Merkel cell polyomavirus large T antigen disrupts lysosome clustering by translocating human Vam6p from the cytoplasm to the nucleus

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication

Ratios of BB and DD Meson Decay Constants in Relativistic Quark Model

We calculate the ratios of $B$ and $D$ meson decay constants by applying the variational method to the relativistic hamiltonian of the heavy meson. We adopt the Gaussian and hydrogen-type trial wave functions, and use six different potentials of the potential model. We obtain reliable results for the ratios, which are similar for different trial wave functions and different potentials. The obtained ratios show the deviation from the nonrelativistic scaling law, and they are in a pretty good agreement with the results of the Lattice calculations.Comment: 13 pages, 1 Postscript figur

Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~ 45.9 kDa, ~ 37 kDa, and ~ 83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50 > 100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24 h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24 h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24 h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24 h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted

The Structure of a Rigorously Conserved RNA Element within the SARS Virus Genome

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold

Relativistic Calculation of the Meson Spectrum: a Fully Covariant Treatment Versus Standard Treatments

A large number of treatments of the meson spectrum have been tried that consider mesons as quark - anti quark bound states. Recently, we used relativistic quantum "constraint" mechanics to introduce a fully covariant treatment defined by two coupled Dirac equations. For field-theoretic interactions, this procedure functions as a "quantum mechanical transform of Bethe-Salpeter equation". Here, we test its spectral fits against those provided by an assortment of models: Wisconsin model, Iowa State model, Brayshaw model, and the popular semi-relativistic treatment of Godfrey and Isgur. We find that the fit provided by the two-body Dirac model for the entire meson spectrum competes with the best fits to partial spectra provided by the others and does so with the smallest number of interaction functions without additional cutoff parameters necessary to make other approaches numerically tractable. We discuss the distinguishing features of our model that may account for the relative overall success of its fits. Note especially that in our approach for QCD, the resulting pion mass and associated Goldstone behavior depend sensitively on the preservation of relativistic couplings that are crucial for its success when solved nonperturbatively for the analogous two-body bound-states of QED.Comment: 75 pages, 6 figures, revised content

An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC50 for RT was 0.08 ± 0.004 ng/mL whereas the IC50 for RT + 100 μM eGCG was 3.02 ± 0.572 ng/mL, indicating that eGCG mediated a significant (p < 0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC50 values were obtained for RT (0.54 ± 0.024 ng/mL) and RT + 100 μM eGCG (0.68 ± 0.235 ng/mL) again using 100 μM eGCG and was significant (p = 0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100 μg/mL (i.e. 178 and 223 μM respectively) of eGCG mediating a significant (p = 0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4 ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10 ng/mL and 5 ng/mL of RT was used. The addition of 1000 μM and 100 μM eGCG mediated a significant (p = 0.0015 and < 0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1 μg eGCG. Further, eGCG (100 μM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p = 0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin

Wave functions and decay constants of BB and DD mesons in the relativistic potential model

With the decay constants of $D$ and $D_s$ mesons measured in experiment recently, we revisit the study of the bound states of quark and antiquark in $B$ and $D$ mesons in the relativistic potential model. The relativistic bound state wave equation is solved numerically. The masses, decay constants and wave functions of $B$ and $D$ mesons are obtained. Both the masses and decay constants obtained here can be consistent with the experimental data. The wave functions can be used in the study of $B$ and $D$ meson decays.Comment: more discussion added, to appear in EPJ

Construction and physiochemical characterisation of a multi-composite, potential oral vaccine delivery system (VDS)

An increasing human population requires a secure food supply and a cost effective, oral vaccine delivery system for livestock would help facilitate this end. Recombinant antigen adsorbed onto silica beads and coated with myristic acid, was released (∼15% (w/v)) over 24 h at pH 8.8. At pH 2, the myristic acid acted as an enteric coating, protecting the antigen from a variety of proteases. The antigen adsorbed onto silica particles, coated in myristic acid had a conserved secondary structure (measured by circular dichroism (CD) spectroscopy) following its pH-triggered release. Small angle neutron scattering (SANS) was used to measure the thickness of the adsorbed antigen, finding that its adsorbed conformation was slightly greater than its solution radius of gyration, i.e. 120–160 Å. The addition of myristic acid led to a further increase in particle size, with scattering data consistent with an acid thickness slightly greater than a monolayer of fully extended alkyl chains and a degree of hydration of around 50%. Whilst adsorbed onto the silica and coated in myristic acid, the protein was stable over 14 days at 42 °C, indicating a reduced need for cold chain storage. These data indicate that further investigation is warranted into the development of this technology

Accounting for International War: The State of the Discipline

In studies of war it is important to observe that the processes leading to so frequent an event as conflict are not necessarily those that lead to so infrequent an event as war. Also, many models fail to recognize that a phenomenon irregularly distributed in time and space, such as war, cannot be explained on the basis of relatively invariant phenomena. Much research on periodicity in the occurrence of war has yielded little result, suggesting that the direction should now be to focus on such variables as diffusion and contagion. Structural variables, such as bipolarity, show contradictory results with some clear inter-century differences. Bipolarity, some results suggest, might have different effects on different social entities. A considerable number of studies analysing dyadic variables show a clear connection between equal capabilities among contending nations and escalation of conflict into war. Finally, research into national attributes often points to strength and geographical location as important variables. In general, the article concludes, there is room for modest optimism, as research into the question of war is no longer moving in non-cumulative circles. Systematic research is producing results and there is even a discernible tendency of convergence, in spite of a great diversity in theoretical orientations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69148/2/10.1177_002234338101800101.pd