Yeast Platforms for Production and Screening of Bioactive Derivatives of Rauwolscine.

Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses.

We present a selection design that couples S-adenosylmethionine-dependent methylation to growth. We demonstrate its use in improving the enzyme activities of not only N-type and O-type methyltransferases by 2-fold but also an acetyltransferase of another enzyme category when linked to a methylation pathway in Escherichia coli using adaptive laboratory evolution. We also demonstrate its application for drug discovery using a catechol O-methyltransferase and its inhibitors entacapone and tolcapone. Implementation of this design in Saccharomyces cerevisiae is also demonstrated.

Using multidose eyedrops in a health care setting: a policy and procedural approach to safe and effective treatment of patients.

IMPORTANCE: Quality and safety of eyedrop use for patients treated in a health care setting play a vital role in the delivery of health care. OBJECTIVE: To describe the development of a policy and procedural approach to the use of multidose eyedrops in multiple patients, approved and accepted by The Joint Commission in compliance with preferred practice standards in ophthalmology, the safe handling and administration of multidose eyedrops, and cost benefits of a multidose eyedrop approach. DESIGN, SETTING, AND PATIENTS: Using a policy and procedural approach, we petitioned The Joint Commission for approval and evaluated the cost benefits of implementation of a multidose process for eyedrop administration in patients undergoing surgery and treatment at the Utah Valley Regional Medical Center, Provo. RESULTS: The Joint Commission approved our policy and procedural approach, implemented in April 2012. Cost savings to both patients and the facility were significant. Costs to patients undergoing a single cataract operation were decreased as much as $283.85. Costs to the facility were decreased by $330.91 per cataract case. CONCLUSIONS AND RELEVANCE: Approval of our policy and processes indicates that The Joint Commission validates our policy and its adherence to accepted preferred practice guidelines of safe handling and administration of multidose eyedrops and establishes precedence that may be followed by other eye care facilities and health care organizations in the future. Our policy provides a safe and effective process for administering eyedrop medications to patients as well as controlling excessive health care costs to both patients and health care facilities.

Ca2+ induces spontaneous dephosphorylation of a novel P5A-type ATPase.

P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mM) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.

Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley.

The receptor-like protein kinases (RLKs) constitute a large and diverse group of proteins controlling numerous plant physiological processes, including development, hormone perception and stress responses. The cysteine-rich RLKs (CRKs) represent a prominent subfamily of transmembrane-anchored RLKs. We have identified a putative barley (Hordeum vulgare) CRK gene family member, designated HvCRK1. The mature putative protein comprises 645 amino acids, and includes a putative receptor domain containing two characteristic 'domain 26 of unknown function' (duf26) domains in the N-terminal region, followed by a rather short 17-amino-acid transmembrane domain, which includes an AAA motif, two features characteristic of endoplasmic reticulum (ER)-targeted proteins and, finally, a characteristic putative protein kinase domain in the C-terminus. The HvCRK1 transcript was isolated from leaves inoculated with the biotrophic fungal pathogen Blumeria graminis f.sp. hordei (Bgh). HvCRK1 transcripts were observed to accumulate transiently following Bgh inoculation of susceptible barley. Transient silencing of HvCRK1 expression in bombarded epidermal cells led to enhanced resistance to Bgh, but did not affect R-gene-mediated resistance. Silencing of HvCRK1 phenocopied the effective penetration resistance found in mlo-resistant barley plants, and the possible link between HvCRK1 and MLO was substantiated by the fact that HvCRK1 induction on Bgh inoculation was dependent on Mlo. Finally, using both experimental and in silico approaches, we demonstrated that HvCRK1 localizes to the ER of barley cells. The negative effect on basal resistance against Bgh and the functional aspects of MLO- and ER-localized HvCRK1 signalling on Bgh inoculation are discussed.

One-day bowel preparation with polyethylene glycol 3350: an effective regimen for colonoscopy in children.

