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The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

Drugs which independently inhibit a shared target or pathway can have synthetic activities that result in multiplicative instead of merely additive potencies. This characteristic of drug combinations can be quantified by expressing the potency of the combination as if it were a single agent. We show that by optimizing this quantity we can prospectively design drug cocktails with apparent potencies that far exceed any of its individual components. We illustrate the power of this approach, which is based on statistical design of experiments to select optimal drug combinations, and response surface methodology to determine optimal drug ratios, by building a drug cocktail comprised of three antibodies for treating C. difficile infection that is almost 1000-fold more potent than the current, clinically approved antibody monotherapy. High synthetic activities do not require unusual drug interactions, and therefore may be achievable much more readily than generally appreciated.One-Sentence SummaryA development pathway is described for designing antibody cocktails with potencies that far exceed what is achievable with single antibodies

Arthrospira platensis(commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulina’s genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

Background: The presence of extrahepatic disease, bilobar disease or greater than four hepatic lesions were once considered contraindications to hepatectomy for colorectal metastases but are being reconsidered. We reviewed our experience with resection of extrahepatic disease (EHD) at the time of hepatectomy for metastatic colorectal cancer to determine the impact on perioperative and long-term outcome. Methods: The medical records of 441 patients who underwent hepatectomy for colorectal cancer metastases from 1989 to 2010 were reviewed. Demographics, clinicopathologic characteristics, and outcomes were compared between those with and without extrahepatic disease. Overall survival curves were constructed using the Kaplan Meier method and compared with the log rank test. Multivariate analysis using logistic and Cox proportional hazards regression were used to determine predictors of perioperative mortality and longterm survival, respectively. Results: Fifty-nine (13%) patients presented with EHD at the time of hepatectomy. There were no significant differences between patients with and without EHD with regard to age, gender, disease free interval, or the number and distribution of hepatic metastases. Patients with EHD were as likely to undergo complete (i.e. R0) resection as those with isolated liver metastases (59% vs. 74%. p>0.05). Perioperative mortality in the 441 patients was 2.3%. Only increasing number of liver segments resected and perioperative complications were predictive of perioperative mortality by multivariate analysis. Median overall survival was 20.5 months in patients with EHD compared to 35.2 months in patients without EHD (p

In ripening fruits of tomato (Lycopersicon esculentum L. var 83-G-38), the amounts of cellulose and xyloglucan (XG) remained constant during tissue softening, but the relative molecular weight (Mr) of XG decreased markedly and the Mr of cellulose declined slightly. These changes could have been due to activities of non-specific endo-1,4-[beta]-glucanases and/or buffer-soluble XG endo-transglycosylase, both of which increased when tissue firmness declined most rapidly. Tomato extracts also reduced the viscosity of XG solutions, especially in the presence of added XG oligosac-charides. This depolymerizing (XGase) capacity differed from [beta]-glucanase and XG transglycosylase activity (a) by being almost entirely buffer insoluble, and (b) by declining precipitously during fruit softening. Although it disappeared from ripe fruit, XGase may have functioned in promoting wall loosening at earlier stages of fruit development when its activity was highest. By contrast, during aging of fruit in the ripening-inhibited mutant rin there was no change in Mr of XG or cellulose, and activities of [beta]-glucanases and XG transglycosylase were lower than in wild-type tomato. Nevertheless, some softening of the fruit did take place over time and XG amounts declined, possibly because high XGase activity was maintained in the mutant, unlike in wild-type fruit.

Polymorphism of the dopamine transporter genotype (DAT1) confers a small but significant susceptibility to attention deficit hyperactivity disorder (ADHD). We examined whether the volume of the head of caudate, a striatal structure with high DAT expression that is important for inhibitory function, differs by DAT1 in children diagnosed with the disorder relative to age and IQ matched controls. Volume of the head of caudate was delineated in the right and left hemisphere and compared between 7- and 13-year-old children with and without ADHD (combined type) who were carriers of two (10/10) or one (9/10) copy of the 10-repeat DAT1 allele. Caudate volumes were overall smaller in 10/10 than 9/10 children, particularly in the left than right hemisphere. While DAT1 effects did not vary by ADHD diagnosis, overall caudate volumes were smaller in ADHD relative to control children. Altered caudate development associated with 10-repeat homozygosity of DAT1 may contribute susceptibility to ADHD.