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Martin P.,Max Planck Institute of Immunobiology | Martin P.,University of Geneva | Pardo J.,Max Planck Institute of Immunobiology | Pardo J.,University of Zaragoza | And 7 more authors.
Journal of Biological Chemistry | Year: 2010

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH2-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Eva N.,50 North 1950 West | Eva N.,Utah State University | Durrant G.C.,Metropolitan Water District of Salt Lake and Sandy | Hoyt M.B.,Central Utah Water Conservancy District | And 5 more authors.
Journal / American Water Works Association | Year: 2010

This study evaluated the validity of monitoring for Escherichia coli in lieu of Cryptosporidium to assess the vulnerability of source waters to the presence of Cryptosporidium oocysts. Over a period of seven years, four large water systems in Utah collected Cryptosporidium, E. coli, and turbidity data from seven water treatment plants that treat both reservoir and stream sources. The data represented analyses completed by two US Environmental Protection Agency-approved protozoan laboratories and local state-certified laboratories for E coli. Results of the statistical analyses indicated poor correlation between Cryptosporidium and E. coli and between Cryptosporidium and turbidity in all monitored water sources throughout the entire monitoring period. The analyses indicate that elevated concentrations of E. coli are not indicative of the presence of Cryptosporidium in surface water. Both E. coli and turbidity are poor surrogates for occurrence of Cryptosporidium at treatment plant intakes. Further investigation is needed to establish appropriate surrogates for Cryptosporidium occurrence. Source

Kasper D.C.,Medical University of Vienna | Herman J.,Thermo Fisher Scientific | De Jesus V.R.,Centers for Disease Control and Prevention | Mechtler T.P.,Medical University of Vienna | And 2 more authors.
Rapid Communications in Mass Spectrometry | Year: 2010

Lysozomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the analytical challenges associated with published assays including complex sample preparation and potential interference from excess residual substrate. Solutions to these challenges are presented in the form of on-line two-dimensional chromatography to eliminate off-line liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the use of ultra-high-performance liquid chromatography (UHPLC) to separate excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method. © 2010 John Wiley & Sons, Ltd. Source

Shushan B.,Mass Consultants
Mass Spectrometry Reviews | Year: 2010

Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) technology is emerging as a complementary method to traditional methodology used for clinical applications. Enhanced specificity and high-throughput capabilities are providing significant benefits to clinical diagnostic laboratories conducting routine analyses. This technology is expected to expand rapidly as scientists focus on more complicated challenges that can be solved efficiently by adding LC/MS/MS to their arsenal of techniques. © 2010 Wiley Periodicals, Inc. Source

Mechtler T.P.,Medical University of Vienna | Metz T.F.,Medical University of Vienna | Muller H.G.,Medical University of Vienna | Ostermann K.,Medical University of Vienna | And 7 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

The interest in early detection strategies for lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within 4. h including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations, such as liquid-liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. Applying incubation times of 3. h revealed in intra-day CV% values ranging from 4% to 11% for all five enzyme activities, respectively. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, turbulent flow chromatography-ultra high performance liquid chromatography-tandem mass spectrometer assay. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day. © 2012 Elsevier B.V. Source

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