MRI-Based Detection of Alkaline Phosphatase Gene Reporter Activity Using a Porphyrin Solubility Switch

The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with MRI could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water-soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by postmortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets.Raymond and Beverley Sackler FoundationNational Institutes of Health (U.S.) (New Innovator Award Grant DP2-OD2441)MIT-Germany Seed Fund (Grant

Selective accurate-mass-based analysis of 11 oxy-PAHs on atmospheric particultate matter by pressurized liquid extraction followed by highperformance liquid chromatography and magnetic sector mass spectrometry

An innovative analytical method based on high-performance liquid chromatography and atmospheric pressure chemical ionization magnetic sector mass spectrometry was developed and optimized to determine trace concentrations of 11 compounds belonging to the group of the seldom-analyzed oxy-PAHs (phenanthrene-9,10-dione, chrysene-5,6-dione, benzo[a]pyrene-4,5-dione, benzo[a]pyrene-1,6-dione, benzo[a]pyrene-3,6-dione, benzo[a]pyrene-6,12-dione, 4-oxa-benzo[def]chrysene-5-one, pyrene-1-carboxaldehyde, benzo[de]anthracene-7-one, benzo[a]anthracene-7,12-dione, and napthacene-5,12-dione) on airborne particulate matter (PM10). The mass spectrometer was operated in multiple ion detection mode, allowing for selective accurate mass detection (mass resolution of 12,000 full width at half maximum) of the oxy-PAHs characteristic ions. Optimization of both the vaporizer (450 degrees C) and capillary temperature (350 degrees C) resulted into instrumental detection limits in the range between 7 (benzo[a]pyrene-1,6-dione) and 926 pg (benzo[a]anthracene-7,12-dione). The advanced pressurized liquid extraction (PLE) and the more traditionally used ultrasonic extraction (USE) were compared using ethyl acetate as an extraction solvent. For both techniques, high recoveries from spiked quartz fiber filters (PLE, 82-110%; USE, 67-97%) were obtained. Recoveries obtained from real PM10 samples were also high (76-107%), and no significant matrix effects (ME) on the ionization process (enhancement or suppression) were found (ME, 89-123%). Method limits of quantification (S/N=10) were in the range between 2 and 336 pg/m(3). This method was used to analyze real PM samples collected at several urban and rural locations in the Antwerp area. For the first time, concentrations for Belgium are provided. Concentrations of individual oxy-PAHs are in the lower pictograms per cubic meter to 6 ng/m(3) range. High concentration differences between individual compounds are found as exemplified by the 75th percentile of the phenanthrene-9,10-dione and benzo[de]anthracene-7-one concentrations being a factor of 4 to 22 higher compared with the other target oxy-PAHs