De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wildtype (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs. (C) 2008 Elsevier B.V. All rights reserved.

Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.

Remarks on common hypercyclic vectors

We treat the question of existence of common hypercyclic vectors for families of continuous linear operators. It is shown that for any continuous linear operator T on a complex Fréchet space X and a set ? ? R+ × C which is not of zero three-dimensional Lebesgue measure, the family {a T + b I : (a, b) ? ?} has no common hypercyclic vectors. This allows to answer negatively questions raised by Godefroy and Shapiro and by Aron. We also prove a sufficient condition for a family of scalar multiples of a given operator on a complex Fréchet space to have a common hypercyclic vector. It allows to show that if D = {z ? C : | z | < 1} and f ? H8 (D) is non-constant, then the family {z Mf{star operator} : b- 1 < | z | < a- 1} has a common hypercyclic vector, where Mf : H2 (D) ? H2 (D), Mf f = f f, a = inf {| f (z) | : z ? D} and b = sup {| f (z) | : | z | ? D}, providing an affirmative answer to a question by Bayart and Grivaux. Finally, extending a result of Costakis and Sambarino, we prove that the family {a Tb : a, b ? C {set minus} {0}} has a common hypercyclic vector, where Tb f (z) = f (z - b) acts on the Fréchet space H (C) of entire functions on one complex variable.

On the set of hypercyclic vectors for the differentiation

Let D be the differentiation operator Df = f' acting on the Fréchet space H of all entire functions in one variable with the standard (compact-open) topology. It is known since the 1950’s that the set H(D) of hypercyclic vectors for the operator D is non-empty. We treat two questions raised by Aron, Conejero, Peris and Seoane-Sepúlveda whether the set H(D) contains (up to the zero function) a non-trivial subalgebra of H or an infinite-dimensional closed linear subspace of H. In the present article both questions are answered affirmatively.

Comparing large graphs efficiently by margins of feature vectors

Measuring the structural similarity of graphs is a challenging and outstanding problem. Most of the classical approaches of the so-called exact graph matching methods are based on graph or subgraph isomorphic relations of the underlying graphs. In contrast to these methods in this paper we introduce a novel approach to measure the structural similarity of directed and undirected graphs that is mainly based on margins of feature vectors representing graphs. We introduce novel graph similarity and dissimilarity measures, provide some properties and analyze their algorithmic complexity. We find that the computational complexity of our measures is polynomial in the graph size and, hence, significantly better than classical methods from, e.g. exact graph matching which are NP-complete. Numerically, we provide some examples of our measure and compare the results with the well-known graph edit distance. (c) 2006 Elsevier Inc. All rights reserved.

Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

THE IMPROVED SIGN BIT ENCRYPTION OF MOTION VECTORS FOR H.264/AVC

In this paper, an improved video encryption method for encrypting the sign bit of motion vectors is proposed based on H.264/AVC, which belongs to selective encryption. This method improves upon previous work involving the sign bit encryption of motion vectors by ensuring the four candidates for the encrypted motion vectors are always located in two orthogonal lines. The improved method can provide a much more effective scrambling effect while keeping the encrypted stream format-compliant and the compression ratio unchanged. The combination of the proposed method with encryption of intra prediction modes can further enhance the scrambling effect, especially for the first few frames which are left clear when only the motion vectors are encrypted.

Interpretable semantic vectors from a joint model of brain-and text-based meaning

Vector space models (VSMs) represent word meanings as points in a high dimensional space. VSMs are typically created using a large text corpora, and so represent word semantics as observed in text. We present a new algorithm (JNNSE) that can incorporate a measure of semantics not previously used to create VSMs: brain activation data recorded while people read words. The resulting model takes advantage of the complementary strengths and weaknesses of corpus and brain activation data to give a more complete representation of semantics. Evaluations show that the model 1) matches a behavioral measure of semantics more closely, 2) can be used to predict corpus data for unseen words and 3) has predictive power that generalizes across brain imaging technologies and across subjects. We believe that the model is thus a more faithful representation of mental vocabularies.