Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches

Chlorpyrifos Affects Phenotypic Outcomes in a Model of Mammalian Neurodevelopment: Critical Stages Targeting Differentiation in PC12 Cells

The organophosphate insecticide chlorpyrifos (CPF) adversely affects mammalian brain development through multiple mechanisms. To determine if CPF directly affects neuronal cell replication and phenotypic fate, and to identify the vulnerable stages of differentiation, we exposed PC12 cells, a model for mammalian neurodevelopment, to CPF concentrations spanning the threshold for cholinesterase inhibition (5–50 μM) and conducted evaluations during mitosis and in early and mid-differentiation. In undifferentiated cells, exposure to 5 μM CPF for 1–3 days reduced DNA synthesis significantly without eliciting cytotoxicity. At the same time, CPF increased the expression of tyrosine hydroxylase (TH), the enzymatic marker for the catecholamine phenotype, without affecting choline acetyltransferase (ChAT), the corresponding marker for the cholinergic phenotype. Upon exposure to nerve growth factor (NGF), PC12 cells developed neuritic projections in association with vastly increased TH and ChAT expression accompanying differentiation into the two phenotypes. CPF exposure begun at the start of differentiation significantly reduced ChAT but not TH activity. In contrast, when CPF was added in mid-differentiation (4 days of NGF pretreatment), ChAT was unaffected and TH was increased slightly. Thus, CPF exerts stage-specific effects, reducing DNA synthesis in the undifferentiated state, impairing development of the cholinergic phenotype at the start of differentiation, and promoting expression of the catecholaminergic phenotype both in undifferentiated and differentiated cells. CPF administration in vivo produces deficits in the number of neurons and cholinergic function, and because we were able to reproduce these effects in vitro, our results suggest that CPF directly influences the phenotypic fate of neuronal precursors

Fielding the NIF Cryogenic Ignition Target

The United States Department of Energy has embarked on a campaign to conduct credible fusion ignition experiments on the National Ignition Facility (NIF) at the Lawrence Livermore National Laboratory in 2010. The target assembly specified for this campaign requires the formation of a deuterium/tritium (DT) fuel ice layer on the inside of a 2 millimeter diameter capsule positioned at the center of a 9 millimeter long by 5 millimeter diameter cylinder, called a hohlraum. The ice layer requires micrometer level accuracy and must be formed and maintained at temperatures below 19 K. At NIF shot time, the target must be positioned at the center of the NIF 10 meter diameter target chamber, aligned to the laser beam lines and held stable to less than 7 micrometers rms. We have completed the final design and are integrating the systems necessary to create, characterize and field the cryogenic target for ignition experiments. These designs, with emphasis on the challenges of fielding a precision cryogenic positioning system will be presented

Ignition Target Fabrication and Fielding for the National Ignition Facility

Continued advances in the design of ignition targets have stimulating new development paths for target fabrication, with potentially important simplifications for fielding cryogenic ignition targets for the National Ignition Facility. Including graded dopants in ablators as well as optimizing capsule and fuel layer dimensions increase implosion stability. This has led to developments of micron-scale fill tubes to fill and field the targets. Rapid progress has been made in development of the graded dopant layers in capsules as well as their characterization, in fabrication methods for micro-fill-tubes, and in fuel fill control with these fill tubes. Phase-contrast x-ray radiography has allowed characterization of fuel layers in beryllium targets. This target development program includes participation from General Atomics, Lawrence Livermore National Laboratory, and Los Alamos National Laboratory

National Ignition Facility Target Design and Fabrication

The current capsule target design for the first ignition experiments at the NIF Facility beginning in 2009 will be a copper-doped beryllium capsule, roughly 2 mm in diameter with 160-{micro}m walls. The capsule will have a 75-{micro}m layer of solid DT on the inside surface, and the capsule will driven with x-rays generated from a gold/uranium cocktail hohlraum. The design specifications are extremely rigorous, particularly with respect to interfaces, which must be very smooth to inhibit Rayleigh-Taylor instability growth. This paper outlines the current design, and focuses on the challenges and advances in capsule fabrication and characterization; hohlraum fabrication, and D-T layering and characterization

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion