association study of a functional variant in intron 8 of the dopamine transporter gene and migraine susceptibility

Migraine is a common, genetically influenced neurovascular disorder. The dopamine transporter gene is a candidate for migraine association studies. This study tested a functionally linked variable number tandem repeat (VNTR) in intron 8 of the dopamine transporter gene (DATInt8) in 550 migraine cases (401 with aura, 149 without aura) and 550 non-migraine controls. Chi-squared analysis of the DATInt8 revealed that the allele and genotype frequency distributions for migraine cases (including subtype analysis) and controls were not different (P > 0.1). These findings offer no evidence for an association of the DATInt8 with migraine with and without aura and therefore do not implicate the dopamine transporter gene as a modifier of migraine risk.

GEF-H1-RhoA signaling pathway mediates LPS-induced NF-κB transactivation and IL-8 synthesis in endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (Guanine-nucleotide exchange factor-H1)-RhoA signalling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signalling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.

Morphology changes and mechanistic aspects of the electrochemically-induced reversible solid-solid transformation of microcrystalline TCNQ into Co TCNQ 2-based materials (TCNQ= 7, 7, 8, 8-Tetracyanoquinodimethane)

The chemically reversible solid−solid phase transformation of a TCNQ-modified glassy carbon, indium tin oxide, or metal electrode into Co\[TCNQ]2(H2O)2 material in the presence of Co2+(aq) containing electrolytes has been induced and monitored electrochemically. Voltammetric data reveal that the TCNQ/Co\[TCNQ]2(H2O)2 interconversion process is independent of electrode material and identity of cobalt electrolyte anion. However, a marked dependence on electrolyte concentration, scan rate, and method of electrode modification (drop casting or mechanical attachment) is found. Cyclic voltammetric and double potential step chronoamperometric measurements confirm that formation of Co\[TCNQ]2(H2O)2 occurs through a rate-determining nucleation and growth process that initially involves incorporation of Co2+(aq) ions into the reduced TCNQ crystal lattice at the TCNQ|electrode|electrolyte interface. Similarly, the reverse (oxidation) process, which involves transformation of solid Co\[TCNQ]2(H2O)2 back to parent TCNQ crystals, also is controlled by nucleation−growth kinetics. The overall chemically reversible process that represents this transformation is described by the reaction:  2TCNQ0(s) + 2e- + Co2+(aq) + 2H2O \[Co(TCNQ)2(H2O)2](s). Ex situ SEM images illustrated that this reversible TCNQ/Co\[TCNQ]2(H2O)2 conversion process is accompanied by drastic size and morphology changes in the parent solid TCNQ. In addition, different sizes of needle-shaped nanorod/nanowire crystals of Co\[TCNQ]2(H2O)2 are formed depending on the method of surface immobilization.

Electrocrystallization of phase I, CuTCNQ (TCNQ= 7, 7, 8, 8-tetracyanoquinodimethane), on indium tin oxide and boron-doped diamond electrodes

The electrochemical reduction of TCNQ to TCNQ•- in acetonitrile in the presence of [Cu(MeCN)4]+ has been undertaken at boron-doped diamond (BDD) and indium tin oxide (ITO) electrodes. The nucleation and growth process at BDD is similar to that reported previously at metal electrodes. At an ITO electrode, the electrocrystallization of more strongly adhered, larger, branched, needle-shaped phase I CuTCNQ crystals is detected under potential step conditions and also when the potential is cycled over the potential range of 0.7 to −0.1 V versus Ag/AgCl (3 M KCl). Video imaging can be used at optically transparent ITO electrodes to monitor the growth stage of the very large branched crystals formed during the course of electrochemical experiments. Both in situ video imaging and ex situ X-ray diffraction and scanning electron microscopy (SEM) data are consistent with the nucleation of CuTCNQ taking place at a discrete number of preferred sites on the ITO surface. At BDD electrodes, ex situ optical images show that the preferential growth of CuTCNQ occurs at the more highly conducting boron-rich areas of the electrode, within which there are preferred sites for CuTCNQ formation.