BACKGROUND: Polyethylene glycol (PEG) 3350 is commonly used and has been proven safe and effective for the treatment of chronic constipation and as a 4-day bowel preparation in children. A 1-day PEG 3350 bowel preparation regimen has been recently developed for adults; however, data regarding its use in children are lacking. OBJECTIVE: To evaluate the safety and effectiveness of a 1-day PEG 3350 regimen for bowel preparation in children before colonoscopy. DESIGN: Retrospective review. SETTING: Tertiary-care center. PATIENTS: This study involved all children prescribed a 1-day PEG 3350 bowel preparation regimen before colonoscopy at our center in 2008. INTERVENTION: We reviewed medical records of patients (< or = 18 years of age) who underwent colonoscopy during 2008 and received the 1-day bowel preparation regimen. MAIN OUTCOME MEASUREMENTS: Adequate preparation for colonoscopy, success of colonoscopy, and factors associated with inadequate bowel preparation. RESULTS: Inclusion criteria were met by 272 patients. The median age of the children receiving the 1-day PEG 3350 preparation regimen was 13.7 years (range 1.08-17.92 years). Fifty-two percent were male; 48% were female. The most common indications for colonoscopy included abdominal pain (65%), bloody stools (29%), diarrhea (21%), and weight loss (18%). The 1-day bowel preparation regimen was effective in 253 patients (93%). The indication for colonoscopy, the age of the child, or a history of constipation did not significantly alter the success rate of colonoscopy. LIMITATIONS: A retrospective study at one tertiary-care center. CONCLUSION: The 1-day PEG 3350 bowel preparation regimen is safe and effective and should be considered for use as preparation for colonoscopy in children.

Third- and fourth-generation fluoroquinolones: retrospective comparison of endophthalmitis after cataract surgery performed over 10 years.

PURPOSE: To determine differences in endophthalmitis rates with prophylactic use of third- versus fourth-generation fluoroquinolones in cataract surgery. SETTING: University hospitals. METHODS: This retrospective cross-sectional (prevalence) study looked at patients who had phacoemulsification at a university eye center over a 10-year period. A nosocomial infectious reporting database was used to report endophthalmitis occurrences. The following were performed: a retrospective analysis of prospectively collected data to establish endophthalmitis rates, a prevalence analysis of the postoperative quinolone antibiotic prescribed, and a comparative analysis of endophthalmitis rate versus postoperative quinolone prescribed for all reported endophthalmitis cases. The main outcome measure was occurrence of endophthalmitis after cataract surgery. RESULTS: From January 1997 to December 2007, 29276 patients had phacoemulsification cataract surgery. Forty cases of postoperative bacterial endophthalmitis were reported. The endophthalmitis rate from January 1997 to August 2003 associated with use of third-generation fluoroquinolones (ciprofloxacin, ofloxacin) was 0.197% (33/16710). The rate from September 2003 to December 2007 associated with fourth-generation fluoroquinolones (gatifloxacin, moxifloxacin) was 0.056% (7/12566). The difference between third- and fourth-generation drugs was statistically significant (P = .0011). Of fourth-generation fluoroquinolone infections, 0.015% (1/6651) and 0.1% (6/5915) were associated with gatifloxacin and moxifloxacin, respectively. The difference between drugs was statistically significant (P = .040). CONCLUSIONS: The differences in the pharmacokinetic and pharmacodynamic properties of quinolone antibiotics may affect the endophthalmitis incidence after cataract surgery. The significant difference in endophthalmitis rates between gatifloxacin and moxifloxacin requires further study.

A retrospective study of endophtalmitis rates comparing quinolone antibiotics.

PURPOSE: To compare endophthalmitis rates between topical quinolone antibiotics over a four-year period in a university setting. DESIGN: Retrospective, cross-sectional (prevalence) study. STUDY POPULATION: Nine thousand seventy-nine patients who underwent a phacoemulsification procedure at a University Eye Center. INTERVENTIONS: The following interventions were conducted: use of a nosocomial infectious reporting database retrospective analysis of prospectively collected data to establish endophthalmitis rates; prevalence analysis of postoperative quinolone antibiotics; analysis of endophthalmitis rate vs postoperative quinolone prescribed. MAIN OUTCOME MEASURES: Occurrence of endophthalmitis after cataract surgery. RESULTS: During a four-year period involving 9079 phacoemulsification procedures, 26 cases of bacterial postoperative endophthalmitis were reported (rate = 0.286%). Eight of the 26 cases were considered complicated; 18 cases were uncomplicated. Among cases performed during this period, postoperative prescription volumes for ciprofloxacin (Ciloxan, Alcon Laboratories, Inc., Fortworth, Texas) and ofloxacin (Ocuflox, Allergan, Inc., Irvine, California) antibiotic drops were nearly identical, with 4538 patients receiving ciprofloxacin (49.98%) and 4541 patients receiving ofloxacin (50.02%). There were 22 endophthalmitis patients (85%) who had used topical ciprofloxacin and 4 patients (15%) who had used topical ofloxacin postoperatively. The difference in infectious rates between antibiotics was highly significant (P < .00026). For uncomplicated cases, 14 patients received ciprofloxacin and 4 patients received ofloxacin. This difference was also significant (P < .015). CONCLUSIONS: Differences in pharmacokinetic and pharmacodynamic properties exist among quinolone antibiotics, which may affect endophthalmitis incidence following cataract surgery.