A Single Antibody based ELISA for the N-terminal sequence of BAG-75, a New Biomarker for Bone Formation [abstract]

Biomedical Tissue Engineering, Biomaterials, & Medical Devices Poster SessionBone acidic glycoprotein-75 (BAG-75) is a secreted product of osteoblastic cells localized predominantly to areas of new bone formation. We have identified the N-terminal sequence of BAG-75 as LPVARYQNTEEEE and shown that anti-peptide antibodies against residues #3-13 only recognize the 75 kDa precursor and apparent 50 kDa fragment in serum and in osteoblastic cultures. Formation of the 50 kDa fragment is blocked by AEBSF, a serine protease inhibitor which we also showed blocks mineralization in osteoblastic cultures. Measurement of BAG-75 and its fragment concentration in serum represents a new method to estimate the rate of new bone formation in vivo. Our purpose was to establish an anti-VARYQNTEEEE peptide antibody based ELISA test to measure cross-reactive proteins released from bone into blood. Western blotting was performed using young rat serum from different ages, rats subjected to ovariectomy (OVX) or sham surgery, and normal human serum. Immunoreactive 50 kDa fragment peaked at 18 days after birth which parallels bone formation. Ovariectomized rats displayed a peak of 50 kDa immunoreactivty at 21 days after surgery which corresponds to a spike in bone formation in this model (~2.5-fold above controls). Comparable assays for osteocalcin showed only a 39% increase. Also, human serum contains a 50 kDa protein which cross-reacts with anti-VARYQNTEEEE antibodies. We then established a competitive 96-well ELISA using anti-peptide antibody and new sera at 21 days from ovariectomized or sham rats, a model for stimulated bone formation. VARYQNTEEEE peptide conjugated to keyhole limpet hemocyanin (KLH) was used as the bound antigen. KLH-peptide amount, primary antibody concentration, secondary antibody concentration, and blocking agent were optimized in a series of experiments. Optimal conditions were determined to be 2 µg input KLH-peptide per well, 1/5,000 dilution of primary anti-VARYQNTEEEE antibody, 1/10,000 dilution of secondary antibody, and gelatin as a blocking agent. Sera from OVX rats and sham-operated controls were compared to the standard curve (r = 0.9923) created with free KLH-peptide as competitor to determine the equivalent amount of KLH-peptide present. OVX sera (n=3) contained an average 2.6 x 10-4 (+/- 1.4 x 10-4) µg peptide equivalent versus 1.05 x 10-4 (+/- 0.68 x 10-4) µg for sham sera (n=3). The difference was not significant (t-test, p=0.157), however, doubling the sample size is predicted to yield significance. Conclusions: A. Cross-reactive 75 kDa and 50 kDa proteins are present in human and rat serum and increase in concentration when bone formation is stimulated. B. A new, single antibody based ELISA assay was established to quantitate antigen released from bone into blood. C. In contrast to other commercial bone formation assays (collagen peptides and osteocalcin), the size of cross-reactive protein (>50 kDa) should preclude kidney filtration and facilitate measurement. D. This serum biomarker undergoes a 2-3 fold average increase within 3 weeks after simulation of bone. This test may be useful to monitor the early response to stimulatory therapy in osteoporosis patients or to repressive glucocorticoid therapy in sarcoidosis patients. Currently, a 1% change in bone mineral density requires 12-18 months to detect by x-ray methods

Vegetated treatment area (VTAs) efficiencies for E. coli and nutrient removal on small-scale swine operations

As small-scale animal feeding operations work to manage their byproducts and avoid regulation, they need practical, cost-effective methods to reduce environmental impact. One such option is using vegetative treatment areas (VTAs) with perennial grasses to treat runoff; however, research is limited on VTA effectiveness as a waste management alternative for smaller operations. This study evaluated the efficiencies of VTAs in reducing bacteria and nutrient runoff from small-scale swine operations in three counties in Central Texas. Based on 4 yr of runoff data, the Bell and Brazos VTAs significantly reduced loads and concentrations of E. coli and nutrients (except NO3-N) and had treatment efficiencies of 73–94%. Most notably, the Bell VTA reduced loads of E. coli, NH4-N, PO4-P, total N, and total P similar to that of the background (control). In spite of significant reductions, runoff from the Brazos VTA had higher concentrations and loads than the control site, especially following installation of concrete pens and increased pen washing, which produced standing water and increased E. coli and nutrient influx. The Robertson VTA produced fewer significant reductions and had lower treatment efficiencies (29–69%); however, E. coli and nutrient concentrations and loads leaving this VTA were much lower than observed at the Bell and Brazos County sites due to alternative solids management and enclosed pens. Based on these results and previous research, VTAs can be practical, effective waste management alternatives for reducing nutrient and bacteria losses from small-scale animal operations, but only if properly designed and managed. Keywords: Bacteria, Nutrients, Swine manure, Waste management, Water qualit

Traditional marijuana, high-potency cannabis and synthetic cannabinoids:increasing risk for psychosis

Epidemiological evidence demonstrates that cannabis use is associated with an increased risk of psychotic outcomes, and confirms a dose‐response relationship between the level of use and the risk of later psychosis. High‐potency cannabis and synthetic cannabinoids carry the greatest risk. Experimental administration of tetrahydrocannabinol, the active ingredient of cannabis, induces transient psychosis in normal subjects, but this effect can be ameliorated by co‐administration of cannabidiol. This latter is a constituent of traditional hashish, but is largely absent from modern high‐potency forms of cannabis. Argument continues over the extent to which genetic predisposition is correlated to, or interacts with, cannabis use, and what proportion of psychosis could be prevented by minimizing heavy use. As yet, there is not convincing evidence that cannabis use increases risk of other psychiatric disorders, but there are no such doubts concerning its detrimental effect on cognitive function. All of the negative aspects are magnified if use starts in early adolescence. Irrespective of whether use of cannabis is decriminalized or legalized, the evidence that it is a component cause of psychosis is now sufficient for public health messages outlining the risk, especially of regular use of high‐potency cannabis and synthetic cannabinoids