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

Dynamic double directional propagation channel analysis with dual circular arrays

Dynamic double directional propagation channel analysis with dual circular arrays. (pp. 6 p). Peer reviewed versio

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

Following the rivers: historical reconstruction of California voles Microtus californicus (Rodentia: Cricetidae) in the deserts of eastern California

The California vole, Microtus californicus, restricted to habitat patches where water is available nearly year-round, is a remnant of the mesic history of the southern Great Basin and Mojave deserts of eastern California. The history of voles in this region is a model for species-edge population dynamics through periods of climatic change. We sampled voles from the eastern deserts of California and examined variation in the mitochondrial cytb gene, three nuclear intron regions, and across 12 nuclear microsatellite markers. Samples are allocated to two mitochondrial clades: one associated with southern California and the other with central and northern California. The limited mtDNA structure largely recovers the geographical distribution, replicated by both nuclear introns and microsatellites. The most remote population, Microtus californicus scirpensis at Tecopa near Death Valley, was the most distinct. This population shares microsatellite alleles with both mtDNA clades, and both its northern clade nuclear introns and southern clade mtDNA sequences support a hybrid origin for this endangered population. The overall patterns support two major invasions into the desert through an ancient system of riparian corridors along streams and lake margins during the latter part of the Pleistocene followed by local in situ divergence subsequent to late Pleistocene and Holocene drying events. Changes in current water resource use could easily remove California voles from parts of the desert landscape

Synthesis and Single Crystal X-Ray Crystallographic Analysis of 2-oxo-2,3- dihydropyrimidin-1-ium{trichloridopyrimidin-2(1H)-one} Cobaltate (II) [H2pymo][CoCl3(Hpymo)]

Mechanochemical synthetic method was employed to synthesise bis(2-oxo-2,3-dihydropyrimidin-1-ium) tetrachlorocobaltate(II) [H2pymo]2[CoCl4]. The compound was re-crystallized in diethyl ether to obtain a suitable single crystal for X-ray diffraction analysis which revealed a molecule crystallizes in the orthorhombic crystal system with the chiral space group P212121 and a Flack parameter value of 0.514 (13). The asymmetric unit of the cell contains one discrete [H2pymo]+ cation and a [CoCl3(Hpymo)]- anion which are bonded to each other through N—H···O and N—H···Cl interactions.Keywords: Crystal Engineering, Mechanochemical, Single Crystal X-ray Diffractio

A novel precursor towards buffer layer materials: the first solution based CVD of zinc oxysulfide

We report the first solution based deposition of zinc oxysulfide, Zn(O,S), thin films via aerosol-assisted chemical vapour deposition (AACVD) facilitated by the use of a specifically designed precursor: [Zn8(SOCCH3)12S2] (1). This buffer layer material, synthesised from the dual source AACVD reaction of 1 with ZnEt2 and MeOH was analysed via X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM), Hall effect measurements and UV/vis spectroscopy. The film was highly transparent (490%), conductive (r = 0.02998 O cm) and had a high charge carrier concentration (1.36 1019 cm3 ), making it a good contendor as a buffer layer in thin film photovoltaics. In an additional study, large area films were deposited and mapped to correlate compositional variation to optoelectronic propertie

The changing shape of patients with idiopathic pulmonary fibrosis

This is the author accepted manuscript. The final version is available from BMJ via the DOI in this record

Numerical evaluation of shear strength of CFS shear wall panels for different height-to-width ratios

This paper presents a numerical evaluation of the shear strength of Cold Formed Steel Shear Wall Panels (CFS-SWPs) having 1.33:1 and 1:1 height-to-width aspect ratios with 0.76 mm steel plate sheathing thickness and 1:4,  1.33:1 and 1:1  height-to-width aspect ratios with 0.46 mm steel plate sheathing thickness, which are not provided by AISI S400. For this purpose, shell finite element (FE) models, validated with test results, are completed in ABAQUS v2018 with nonlinear geometry, material and connection. A good agreement is achieved between experimental and numerical results in terms of shear strength-lateral displacement and failure modes.It is concluded that, for a fixed height-to-width aspect ratio, the shear strength of SWPs having different screws spacing varying from 50.4 mm up to 152.4 can be assessed by interpolation using this FE method. However, by interloping the shear strength from 4:1 to 1:1 height-to-width aspect ratio, the shear strength can be underestimated; hence, it is more economical for practicing engineers to use the shear strength assessed by this proposed FE method for 1.33:1 and 1:1 height-to-width aspect ratios. Moreover, the effect of the sheathing thickness having 0.46 mm is evaluated and proposed as it lacks in data provided by the code (i.e., AISI S400)