Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

Flowers of the hop plant provide both bitterness and “hoppy” flavor to beer. Hops are, however, both a water and energy intensive crop and vary considerably in essential oil content, making it challenging to achieve a consistent hoppy taste in beer. Here, we report that brewer’s yeast can be engineered to biosynthesize aromatic monoterpene molecules that impart hoppy flavor to beer by incorporating recombinant DNA derived from yeast, mint, and basil. Whereas metabolic engineering of biosynthetic pathways is commonly enlisted to maximize product titers, tuning expression of pathway enzymes to affect target production levels of multiple commercially important metabolites without major collateral metabolic changes represents a unique challenge. By applying state-of-the-art engineering techniques and a framework to guide iterative improvement, strains are generated with target performance characteristics. Beers produced using these strains are perceived as hoppier than traditionally hopped beers by a sensory panel in a double-blind tasting.

No abstract

ICI 200,880 and its close structural analog, ICI 200,355, are representatives of a new chemical class of inhibitors of human neutrophil elastase (HNE). Both compounds are substituted tripeptide ketones, which demonstrated competitive kinetics versus HNE, with identical Ki values of 5.0 x 10(-10) M. The selectivity of ICI 200,880 for HNE versus a variety of enzymes ranged from 150-fold [relative to porcine pancreatic elastase (PPE)] to greater than 360,000-fold in favor of HNE. The compound effectively inhibited HNE-hydrolysis of bovine ligamentum nuchae elastin. In pharmacokinetic studies, ICI 200,880 and ICI 200,355 displayed long retention times when administered directly to the lung and were rapidly eliminated after intravenous administration. Pretreatment of hamsters with either inhibitor before intratracheal administration of HNE produced dose- and time-dependent inhibition of enzyme-induced increases in lung weight, total lavageable red cells, and total lavageable white cells. Aerosol administration of ICI 200,880 produced similar results. Subcutaneous administration of either 50 or 100 mumol/kg (twice/day) of ICI 200,880 for 14 or 28 days prevented the time-dependent increase in alveolar diameter produced by a single intratracheal dose of PPE when compound dosing was initiated 24 h after the enzyme. Treatment of hamsters with the same protocol and doses of ICI 200,880 for 8 wk prevented the destructive lesion induced by a single intratracheal dose of HNE. It is concluded that ICI 200,880 and ICI 200,355 have biochemical, pharmacokinetic, and pharmacologic profiles that make them useful therapeutic agents for understanding the role of HNE in various diseases. ICI 200,880 is presently being evaluated in humans.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.