Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed

A mathematical model quantifies proliferation and motility effects of TGF--β\beta on cancer cells

Transforming growth factor (TGF) $\beta$ is known to have properties of both a tumor suppressor and a tumor promoter. While it inhibits cell proliferation, it also increases cell motility and decreases cell--cell adhesion. Coupling mathematical modeling and experiments, we investigate the growth and motility of oncogene--expressing human mammary epithelial cells under exposure to TGF--$\beta$. We use a version of the well--known Fisher--Kolmogorov equation, and prescribe a procedure for its parametrization. We quantify the simultaneous effects of TGF--$\beta$ to increase the tendency of individual cells and cell clusters to move randomly and to decrease overall population growth. We demonstrate that in experiments with TGF--$\beta$ treated cells \textit{in vitro}, TGF--$\beta$ increases cell motility by a factor of 2 and decreases cell proliferation by a factor of 1/2 in comparison with untreated cells.Comment: 15 pages, 4 figures; to appear in Computational and Mathematical Methods in Medicin

Only connect: addressing the emotional needs of Scotland's children and young people

A report on the SNAP (Scottish Needs Assessment Programme) Child and Adolescent Mental Health Phase Two survey. It describes a survey of a wide range of professionals working with children and young people in Scotland, and deals with professional perspectives on emotional, behavioural and psychological problems. Conclusions and recommendations are presented

The neural correlates of emotion processing in juvenile offenders

Individuals with severe antisocial behaviour often demonstrate abnormalities or difficulties in emotion processing. Antisocial behaviour typically onsets before adulthood and is reflected in antisocial individuals at the biological level. We therefore conducted a brain-based study of emotion processing in juvenile offenders. Male adolescent offenders and age-matched non-offenders passively viewed emotional images whilst their brain activity was recorded using electroencephalography. The early posterior negativity (EPN) and the late positive potential (LPP) components were used as indices of emotion processing. For both juvenile offenders and non-offenders, the EPN differentiated unpleasant images from other image types, suggesting that early perceptual processing was not impaired in the offender group. In line with normal emotion processing, the LPP was significantly enhanced following unpleasant images for non-offenders. However, for juvenile offenders, the LPP did not differ across image categories, indicative of deficient emotional processing. The findings indicated that this brain-based hypo-reactivity occurred during a late stage of cognitive processing and was not a consequence of atypical early visual attention or perception. This study is the first to show attenuated emotion processing in juvenile offenders at the neural level. Overall, these results have the potential to inform interventions for juvenile offending

Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE

The Real-Time Evolution of Sakurai's Star (V4334 Sgr) and other (V)LTP Objects

We report on the progress of our on-going campaign to monitor the evolution of the VLTP objects V4334 Sgr and V605 Aql, as well as the suspected (V)LTP object CK Vul. V4334 Sgr does not show signs of increased ionization compared to our previous observations in 2004. We obtained the first radio detection of V605 Aql, indicating a strong increase in radio flux since 1987. We also present the first radio detection of CK Vul and discuss the expansion of the material ejected during the 1670 event.Comment: 4 pages, 2 figures in eps format, IAU symp. 234: `Planetary Nebulae in our Galaxy and Beyond', eds. M. J. Barlow & R. H. Mende