Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7ß-Hydroxylation of Lithocholic Acid.

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7ß-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7ß-position but LCA is exclusively hydroxylated at the 6ß-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

Antimicrobial efficacy of amine fluoride based tooth gels compared to a toothpaste in a phase 2/step 2 in-vitro test model.

INTRODUCTION: The aim of the present study was to determine the antimicrobial effect of various gel formulations on plaque formation; different tooth gels were compared to a toothpaste containing comparable antimicrobial ingredients with regard to its microbiocidal activity. The study was conducted under the assumption, that a chief requirement for the prevention of plaque formation is the combination of mechanical removal and antimicrobial activity, and not the sole capability of mechanical plaque removal. METHODS: Ledermix(®) fluoride gel as commercially available with preservative, and without preservative and perfume oils, Elmex(®) gelée, and Meridol(®) toothpaste were tested in a standardized in-vitro test modification of the quantitative suspension test EN 1040. Instead of testing in a suspension, the respective product was directly placed on a standardized contaminated sterile stainless steel disk without adding any bio-burden. 50% egg yolk in Aqua dest. was used as a neutralizer. RESULTS: Within 1 min, Elmex(®) gelée showed a RF >5 log(10) against S. pyogenes and S. sanguinis. Against S. mutans, a log(10) RF of ≥5 was achieved after 2 min, against C. albicans after 5 min, and against P. aeruginosa after 10 min S. aureus was the most difficult organisms to be reduced. After an application time of 10 min, only a log(10) RF of 2.4 was achieved. Ledermix exceeded the antimicrobial efficacy of Elmex(®) gelée against S. mutans and C. albicans and was already effective against these organisms after 1 min, but did not show the same antimicrobial efficacy as Elmex(®) gelée against P. aeruginosa. Similar to Elmex(®) gelée, a required reduction of >5 log(10) for antimicrobials under no organic challenge was not achieved against S. aureus. Ledermix(®) fluoride gel without preservatives and Ledermix(®) fluoride gel without preservatives and perfume oil did not show the antimicrobial efficacy of the standard Ledermix(®) fluoride gel formulation, indicating that the observed antimicrobial efficacy is chiefly based on the preservative, and possibly the perfume oil. Compared to the tested gels, Meridol(®) toothpaste was less effective and reached any antimicrobial effect >5 log(10) only against S. sanguinis after 10 min. CONCLUSION: All unmodified tested gels showed an antimicrobial effect. Because no relevant antimicrobial efficacy against plaque forming bacteria was achieved within 2 min, in practice, an anti-plaque forming effect based on the antimicrobial action of gels cannot be assumed when used in the oral cavity. However, the results of the present study indicate that the antimicrobial efficacy of gels is determined by their formulation and that for the prevention of plaque formation the combination of mechanical removal and antimicrobial activity is not the chief requirement only, but a sustained antimicrobial effect may be of greater importance.

The use of concentrated heat after insect bites/stings as an alternative to reduce swelling, pain, and pruritus: an open cohort-study at German beaches and bathing-lakes.

BACKGROUND: Swelling, pain, and pruritus are the most relevant symptoms after insect bites/stings. Glucocorticoids and antihistamines are well established in insect sting treatment. Bite Away(®) is a CE-certified medical device of class 2A (noninvasive device intended for administration to the body, which exchanges energy with the patient in a therapeutic manner) to reduce swelling, pruritus, and pain after insect bites/stings via non-invasive administration of concentrated heat to the skin. We therefore performed a prospective, non-interventional, single-arm cohort study with 146 volunteers using the visual analog scale (VAS) for insect bites/stings to study the reduction of swelling, pruritus, and pain. Demographic data, time from insect sting to treatment, number and duration of administrations of concentrated heat, relevant symptoms, and the development of a VAS score of swelling, pruritus, and pain on baseline, after 2, 5, and 10 minutes after administration, were registered. RESULTS: In total 146 subjects with a mean age of 29.4 ± 20.7 years (range 2-81) were enrolled in the study. Ninety-three (63.7%) of the subjects were stung by wasps, 33 (22.6%) of the subjects were bitten by mosquitoes, and eight suffered bee stings (5.3%). VAS score swelling decreased with statistical significance after the use of Bite Away(®) from 4 before treatment to 2 and 1 after 2-5 and 10 minutes, respectively. VAS pain score was 6 before treatment, 2 after 2 minutes, 1 after 5 minutes, and 0 after 10 minutes (median). VAS pruritus score was only available for 52 (35.2%) of the patients. The score decreased from 5 before treatment, to 2 after 2 minutes, and 0 after 5 and 10 minutes. CONCLUSIONS: Locally administrated concentrated heat leads to fast amelioration of symptoms. Usually an absence of symptoms is noticeable 10 minutes after administration. Pain reduction is the dominant effect. Compared with alternatives of pruritus and pain treatment after insect bites/stings, Bite Away(®) seems to be the fastest treatment option available.

Ceftriaxone causes drug-induced immune thrombocytopenia and hemolytic anemia: characterization of targets on platelets and red blood cells.

BACKGROUND: Ceftriaxone, a third-generation cephalosporin, has been reported to occasionally cause fatal drug-induced immune hemolytic anemia (DIHA). A clinical and serologic analysis of the first two patients with severe drug-induced thrombocytopenia (DITP) due to ceftriaxone and one patient with fatal DIHA is reported. STUDY DESIGN AND METHODS: Sera were assessed by the IAT, EIA, glycoprotein (GP)-specific immunoassay, flow cytometry, and immunoprecipitation using transfectants expressing GPIIb/IIIa and GPIb/IX and with different cephalosporins. RESULTS: Sera from Patients 1 and 2 reacted strongly with PLTs in the presence of the drug, but not with RBCs. The binding sites of the drug-dependent antibodies (DDAbs) could be localized to GPIIb/IIIa and GPIb/IX, respectively. Inhibition studies indicated that DDAbs recognized epitopes residing on the GPIIb/IIIa complex and on the GPIX subunit, respectively. No cross-reactivity was observed with other cephalosporin derivatives. Serum 3 showed strong agglutination with RBCs of Rh(null) phenotype in the presence of ex-vivo metabolites of ceftriaxone, but no cross-reactivity with PLTs. CONCLUSIONS: The first two cases of severe DITP and a third patient with DIHA are reported. DDAbs from all patients showed individual reaction patterns and clear cell lineage specificity. In addition, the DDAbs were dependent on the substitution at position 3 of the ceftriaxone molecule. Epitopes on GPIIb/IIIa and GPIX were involved on PLTs. The Rh protein was not the only target of DDAbs on RBCs.

Selective removal of circulating immune complexes from patient plasma.

The principle of a patient-specific immunoadsorber (PsIA) is demonstrated. Studies with model systems (HSA/anti-HSA) on immobilization, stability, and leakage form the basis for the presented fast-performance liquid chromatography (FPLC) and batch experiments, which were conducted using two different protein A adsorbers and autologous and heterologous PsIA systems. Experiments to determine the binding capacity of protein A adsorbers and PsIAs are described. In all experiments, the adsorption of plasma IgG, total protein, and C1q and C3d circulating immune complexes were measured. Plasma of patients with autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus) was investigated. Analysis was performed in both the initial plasma and the flow-through or supernatant. Results of the investigations using FPLC and batch experiments were compared. Autologous PsIA systems are suitable for the selective removal of elevated levels of circulating immune complexes in the plasma.