Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.

Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes. Oil red O uptake into fat droplets and adiponectin production were used as biomarkers of adipogenesis. Positive peptides were formulated in either collagen I or hyaluronan (Orthovisc) gels then assessed for their adipogenic potential in vivo following injection into dorsal rat skin and mammary fat pads. Fat content was quantified and characterized using micro CT imaging, morphometry, histology, RT-PCR and ELISA analyses of adipogenic gene expression. Injection of screened peptides increased dorsal back subcutaneous fat pad area (208.3 ± 10.4 mm2versus control 84.11 ± 4.2 mm2; p < 0.05) and mammary fat pad size (45 ± 11 mg above control background, p = 0.002) in female rats. This effect lasted >5 weeks as detected by micro CT imaging and perilipin 1 mRNA expression. RHAMM expression suppresses while blocking peptides promote expression of PPARγ, C/EBP and their target genes. Blocking RHAMM function by peptide injection or topical application is a novel and minimally invasive method for potentially promoting subcutaneous adipogenesis in lipodystrophic diseases and a complementary tool to subcutaneous fat augmentation techniques.

Determination of bisphenol A in sewage effluent and sludge by solid-phase and supercritical fluid extraction and gas chromatography/mass spectrometry.

Methods have been developed for the determination of bisphenol A (BPA) residues in municipal sewage and sludge samples. BPA in wastewater samples was enriched with a C18 solid-phase extraction cartridge, eluted with acetone, and converted to the pentafluoropropionyl derivative. For sludge samples, BPA was acetylated and extracted with supercritical carbon dioxide. In both cases, BPA-d16 was used as a surrogate to monitor extraction efficiency. Final analyses of derivatized sample extracts were performed by gas chromatography/mass spectrometry operating in the electron impact mode. For water samples, mean recoveries and standard deviations were 89 +/- 6, 94 +/- 4, and 85 +/- 7% at fortification levels of 1, 0.1, and 0.025 microg/L, respectively, with a method detection limit of 0.006 microg/L. For solid waste samples, mean recoveries and standard deviations were 93 +/- 5 and 92 +/- 6% at fortification levels of 2.5 and 0.25 microg/g, respectively, and the method detection limit was 0.05 microg/g. For the Canadian samples under investigation, concentrations of BPA ranged from 49.9 to 0.031 microg/L in sewage influent and effluent, and from 36.7 to 0.104 microg/g in sludge.

Determination of 17 beta-estradiol and its metabolites in sewage effluent by solid-phase extraction and gas chromatography/mass spectrometry.

The paper describes a simple and quantitative method for monitoring non-conjugated 17 beta-estradiol (E2) and its metabolites estrone (E1) and estriol (E3) as environmental contaminants in municipal sewage effluents. Estrogens were preconcentrated and cleaned up by solid-phase extraction using a reversed-phase C18 cartridge. They were derivatized with pentafluoropropionic acid anhydride, and the products were analyzed by gas chromatography/mass spectrometry. Recoveries from spiked distilled water and sewage were better than 87% at fortification levels of 100 and 20 ng/L. For a 1 L sewage sample and a concentration factor of 5000, detection limits were 5 ng/L for E1 and E2 and 10 ng/L for E3. In a brief survey of Canadian wastewater, these estrogens were detected in many raw sewage and effluent samples at concentrations ranging from 6 to 109 ng/L for E1, from < 5 to 15 ng/L for E2, and from < 10 to 250 ng/L for E3.

Determination of resin and fatty acids in sediments near pulp mill locations.

A gas chromatographic method for the determination of resin and fatty acids in sediments is described. In this procedure, the sediment sample was air-dried and soxhlet-extracted with a mixture of acetone--methanol (88:12, v/v) in the presence of hydrochloric acid. The acids extracted were converted into their pentafluorobenzyl esters and were then cleaned up on a deactivated silica gel column. Final analysis was performed on either a DB-17 or a DB-5 capillary column with electron-capture detection. Quantitative recovery was obtained from fortified sediments for all acids except palustric, neoabietic and levopimaric acids. The detection limit of all acids in this method was 0.1 micrograms/g based on 1 g of sample. This procedure has been successfully applied to the monitoring of resin and fatty acids in sediment samples collected in the vicinity of several Canadian pulp mill locations.