Study of Legionella pneumophila treatment with copper in drinking water by single cell-ICP-MS.

Legionella pneumophila is a persistent opportunistic pathogen that poses a significant threat to domestic water systems. Previous studies suggest that copper (Cu) is an effective antimicrobial in water systems. A rapid and sensitive quantification method is desired to optimize the conditions of L. pneumophila treatment by Cu and to better understand the interaction mechanisms between Cu and cells. In this study, we developed a highly sensitive single cell (SC)-ICP-MS method to monitor L. pneumophila cell concentration and track their uptake of Cu. The SC-ICP-MS method showed excellent sensitivity (with a cell concentration detection limit of 1000 cells/mL), accuracy (good agreement with conventional hemocytometry method), and precision (relative standard deviation < 5%) in drinking water matrix. The cupric ions (Cu2+) treatment results indicated that the total L. pneumophila cell concentration, Cu mass per cell, colony-forming unit counting, and Cu concentration in supernatant all exhibited a dose-dependent trend, with 800-1200 µg/L reaching high disinfection rates in drinking water. The investigation of percentages of viable and culturable, viable but nonculturable (VBNC), and lysed cells suggested there always were VBNC present at any Cu concentration. Experimental results of different Cu2+ treatment times further suggested that L. pneumophila cells developed an antimicrobial resistant mechanism with the prolonged Cu exposure. This is the first quantification study on the interactions of Cu and L. pneumophila in drinking water using SC-ICP-MS.

Repeated Low-Level Blast Exposure Alters Urinary and Serum Metabolites.

Repeated exposure to low-level blast overpressures can produce biological changes and clinical sequelae that resemble mild traumatic brain injury (TBI). While recent efforts have revealed several protein biomarkers for axonal injury during repetitive blast exposure, this study aims to explore potential small molecule biomarkers of brain injury during repeated blast exposure. This study evaluated a panel of ten small molecule metabolites involved in neurotransmission, oxidative stress, and energy metabolism in the urine and serum of military personnel (n = 27) conducting breacher training with repeated exposure to low-level blasts. The metabolites were analyzed using HPLC-tandem mass spectrometry, and the Wilcoxon signed-rank test was used for statistical analysis to compare the levels of pre-blast and post-blast exposures. Urinary levels of homovanillic acid (p < 0.0001), linoleic acid (p = 0.0030), glutamate (p = 0.0027), and serum N-acetylaspartic acid (p = 0.0006) were found to be significantly altered following repeated blast exposure. Homovanillic acid concentration decreased continuously with subsequent repeat exposure. These results suggest that repeated low-level blast exposures can produce measurable changes in urine and serum metabolites that may aid in identifying individuals at increased risk of sustaining a TBI. Larger clinical studies are needed to extend the generalizability of these findings.

Development of a HPLC-MS/MS method for assessment of thiol redox status in human tear fluids.

Oxidative stress is reported to be part of the pathology of many ocular diseases. For the diagnosis of ocular diseases, tear fluid has unique advantages. Although numerous analytical methods exist for the measurement of different types of biomolecules in tear fluid, few have been reported for comprehensive understanding of oxidative stress-related thiol redox signaling. In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine a panel of twelve metabolites that systematically covered several thiol metabolic pathways. With optimization of MS/MS parameters and HPLC mobile phases, this method was sensitive (LOQ as low as 0.01 ng/ml), accurate (80-125% spike recovery) and precise (<10% RSD). This LC-MS/MS method combined with a simple tear fluid collection with Schirmer test strip followed by ultrafiltration allowed the high-throughput analysis for efficient determination of metabolites associated with thiol redox signaling in human tear fluids. The method was then applied to a small cohort of tear fluids obtained from healthy individuals. The method presented here provides a new technique to facilitate future work aiming to determine the complex thiol redox signaling in tear fluids for accurate assessment and diagnosis of ocular diseases.

Simultaneous Determination of Eight Urinary Metabolites by HPLC-MS/MS for Noninvasive Assessment of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a serious public health concern for which sensitive and objective diagnostic methods remain lacking. While advances in neuroimaging have improved diagnostic capabilities, the complementary use of molecular biomarkers can provide clinicians with additional insight into the nature and severity of TBI. In this study, a panel of eight metabolites involved in distinct pathophysiological processes related to concussion was quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Specifically, the newly developed method can simultaneously determine urinary concentrations of glutamic acid, homovanillic acid, 5-hydroxyindoleacetic acid, methionine sulfoxide, lactic acid, pyruvic acid, N-acetylaspartic acid, and F2α-isoprostane without intensive sample preparation or preconcentration. The method was systematically validated to assess sensitivity (method detection limits: 1-20 µg/L), accuracy (81-124% spike recoveries in urine), and reproducibility (relative standard deviation: 4-12%). The method was ultimately applied to a small cohort of urine specimens obtained from healthy college student volunteers. The method presented here provides a new technique to facilitate future work aiming to assess the clinical efficacy of these putative biomarkers for noninvasive assessment of TBI.