Development of a minigenome cassette for Lettuce necrotic yellows virus: A first step in rescuing a plant cytorhabdovirus

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product

Antibody degradation in tobacco plants: a predominantly apoplastic process.

BACKGROUND: Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. RESULTS: In this study, we investigated the dynamics of the assembly and breakdown of a human IgG(1)κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space. CONCLUSIONS: The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions

The Interaction of Venus-like, M-dwarf Planets with the Stellar Wind of Their Host Star

We study the interaction between the atmospheres of Venus-like, non-magnetized exoplanets orbiting an M-dwarf star, and the stellar wind using a multi-species Magnetohydrodynaic (MHD) model. We focus our investigation on the effect of enhanced stellar wind and enhanced EUV flux as the planetary distance from the star decreases. Our simulations reveal different topologies of the planetary space environment for sub- and super-Alfvenic stellar wind conditions, which could lead to dynamic energy deposition in to the atmosphere during the transition along the planetary orbit. We find that the stellar wind penetration for non-magnetized planets is very deep, up to a few hundreds of kilometers. We estimate a lower limit for the atmospheric mass-loss rate and find that it is insignificant over the lifetime of the planet. However, we predict that when accounting for atmospheric ion acceleration, a significant amount of the planetary atmosphere could be eroded over the course of a billion years.Comment: 13 pages, 7 figures, accepted to Ap

Recombination Induced Softening and Reheating of the Cosmic Plasma

The atomic recombination process leads to a softening of the matter equation of state as reflected by a reduced generalized adiabatic index, with accompanying heat release. We study the effects of this recombination softening and reheating of the cosmic plasma on the ionization history, visibility function, Cold Dark Matter (CDM) transfer function, and the Cosmic Microwave Background (CMB) spectra. The resulting modifications of the CMB spectrm is 1/10 of WMAP's current error and is comparable to PLANCK's error. Therefore, this effect should be considered when data with higher accuracy are analysed.Comment: 11 pages, 6 figures, Accepted for publication in Monthly Notices of the Royal Astronomical Society; as advised by referee, omit high-baryon mode

Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products.

Plant molecular farming (PMF) is a convenient and cost-effective way to produce high-value recombinant proteins that can be used in the production of a range of health products, from pharmaceutical therapeutics to cosmetic products. New plant breeding techniques (NPBTs) provide a means to enhance PMF systems more quickly and with greater precision than ever before. However, the feasibility, regulatory standing and social acceptability of both PMF and NPBTs are in question. This paper explores the perceptions of key stakeholders on two European Union (EU) Horizon 2020 programmes-Pharma-Factory and Newcotiana-towards the barriers and facilitators of PMF and NPBTs in Europe. One-on-one qualitative interviews were undertaken with N = 20 individuals involved in one or both of the two projects at 16 institutions in seven countries (Belgium, France, Germany, Italy, Israel, Spain and the UK). The findings indicate that the current EU regulatory environment and the perception of the public towards biotechnology are seen as the main barriers to scaling-up PMF and NPBTs. Competition from existing systems and the lack of plant-specific regulations likewise present challenges for PMF developing beyond its current niche. However, respondents felt that the communication of the benefits and purpose of NPBT PMF could provide a platform for improving the social acceptance of genetic modification. The importance of the media in this process was highlighted. This article also uses the multi-level perspective to explore the ways in which NPBTs are being legitimated by interested parties and the systemic factors that have shaped and are continuing to shape the development of PMF in Europe

Nuclear symmetry potential in the relativistic impulse approximation

Using the relativistic impulse approximation with the Love-Franey \textsl{NN} scattering amplitude developed by Murdock and Horowitz, we investigate the low-energy (100 MeV$\leq E_{\mathrm{kin}}\leq 400$ MeV) behavior of the nucleon Dirac optical potential, the Schr\"{o}dinger-equivalent potential, and the nuclear symmetry potential in isospin asymmetric nuclear matter. We find that the nuclear symmetry potential at fixed baryon density decreases with increasing nucleon energy. In particular, the nuclear symmetry potential at saturation density changes from positive to negative values at nucleon kinetic energy of about 200 MeV. Furthermore,the obtained energy and density dependence of the nuclear symmetry potential is consistent with those of the isospin- and momentum-dependent MDI interaction with $x=0$, which has been found to describe reasonably both the isospin diffusion data from heavy-ion collisions and the empirical neutron-skin thickness of $^{208}$Pb.Comment: 8 pages, 5 figures, revised version to appear in PR

Collective multipole excitations in a microscopic relativistic approach

A relativistic mean field description of collective excitations of atomic nuclei is studied in the framework of a fully self-consistent relativistic random phase approximation (RRPA). In particular, results of RRPA calculations of multipole giant resonances and of low-lying collective states in spherical nuclei are analyzed. By using effective Lagrangians which, in the mean-field approximation, provide an accurate description of ground-state properties, an excellent agreement with experimental data is also found for the excitation energies of low-lying collective states and of giant resonances. Two points are essential for the successful application of the RRPA in the description of dynamical properties of finite nuclei: (i) the use of effective Lagrangians with non-linear terms in the meson sector, and (ii) the fully consistent treatment of the Dirac sea of negative energy states.Comment: 10 figures, submitted to Nucl.Phys.