EUS-guided thrombin injection for management of gastric fundal varices.

BACKGROUND AND STUDY AIMS: Cyanoacrylate glue is recommended first-line endoscopic therapy for gastric fundal varices but it is difficult to use and carries a risk of embolization. Thrombin is preferred by many in the UK, but its effectiveness can be difficult to establish at endoscopy and the rate of re-bleeding is higher. Endoscopic ultrasound (EUS) can help assess variceal blood flow and has the potential to improve both targeting and effectiveness of injection therapy. Whereas there is already some data for its use with glue, little data currently exist in relation to its use with thrombin. PATIENTS AND METHODS: We present a series of patients treated with EUS-guided thrombin injection over the last 4 years. Thrombin was injected under EUS guidance with the intention of obliterating flow within the fundal varices. Outcomes reviewed included whether haemostasis was achieved, the dose of thrombin required for endosonographic variceal obliteration, the incidence of re-bleeding, and procedural related adverse events. RESULTS: Eight patients received EUS-guided thrombin: 3 with active bleeding and 5 as elective prevention. In 2/3 (66 %) patients with active bleeding haemostasis was achieved after a single dose with complete variceal obliteration. 1/3 (33 %) had no alteration in blood flow despite 10 000 IU. None of the elective prevention group had further bleeding and obliteration was observed in 4/5 (80 %). A range of 600 to 10 000 IU of thrombin was used and there were no adverse procedure-related outcomes. CONCLUSIONS: Our results are promising and suggest that EUS-guided thrombin injection may have a role in managing bleeding from gastric fundal varices.

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate aldolase to replace 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase.

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbial synthesis of shikimate pathway products provides an alternate strategy to circumvent the competition for phosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and the phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGal aldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, and multiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-fold improvement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the addition of pyruvate to d-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2 contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression of NR8.276-2 in the DAHP synthase and shikimate dehydrogenase-deficient E. coli strain NR7 under fed-batch fermentor-controlled cultivation conditions resulted in synthesis of 13 g/L 3-dehydroshikimic acid in 6.5% molar yield from glucose. Increased coexpression of the irreversible downstream enzyme 3-dehydroquinate synthase increased production of 3-dehydroshikimic acid to 19 g/L in 9.7% molar yield from glucose. Coamplification with transketolase, which increases d-erythrose 4-phosphate availability, afforded 16 g/L 3-dehydroshikimic acid in 8.5% molar yield.

Microbial synthesis of triacetic acid lactone.

Native g2ps1-encoded 2-pyrone synthase (2-PS) from Gerbera hybrida, a mutant Brevibacterium ammoniagenes fatty acid synthase B (FAS-B) and two different mutants of Penicillium patulum 6-methylsalycilic acid synthase (6-MSAS) are examined to identify the best enzyme to recruit for the microbial synthesis of triacetic acid lactone (TAL). To identify the best microbial host for these evaluations, the native TAL-synthesizing activity of g2ps1-encoded 2-PS is expressed in recombinant Escherichia coli and Saccharomyces cerevisiae constructs. Five-fold higher expression levels of 2-PS are observed in S. cerevisiae. Consequently, microbial synthesis of TAL focuses on S. cerevisiae constructs. Comparison of different promoters for the expression of g2ps1 in S. cerevisiae indicates that the alcohol dehydrogenase II promoter (P(ADH2)) affords the highest expression levels of 2-PS. As a result, the genes encoding the various TAL-synthesizing enzyme activities are expressed in S. cerevisiae from a P(ADH2) promoter. To extend TAL-synthesizing activity beyond g2ps1-encoded 2-PS, the ketoreductase domains of fasB-encoded FAS-B and 6-MSAS-encoded 6-MSAS are modified using a single mutation. Modification of the nicotinamide cofactor-binding site of 6-MSAS with a triple mutation is also examined. Separate S. cerevisiae constructs expressing native g2ps1, mutant Y2226F fasB, mutant Y1572F 6-MSAS, and mutant G1419A-G1421P-G1424A 6-MSAS are cultured under the same fermentor-controlled conditions. The highest concentration (1.8 g/L) and yield (6%) of TAL are synthesized from glucose by S. cerevisiae expressing the Y1572F mutant of 6-MSAS.

Rational pathway engineering of type I fatty acid synthase allows the biosynthesis of triacetic acid lactone from D-glucose in vivo.

Metabolic pathway engineering is a powerful tool to synthesize structurally diverse and complex chemicals via genetic manipulation of multistep catalytic systems involved in cell metabolism. Here, we report the rational design of a fatty acid biosynthetic pathway, Brevibacterium ammoniagenes fatty acid synthase B (FAS-B), that allows the microbial synthesis of triacetic acid lactone (TAL) from an inexpensive feedstock, d-glucose. TAL can be chemically converted to phloroglucinol, which is a core structure for the synthesis of various high value bioactive compounds and energetic compounds such as 1,3,5-triamino-2,4,6-trinitrobenzene (TATB). Synthesis of phloroglucinol from d-glucose using this combined biological and chemical synthesis may offer significant advantages over the current phloroglucinol manufacture, including environmental friendliness and reduction in the cost of phloroglucinol. More importantly, it represents a novel strategy for the benzene-free synthesis of aromatic chemicals.

Antioxidant activity of 3-dehydroshikimic acid in liposomes, emulsions, and bulk oil.

The antioxidant activity of 3-dehydroshikimic acid (DHS), an intermediate in the biosynthesis of aromatic amino acids, was evaluated in three assay systems: bulk oil (lard), liposomes, and a 10% corn oil-in-water emulsion. Upon initiation of peroxidation in the liposome or emulsion systems, DHS exhibited weak antioxidant activity. In contrast, DHS displayed strong antioxidant activity in lard, suppressing peroxidation with activity comparable to that of tert-butylhydroquinone, propyl gallate, and gallic acid and superior to that of alpha-tocopherol. Two major DHS oxidation products, gallic acid and protocatechuic acid, were identified by gas chromatography/mass spectral analysis of lard extracts; both compounds are effective antioxidants in the bulk oil system. In the liposome system, DHS remained intact throughout the assay period. A small amount of gallic acid was observed in extracts of the emulsion; however, protocatechuic acid was not detected. A mechanism to explain the different activities of DHS in the three lipid systems is proposed.

Kanosamine biosynthesis: a likely source of the aminoshikimate pathway's nitrogen atom.

The biosynthetic source of the nitrogen atom incorporated into the aminoshikimate pathway has remained a question for some time. 3-Amino-3-deoxy-D-fructose 6-phosphate has previously been demonstrated to be a precursor to 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid via the inferred intermediacy of 1-deoxy-1-imino-D-erythrose 4-phosphate in Amycolatopsis mediterranei cell-free extract. This investigation examines the possibility that the natural product kanosamine might be a precursor to 3-amino-3-deoxy-D-fructose 6-phosphate. Kanosamine 6-phosphate was synthesized by a chemoenzymatic route and incubated in A. mediterranei cell-free lysate along with D-ribose 5-phosphate and phosphoenolpyruvate. Formation of 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid was observed. Subsequent incubation in A. mediterranei cell-free lysate of glutamine and NAD with UDP-glucose resulted in the formation of kanosamine. The bioconversion of UDP-glucose into kanosamine along with the bioconversion of kanosamine 6-phosphate into 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid suggests that kanosamine biosynthesis is the source of the aminoshikimate pathway's nitrogen atom.