Various "legal high" products were tested for synthetic cannabinoids and synthetic stimulants to qualitatively determine the active ingredient(s). Ultra-performance liquid chromatography with accurate mass time-of-flight mass spectrometry (UPLC-TOF) was used to monitor the non-biological specimens utilizing a customized panel of 65+ compounds comprised of synthetic cannabinoids, synthetic stimulants and other related drugs. Over the past year, the United States Drug Enforcement Agency has controlled five synthetic cannabinoid compounds (JWH-018, JWH-073, JWH-200, CP-47,497 and CP-47,497-C8) and three synthetic stimulant compounds (3,4-methylenedioxypyrovalerone, mephedrone and methylone) that were previously reported to be detected in these legal high products. Through our analyses of first and second generation products, it was shown that many of these banned substances are no longer used and have been replaced by other derivatives that are federally legal. Since enactment of the federal bans on synthetic cannabinoids and synthetic stimulants, 4.9% of the products analyzed at our facility contained at least one controlled substance. The remaining 95.1% of products contained only uncontrolled drugs. We demonstrate the UPLC-TOF methodology to be a powerful tool in the qualitative identification of these designer drugs, thus enabling a laboratory to keep current with the drugs that are being sold as these designer products.

The bZIP proto-oncoprotein c-Fos activates transcription of a wide variety of genes involved in cell growth. The C-terminal activation domain of c-Fos is functionally independent of the remainder of the protein. Fos-AD corresponds to the C-terminal activation domain of human c-Fos (residues 216-380). Fos-AD suppresses (squelches) transcription in vitro, as expected for a functional activation domain lacking a DNA-binding domain. Fos-AD is unstructured and highly mobile, as demonstrated by circular dichroism spectra indicative of unfolded proteins, a lack of (1)H chemical shift dispersion, and negative (1)H-(15)N heteronuclear nuclear Overhauser effects. The hydrodynamic properties of Fos-AD are also consistent with an extended structure. We conclude that the C-terminal domain of human c-Fos is biologically active yet intrinsically disordered. Our results suggest that conformational disorder is an integral aspect of the diverse contributions to transcriptional regulation by c-Fos.

Synthetic cannabinoids have been detected in various herbal blends sold legally in convenience stores, smoke shops, and on the Internet. Many of these compounds have extreme forensic significance. We developed and validated a rapid ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of trace concentrations of two of these compounds, JWH-018 and JWH-073, in human blood. Samples underwent liquid-liquid extraction at pH 10.2 into ethyl ether. Tandem mass spectrometry was performed in positive electrospray ionization mode with multiple reaction monitoring using two transitions and one calculated ion transition ratio for each analyte. Deuterated analogs were used as internal standards. Total run time was 2.6 min. The linear dynamic range was 0.05-50 ng/mL with a limit of detection of 0.01 ng/mL for each analyte. Intra-run imprecision (at two different concentration levels, 2 and 8 ng/mL) was 3.9-10.3% for JWH-018 and 3.5-6.2% for JWH-073. Inter-run imprecision was 6.5-7.2% for JWH-018 and 4.8-5.5% for JWH-073. Intra-run accuracy was 95.9-112.7% for JWH-018 and 92.6-104.7% for JWH-073. Inter-run accuracy was 99.1-107.0% for JWH-018 and 97.7-102.0% for JWH-073. Carryover, exogenous drug interferences, ion suppression and matrix selectivity were also assessed. The method has been applied to postmortem forensic casework received by the laboratory and has proven to be robust and reliable. Concentrations of authentic samples have ranged from 0.1-199 ng/mL for JWH-018 and 0.1-68.3 ng/mL for JWH-073.

Osmol gap can be used as a surrogate marker for serum propylene glycol concentration. In critically ill patients receiving lorazepam for sedation, an osmol gap above 10 was associated with concentrations previously reported to cause toxicity.

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of histonemodifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.Chromatin functions to compact and organize DNA in the nucleus of eukaryotic cells in a manner that allows regulated access to genes for transcription and DNA replication. The role of nucleosomes in transcriptional regulation has become a major area of study. From in vitro studies, it is clear that nucleosomes can repress transcription by RNA polymerase II (1-3). Further confirmation that nucleosomes play a prominent role in gene regulation came from studies showing that histone H4 depletion in yeast cells results in nucleosome loss and transcription derepression of several RNA polymerase II-transcribed genes (4, 5). Barring artificial loss, nucleosomes must be reconfigured prior to transcriptional activation of these genes. Recently, many yeast activities that remodel chromatin have been identified, including the SWI/SNF, INO80, ISW1, ISW2, and RSC complexes (reviewed in Ref. 6). All of these activities contain a DNA-or chromatin-dependent ATPase subunit required for remodeling. In addition, many transcriptional activators recruit histone acetyltransferase activities, which in yeast include the ADA, Spt-Ada-Gcn5 acetyltransferase (SAGA), NuA3, and NuA4 complexes (7,8).The chromatin structure of the yeast PHO5 promoter regulates RNA polymerase II transcription of the PHO5 gene, which encodes the major, secreted acid phosphatase in yeast (9). Under repressive conditions (adequate phosphate) the PHO5 promoter is bound in an array of positioned nucleosomes (10). Activation of PHO5 through phosphate starvation is accompanied by a loss or reconfiguration of four nucleosomes from the promot...