Characterisation of two distinctly different processes associated with the electrocrystallization of microcrystals of phase I CuTCNQ (TCNQ= 7, 7, 8, 8-tetracyanoquinodimethane)

Semi-conducting phase I CuTCNQ (TCNQ = 7,7,8,8-tetracyanoquinodimethane), which is of considerable interest as a switching device for memory storage materials, can be electrocrystallized from CH3CN via two distinctly different pathways when TCNQ is reduced to TCNQ˙− in the presence of [Cu(MeCN)4]+. The first pathway, identified in earlier studies, occurs at potentials where TCNQ is reduced to TCNQ˙− and involves a nucleation–growth mechanism at preferred sites on the electrode to produce arrays of well separated large branched needle-shaped phase I CuTCNQ crystals. The second pathway, now identified at more negative potentials, generates much smaller needle-shaped phase I CuTCNQ crystals. These electrocrystallize on parts of the surface not occupied in the initial process and give rise to film-like characteristics. This process is attributed to the reduction of Cu+[(TCNQ˙−)(TCNQ)] or a stabilised film of TCNQ via a solid–solid conversion process, which also involves ingress of Cu+via a nucleation–growth mechanism. The CuTCNQ surface area coverage is extensive since it occurs at all areas of the electrode and not just at defect sites that dominate the crystal formation sites for the first pathway. Infrared spectra, X-ray diffraction, surface plasmon resonance, quartz crystal microbalance, scanning electron microscopy and optical image data all confirm that two distinctly different pathways are available to produce the kinetically-favoured and more highly conducting phase I CuTCNQ solid, rather than the phase II material.

Electrochemically-induced TCNQ/Mn TCNQ 2 (H2O) 2 (TCNQ= 7, 7, 8, 8-Tetracyanoquinodimethane) solid-solid interconversion : two voltammetrically distinct processes that allow selective generation of nanofiber or nanorod network morphologies

Unlike the case with other divalent transition metal M\[TCNQ](2)(H(2)O)(2) (M = Fe, Co, Ni) analogues, the electrochemically induced solid-solid phase interconversion of TCNQ microcrystals (TCNQ = 7,7,8,8-tetracyanoquinodimethane) to Mn\[TCNQ](2)(H(2)O)(2) occurs via two voltammetrically distinct, time dependent processes that generate the coordination polymer in nanofiber or rod-like morphologies. Careful manipulation of the voltammetric scan rate, electrolysis time, Mn(2+)((aq)) concentration, and the method of electrode modification with solid TCNQ allows selective generation of either morphology. Detailed ex situ spectroscopic (IR, Raman), scanning electron microscopy (SEM), and X-ray powder diffraction (XRD) characterization clearly establish that differences in the electrochemically synthesized Mn-TCNQ material are confined to morphology. Generation of the nanofiber form is proposed to take place rapidly via formation and reduction of a Mn-stabilized anionic dimer intermediate, \[(Mn(2+))(TCNQ-TCNQ)(2)(*-)], formed as a result of radical-substrate coupling between TCNQ(*-) and neutral TCNQ, accompanied by ingress of Mn(2+) ions from the aqueous solution at the triple phase TCNQ/electrode/electrolyte boundary. In contrast, formation of the nanorod form is much slower and is postulated to arise from disproportionation of the \[(Mn(2+))(TCNQ-TCNQ)(*-)(2)] intermediate. Thus, identification of the time dependent pathways via the solid-solid state electrochemical approach allows the crystal size of the Mn\[TCNQ](2)(H(2)O)(2) material to be tuned and provides new mechanistic insights into the formation of different morphologies.

Electrochemical formation of porous copper 7, 7, 8, 8-tetracyanoquinodimethane and copper 2, 3, 5, 6-tetrafluoro-7, 7, 8, 8-tetracyanoquinodimethane honeycomb surfaces with superhydrophobic properties

The electrochemical formation of highly porous CuTCNQ (TCNQ = 7,7,8,8-tetracyanoquinodimethane) and CuTCNQF4 (TCNQF4 = 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane) materials was undertaken via the spontaneous redox reaction between a porous copper template, created using a hydrogen bubbling template technique, and an acetonitrile solution containing TCNQ or TCNQF4. It was found that activation of the surface via vigorous hydrogen evolution that occurs during porous copper deposition and TCNQ mass transport being hindered through the porous network of the copper template influenced the growth of CuTCNQ and CuTCNQF4. This approach resulted in the fabrication of a honeycomb layered type structure where the internal walls consist of very fine crystalline needles or spikes. This combination of microscopic and nanoscopic roughness was found to be extremely beneficial for anti-wetting properties where superhydrophobic materials with contact angles as high as 177° were created. Given that CuTCNQ and CuTCNQF4 have shown potential as molecular based electronic materials in the area of switching and field emission, the creation of a surface that is moisture resistant may be of applied interest.

Effect of Imidazolium-based Ionic Liquids on the nanoscale morphology of CuTCNQ (TCNQ= 7, 7, 8, 8-tetracyanoquinodimethane) metal-organic semiconductors

We demonstrate for the first time the ionic-liquid-mediated synthesis of nanostructured CuTCNQ by the simple immersion of copper in a solution of TCNQ where the viscosity of the medium significantly impacts the corrosion–crystallization process and the final morphology of the material.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67 348 polypeptide encoded by genome segment 8

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid sequence of a ~43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto- catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a ~43K major capsid protein.