Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars.

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates.

Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

Efficacy of Single-stage Revision with Aggressive Debridement Using Intra-articular Antibiotics in the Treatment of Infected Joint Prosthesis.

Prosthetic joint infections (PJI) of the hip and knee are uncommon, but result in significant morbidity and mortality when they do occur. Current management consists of a combination of either single- or two-stage exchange of the prosthesis and/or exchange of polymer components with intravenous (IV) antibiotics (4-6 weeks) and intraoperative debridement of the joint prior to reimplantation. However, failure rate, morbidity, and expense associated with current management are high, especially if the infection involves resistant pathogens and/or osteomyelitis. Also, the current use of systemic antibiotics does not allow for high local concentrations of the drug and biofilm penetration of the infected prosthesis. To overcome these difficulties, we examined the outcomes of aggressive operative debridement of the infected prosthesis. This was achieved through the use of a single-stage revision and administration of high concentrations of local intra-articular antibiotics via Hickman catheters. We present 57 patients with PJI who were treated with intra-articular antibiotics and single-stage revisions. Minimal systemic toxicity was observed along with a 100% microbiologic cure rate and 89% without relapse at 11-month follow-up despite isolation of multidrug resistant pathogens. This is the largest study to date using this method in the treatment of PJI.

Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol.

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads® EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.

Real-time ophthalmoscopic findings of superselective intraophthalmic artery chemotherapy in a nonhuman primate model.

OBJECTIVE: To report real-time ophthalmoscopic findings during superselective intraophthalmic artery chemotherapy (SSIOAC) in a nonhuman primate model. METHODS: Six adult male Rhesus macaques (Macacca mulatta) were randomly assigned to 1 of 2 treatment cohorts: melphalan (5 mg/30 mL) or carboplatin (30 mg/30 mL). Each animal underwent 3 separate SSIOAC procedures at 3-week intervals. Digital retinal images were obtained during each infusion. Intravenous fluorescein angiography was performed immediately after each procedure. RESULTS: All SSIOAC procedures were successfully completed. Toxicities were equally distributed between drug cohorts. Systemic toxicities included mild bone marrow suppression in all animals and anorexia in 1. One animal had greater than 50% narrowing of the treated ophthalmic artery after its second infusion. All 18 procedures (100%) resulted in pulsatile optic nerve and choroid blanching, retinal artery narrowing, and retinal edema. Of the 18 procedures, retinal artery sheathing was found during 17 (94%), and retinal artery precipitates were seen in 10 (56%); choroidal hypoperfusion was seen by fluorescein angiogram in 18 (100%). CONCLUSION: Real-time ophthalmic investigations are useful and, in our nonhuman primate model, indicate prevalent, acute ocular vascular toxicities during SSIOAC. CLINICAL RELEVANCE: Real-time retinal imaging is feasible in a nonhuman primate model of SSIOAC. Application to SSIOAC in children may shed insight into reported vascular toxicities.

Production of Candida antarctica lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in an ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production.

A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. Saccharomyces cerevisiae strains expressing CALB protein or CALB Lyt-1 fusion protein were first grown on 2% (w/v) glucose, producing 9.3 g/L ethanol during fermentation. The carbon source was switched to galactose for GAL1-driven expression, and the CALB and CALB Lyt-1 enzymes expressed were tested for fatty acid ethyl ester (biodiesel) production. The synthetic enzymes catalyzed the formation of fatty acid ethyl esters from ethanol and either corn or soybean oil. It was further demonstrated that a one-step-charging resin, specifically selected for binding to lipase, was capable of covalent attachment of the CALB Lyt-1 enzyme, and that the resin-bound enzyme catalyzed the production of biodiesel. High-level expression of lipase in an ethanologenic yeast strain has the potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel.

Triplex forming oligonucleotides against type α1(I) collagen attenuates liver fibrosis induced by bile duct ligation.

Liver fibrosis is a consequence of chronic liver disorders which lead to the accumulation of extracellular matrix (ECM). Particularly, there is an increased accumulation of collagen in the fibrotic liver. We have therefore used a triplex forming oligonucleotide (TFO) against the type α1(I) collagen and evaluated, whether it can attenuate liver fibrosis induced by common bile duct ligation (CBDL) in rats. There was a significant decrease in hydroxyproline levels and Masson's trichrome staining for collagen in TFO-treated CBDL groups compared to non-treated CBDL group. There was over expression of type α1(I) collagen, α-smooth muscle actin (α-SMA) and TGF-ß1 expression in the CBDL group compared to TFO-treated CBDL group. Also, the serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were less in the TFO treated group compared to non-treated CBDL group. There was also less neutrophils accumulation in TFO treated CBDL group assayed by myeloperoxidase (MPO) assay. These results suggests that TFO can be used to downregulate type 1 collagen gene expression and can alleviate liver fibrosis induced by common bile duct ligation.

Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain.

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast.

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3' untranslated regions (3' UTRs) of target mRNAs. In yeast, Puf3p binds the 3' UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3' UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3' UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3' UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain.