High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Indocyanine green enhanced subthreshold diode-laser micropulse photocoagulation treatment of chronic central serous chorioretinopathy.

PURPOSE: To assess the efficacy and safety of indocyanine green (ICG) dye-enhanced subthreshold diode-laser micropulse (SDM) photocoagulation in patients with chronic central serous chorioretinopathy (CCSC) with no spontaneous resolution 6 months after the onset of the disease. STUDY DESIGN: Interventional prospective non-comparative case series of seven patients presenting with CCSC with well-defined active leaking sites (ALS) suitable for laser treatment and with serous neuroepithelial detachment persisting for 6 or more months. METHODS: SDM treatment was performed 15 minutes after the injection of 25 mg of ICG in 2 cc of 5% glucose solution. ALS were treated with a series of 50 500-ms exposures separated by 500-ms pauses. Each 500-ms exposure delivered a train of 250 micropulses at 10% duty cycle and 500 mW power. ICG angiographic images were taken after the treatment without new ICG injection, to check for the presence of laser-induced spots of background hypofluorescence at the treated leakage sites. RESULTS: Within 7-14 days after treatment, all the patients showed improved visual acuity and reduction of serous neuroepithelial detachment on OCT. No signs of laser lesions were visible at fundus examination and on fluorescein angiography. In a period ranging from 4 to 8 weeks, the neuroepithelial detachment was completely resolved in five patients and reduced in two patients. At the 12-month follow-up visits, no recurrence had occurred in the patients, with resolution of the serous neuro-epithelial detachment, and no worsening of the serous detachment or of VA was noted in the patients with incomplete recovery. CONCLUSIONS: These preliminary results suggest that ICG dye-enhanced SMD photocoagulation appears to be an effective treatment, and can represent a viable approach for the management of CSCC with persistent serous neuroepithelial detachment. Post-treatment ICG angiography, without new ICG dye injection, can be used to verify the placement of the SDM laser applications.

Theoretical bases of non-ophthalmoscopically visible endpoint photocoagulation.

Laser photocoagulation is a photothermal process in which heat is produced by the absorption of laser energy by targeted tissues. The purpose of the treatment is to induce thermal therapeutic damage, which causes biological reactions and ultimately beneficial effects. The current endpoint of laser photocoagulation of the chorioretina is an ophthalmoscopically visible retinal whitening. Retinal blanching is the sign that the retina itself has been thermally damaged and results in a number of undesired adverse events. The mechanisms underpinning the efficacy of laser photocoagulation are still poorly understood. However, recent hypotheses postulate that full thickness retinal damage may not be needed to obtain beneficial therapeutic effectiveness. Preliminary studies with laser photocoagulation on animals demonstrated the ability to create therapeutic lesions confined around the Retinal Pigment Epithelium (RPE) cells without causing apparent damage to the overlying retina. The laser impacts were not visible by slit lamp biomicroscopy at the time of laser delivery. Recent experiments showed that the beneficial effect of retinal photocoagulation is mediated by factors derived from the RPE. Non Ophthalmoscopically Visible Endpoint Photocoagulation (NOVEP) protocols might allow treatments that confine minimal therapeutic damage around the cells of the RPE and minimize the damage to the neurosensory retina.

Crossflow microfiltration of animal cells.

Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s(-1) (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m(2) h (depending on cell size) can be sustained during 20-fold concentration from 2.5 x 10(6) cells/ml, while maintaining high cell viability.

Fractionation of recombinant tumor necrosis factor using hydrophobic and hydrophilic membranes.

Recombinant tumor necrosis factor (TNF) is expressed in Escherichia coli as a soluble intracellular protein. A purification process is described that incorporates a hydrophilic membrane (cellulosic) separation followed by a hydrophobic one (PTFE). The hydrophilic step is a traditional one in that species are separated primarily on the basis of size. The hydrophobic step separates species on the basis of parameters apparently not related to size. By combining these two steps, an increase in TNF purity of 7-10-fold can be achieved with a yield of 50%. The effects of cellular debris and pH on selectivity and recovery of the hydrophobic filtration step are discussed.