Biochemical analyses of human osteoarthritic and periprosthetic synovial fluid.

Biochemical analyses were performed on osteoarthritic and periprosthetic synovial fluid in order to propose changes to lubricant specifications currently outlined in orthopaedic wear testing standards. Osteoarthritic and periprosthetic synovial fluid samples were obtained from the hip and knee joints of 40 patients. The samples in each group were analysed and compared in order to identify differences between the protein concentration, constituent fractions, osmolality, thermal stability and the hyaluronic acid concentration and molecular weight distribution of osteoarthritic and periprosthetic synovial fluid. The average total protein concentration was approximately 30 g/L, which was much higher than the 20 g/L currently specified in the knee wear testing standard; however, the 30 g/L protein concentration matched the recently revised standard for hip simulator wear testing. No significant difference was found between the protein concentration, osmolality, thermal stability, and hyaluronic acid concentration of osteoarthritic and periprosthetic synovial fluid. The clinical data provided should be used to better define the composition of a more clinically relevant lubricant for orthopaedic wear testing.

Enzymatic synthesis of valerena-4,7(11)-diene by a unique sesquiterpene synthase from the valerian plant (Valeriana officinalis).

Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents (e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena-4,7(11)-diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena-4,7(11)-diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next-generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena-4,7(11)-diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (K(m) of ∼ 10 µm and k(cat) of 0.01 s(-1) for both enzymes). The structure of the valerena-4,7(11)-diene produced from the yeast expressing VoTPS2 was further substantiated by (13) C-NMR and GC-MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next-generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.