Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin

Animal health depends on the ability of immune cells to kill invading pathogens, and on the resilience of tissues to tolerate the presence of pathogens. Trueperella pyogenes causes tissue pathology in many mammals by secreting a cholesterol-dependent cytolysin, pyolysin (PLO), which targets stromal cells. Cellular cholesterol is derived from squalene, which is synthesized via the mevalonate pathway enzymes, including HMGCR, FDPS and FDFT1. The present study tested the hypothesis that inhibiting enzymes in the mevalonate pathway to reduce cellular cholesterol increases the resilience of stromal cells to PLO. We first verified that depleting cellular cholesterol with methyl-β-cyclodextrin increased the resilience of stromal cells to PLO. We then used siRNA to deplete mevalonate pathway enzyme gene expression, and used pharmaceutical inhibitors, atorvastatin, alendronate or zaragozic acid to inhibit the activity of HMGCR, FDPS and FDFT1, respectively. These approaches successfully reduced cellular cholesterol abundance, but mevalonate pathway enzymes did not affect cellular resilience equally. Inhibiting FDFT1 was most effective, with zaragozic acid reducing the impact of PLO on cell viability. The present study provides evidence that inhibiting FDFT1 increases stromal cell resilience to a cholesterol-dependent cytolysin

The Human Serum Metabolome

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca

Evaluation of the effects of ultraviolet light on bacterial contaminants inoculated into whole milk and colostrum, and on colostrum immunoglobulin G

Raw milk and colostrum can harbor dangerous microorganisms that can pose serious health risks for animals and humans. According to the USDA, more than 58% of calves in the United States are fed unpasteurized milk. The aim of this study was to evaluate the effect of UV light on reduction of bacteria in milk and colostrum, and on colostrum IgG. A pilot-scale UV light continuous (UVC) flow-through unit (45 J/cm(2)) was used to treat milk and colostrum. Colostrum and sterile whole milk were inoculated with Listeria innocua, Mycobacterium smegmatis, Salmonella serovar Typhimurium, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Acinetobacter baumannii before being treated with UVC. During UVC treatment, samples were collected at 5 time points and bacteria were enumerated using selective media. The effect of UVC on IgG was evaluated using raw colostrum from a nearby dairy farm without the addition of bacteria. For each colostrum batch, samples were collected at several different time points and IgG was measured using ELISA. The UVC treatment of milk resulted in a significant final count (log cfu/mL) reduction of Listeria monocytogenes (3.2 ± 0.3 log cfu/mL reduction), Salmonella spp. (3.7 ± 0.2 log cfu/mL reduction), Escherichia coli (2.8 ± 0.2 log cfu/mL reduction), Staph. aureus (3.4 ± 0.3 log cfu/mL reduction), Streptococcus spp. (3.4 ± 0.4 log cfu/mL reduction), and A. baumannii (2.8 ± 0.2 log cfu/mL reduction). The UVC treatment of milk did not result in a significant final count (log cfu/mL) reduction for M. smegmatis (1.8 ± 0.5 log cfu/mL reduction). The UVC treatment of colostrum was significantly associated with a final reduction of bacterial count (log cfu/mL) of Listeria spp. (1.4 ± 0.3 log cfu/mL reduction), Salmonella spp. (1.0 ± 0.2 log cfu/mL reduction), and Acinetobacter spp. (1.1 ± 0.3 log cfu/mL reduction), but not of E. coli (0.5 ± 0.3 log cfu/mL reduction), Strep. agalactiae (0.8 ± 0.2 log cfu/mL reduction), and Staph. aureus (0.4 ± 0.2 log cfu/mL reduction). The UVC treatment of colostrum significantly decreased the IgG concentration, with an observed final mean IgG reduction of approximately 50%. Development of new methods to reduce bacterial contaminants in colostrum must take into consideration the barriers imposed by its opacity and organic components, and account for the incidental damage to IgG caused by manipulating colostrum