Levels of GHB in hair after regular application.

Gamma hydroxybutyrate (GHB) is a central nervous system depressant that is an approved drug for the treatment of narcolepsy with cataplexy and other syndromes. Due to its dose dependent stimulating, relaxing or sedative effects, illicit abuses include recreational use by young people and cases of drug-facilitated crime (DFC). Since GHB is also produced endogenously, for forensic questions, it is important to be able to differentiate between endogenous GHB and elevated levels due to additional intake. In this study, we measured GHB concentrations in hair of patients with narcolepsy receiving daily GHB treatment. The results were compared to endogenous concentrations and concentrations after chronic intake presented in several former studies. The aim of this study was to investigate whether a regular intake of a known dosage of GHB leads to elevated levels of GHB concentration in hair. We collected hair samples of 19 patients (14 female, 5 male) with narcolepsy under regular GHB treatment and examined the hair samples segmentally by digestion of the hair followed by liquid-liquid extraction and analysis using a Shimadzu LC20 UFLC system coupled with an AB Sciex API 4000 Qtrap tandem mass spectrometer. All volunteers received daily treatment with different doses of sodium oxybate (sodium salt of GHB) ranging between 3 and 9g per night. The observed mean value of GHB concentration in hair was 2.69ng GHB per mg hair for the 5 male participants, 1.56ng/mg for the 14 female participants giving an overall mean value of 1.86ng/mg for all participants. Our results showed no correlation between the daily dose or the duration intake of GHB and the measured concentration of GHB in hair. Although we did find a significant (p<0.01) difference between published endogenous levels of GHB in hair and GHB levels in hair of patients with regular daily GHB intake, the forensic relevance however is disputable. We hypothesise this narrow margin or even overlap to be the reason why analytical results from hair analysis in some cases fail to provide a reliable prove of a single exposition.

Liquid silicone embolism syndrome (SES) in lungs and liver - A case report.

Silicone embolism syndrome (SES) is a well known complication after injection of silicone gel as well as liquid silicone. Rarely, men use physiologic salt solution or liquid silicone injected into the subcutaneous tissue of the scrotum, the penis, the upper genital or the inguinal region. Those men, who call themselves "siliconers", want to get a larger penis and scrotum, also visible when wearing clothes. Injections of liquid silicone in the mentioned regions can lead to liquid silicone embolism in the lungs and also the liver, sometimes eventually leading to death via right heart failure as in the present case. Autopsy revealed "frog spawn"-like vacuoles in the subcutaneous tissue of the genital region and liquid silicone embolism in lungs and liver. Additionally, toxicological analyses revealed different liquid silicones. Smaller oligomers were transported into lung and liver, larger ones showed local enrichment at the injection site. The seized Polydimethylsiloxane (PDMS) could not be detected in abdominal fat, blood or urine, potentially due to low perfusion of fat tissue, the aqueous character of blood and urine or the time span between last injection and death.

Variation of intraindividual levels of endogenous GHB in segmented hair samples.

Gamma-hydroxybutyrate (GHB) belongs to a group of substances that may be used in drug-facilitated crime (DFC). It is also an endogenous substance. There is a dispute whether or not a single exposure to GHB can be detected in hair. The first aim of this study was to develop and validate a method for the sensitive detection of base levels of GHB in hair. The second aim was to collect analytical data of 88 volunteers (62 females/26 males) not claiming any exposure to GHB and discuss the results in the context of the identification of a potential single exposure in cases of DFC. Furthermore hair samples from a male volunteer, who took GHB twice within 8 weeks, were analysed and the results were discussed with regard to mean values of endogenous GHB analysed in this study. Hair was digested under alkaline conditions, and GHB was isolated using liquid-liquid extraction. LC-MS/MS was performed using Electrospray ionization in the negative mode, multiple reaction monitoring, and a deuterated internal standard (GHB-D6). Segmental hair analysis revealed mean concentrations of 0.673ng/mg or 0.676ng/mg (without first segment) in females and 0.935ng/mg or 0.932ng/mg (without first segment) in males. Combined mean values were 0.751ng/mg and 0.752ng/mg (without first segment). In one individual's hair single doses of 2g GHB did not lead to an increase compared to his base levels. The limits of detection and quantitation in human hair were 0.1ng/mg and 0.3ng/mg, respectively. Accuracy at 0,25ng/mg, 2,5ng/mg and 25ng/mg was determined to be 94% or higher for all levels and intra-assay CVs at these concentrations were always lower than 7% (n=5). ß-hydroxybutyrate (BHB) and glycine did not produce an interference. Recovery at 1ng/mg and 25ng/mg GHB was 23% and 13% and Matrix effects were calculated to be 77% and 89% respectively.

Structure and Mechanism of the Sphingopyxin†I Lasso Peptide Isopeptidase.

Lasso peptides are natural products that assume a unique lariat knot topology. Lasso peptide isopeptidases (IsoPs) eliminate this topology through isopeptide bond cleavage. To probe how these enzymes distinguish between substrates and hydrolyze only isopeptide bonds, we examined the structure and mechanism of a previously uncharacterized IsoP from the proteobacterium Sphingopyxis alaskensis RB2256 (SpI-IsoP). We demonstrate that SpI-IsoP efficiently and specifically linearizes the lasso peptide sphingopyxin I (SpI) and variants thereof. We also present crystal structures of SpI and SpI-IsoP, revealing a threaded topology for the former and a prolyl oligopeptidase (POP)-like fold for the latter. Subsequent structure-guided mutational analysis allowed us to propose roles for active-site residues. Our study sheds light on lasso peptide catabolism and expands the engineering potential of these fascinating molecules.

Improved δ-valerolactam templates for the assembly of Aß-miniamyloids by boronic ester formation.

The 6,7,8,8a-cis (all-cis) substituted δ-valerolactams of type 10, 11 and 12 are high-affinity diols for boronic ester formation, superior to the corresponding 6,7-trans analogues 1, 3 and 4. X-ray and NMR structure analysis have identified the differences of the six-membered ring conformations which cause the improved esterification properties of the all-cis stereoisomers. The homooligomeric all-cisδ-valerolactams 46-48 are used as polyol templates for the self-assembly of peptidic oligomers 49-52 by dynamic covalent chemistry. The templates have a diol spacing of approximately 5 Å, suitable for the assembly of branched peptides from the quantitative reaction between the peptide of interest, 2-formylphenylboronic acid and the respective template. According to this strategy, the tetrameric Aß-miniamyloid 52 formed spontaneously from nine individual molecules in a three-component system. A detailed NMR analysis based on the complete sequential assignment of the trimeric Aß(32-40)-miniamyloid 51 identified its three-dimensional structure in solution.

A synthetic adenylation-domain-based tRNA-aminoacylation catalyst.

The incorporation of non-proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20-22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion-protein-based design for synthetic tRNA-aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA-binding domain (Arc1p-C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p-C using flexible linkers and achieved tRNA-aminoacylation with both proteinogenic and non-proteinogenic amino acids. The resulting aminoacyl-tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA-aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non-proteinogenic amino acids.