Generation of metabolites by an automated online metabolism method using human liver microsomes with subsequent identification by LC-MS(n), and metabolism of 11 cathinones

Human liver microsomes (HLMs) are used to simulate human xenobiotic metabolism in vitro. In forensic and clinical toxicology, HLMs are popularly used to study the metabolism of new designer drugs for example. In this work, we present an automated online extraction system we developed for HLM experiments, which was compared to a classical offline approach. Furthermore, we present studies on the metabolism of 11 cathinones; for eight of these, the metabolism has not previously been reported. Metabolites were identified based on MS2 and MS3 scans. Fifty-three substances encompassing various classes of drugs were employed to compare the established offline and the new online methods. The metabolism of each of the following 11 cathinones was studied using the new method: 3,4-methylenedioxy-N-benzylcathinone, benzedrone, butylone, dimethylcathinone, ethylone, flephedrone, methedrone, methylone, methylethylcathinone, naphyrone, and pentylone. The agreement between the offline and the online methods was good; a total of 158 metabolites were identified. Using only the offline method, 156 (98.7%) metabolites were identified, while 151 (95.6%) were identified using only the online method. The metabolic pathways identified for the 11 cathinones included the reduction of the keto group, desalkylation, hydroxylation, and desmethylenation in cathinones containing a methylenedioxy moiety. Our method provides a straightforward approach to identifying metabolites which can then be added to the library utilized by our clinical toxicological screening method. The performance of our method compares well with that of an established offline HLM procedure, but is as automated as possibl

Ecological and economic impacts of wildfires on an Appalachian oak forest in southern West Virginia

Oaks (Quercus spp.) are considered a fire adapted species and fire disturbances throughout the lifecycle have been proposed to be necessary for maintaining oak dominated stands. Wildfire has been blamed for significant losses in sawtimber volume. A total of 164 points were sampled in stands that had experienced between zero to six wildfires over 36 years in Cabwaylingo State Forest (CSF), West Virginia. The wildfires have affected different strata and species of forest vegetation in different ways. While total overstory density decreased from 242 to 38 TPA with increasing fire frequency, oak density was highest in areas of three fires. Although overall basal area decreased from 171 to 52 ft2/ac, oak basal area remained fairly constant at around 60 ft2/ac. Oaks in smaller (\u3c 8 inches) DBH classes were most abundant in areas of two to four wildfires. Total density of sapling sized stems increased with heightened fire occurrence. Oak saplings were significantly highest in density in areas of four fires. Neither oak nor non-oak seedling densities showed significantly differences by fire occurrences. Aggregate oak seedling height, however, was highest in areas that had burned four times. A regeneration assessment method by Steiner et al. (2008) revealed that expected oak stocking 30 years after complete harvest would be 73% in areas of three burns, decreasing with lower or higher fire occurrences. Our results indicate that oak advance reproduction is most competitive over non-oak species in those areas of CSF that have experienced two to four wildfires. Sawtimber volume loss due to cull increased from 181 to 752 Bdft/ac in areas experiencing from zero to six fire occurrences. Total net volume and net dollar value declined with increasing fire frequency ranging from 10,924 to 4,906 Bdft/ac and {dollar}1,208 to 589/ac, respectively; this is equal to a value decline of nearly 54%

Development of a fully automated toxicological LC-MSn screening system in urine using online extraction with turbulent flow chromatography

In clinical toxicology, fast and specific methods are necessary for the screening of different classes of drugs. Therefore, an online extraction high-performance liquid chromatography coupled to mass spectrometry (LC-MSn) screening method using a MS2 and MS3 spectral library for the identification of xenobiotic substances has been developed and validated. Samples were run twice, once native and once after enzymatic hydrolysis. Internal standards and buffer were added to the urine samples. Following centrifugation, the supernatant was injected into the system. Extraction was performed by online turbulent flow chromatography. The chromatographic separation was achieved using a Phenyl/Hexyl column. For detection, a linear ion trap, equipped with an APCI interface, was used and the different compounds were identified using a MS2 and MS3 spectral library containing 356 compounds. The turnaround time to report the results of the screening including hydrolysis was approximately 2h. About 92% of the 356 substances could be identified with a limit of identification below 100ng/ml. The recovery and matrix effect experiments showed suitable results, and in six drug-free urine samples of healthy volunteers analyzed for selectivity, no substances have been identified. Carryover could be well controlled, and the method had a good reproducibility. The comparison of the results of 103 real patient urine samples showed a good agreement between the existing GC-MS and LC-MS methods with offline extraction and the new online extraction LC-MSn screening method. The presented method allows a fast and sensitive analysis of a broad range of compound

Die Rolle der alpha/beta T-Lymphozyten bei der Regulation des Knochenturnover

Das Ziel der hier vorliegenden Arbeit ist es, die Rolle der alpha/beta-T-Lymphozyten in der Regulation des Knochenturnovers anhand biochemischer, densitometrischer und histomorphometrischer Parameter zu untersuchen, um weitere Einblicke in die Verbindung zwischen Immunzellen und Knochenstoffwechsel zu erlangen. In Versuch I wurde die Rolle der alpha/beta-T-Lymophozyten in der Pathogenese der Östrogen- und Androgenmangel-Osteopenie untersucht. Dazu waren 32 weibliche, 6 Monate alte und 32 männliche, 12 Monate alte Fischer-344-Ratten vorgesehen. Jeweils die Hälfte der Tiere wurde entweder mit einer neu etablierten Methode thymektomiert (TX) bzw. scheinoperiert (Sham-TX). Zwei Wochen nach der Operation erhielten die thymektomierten Ratten eine dreimalige intraperitoneale (i.p.) Injektion im Abstand von drei Tagen eines monoklonalen rattenspezifischen alpha/beta-TCR (T-Zell-Rezeptor) gerichteten IgG1-Antikörpers (R73) (20 mg/kg Körpergewicht (KGW)). Die Sham-TX Tiere erhielten 20 mg/kg KGW eines unspezifischen murinen monoklonalen Antikörpers des gleichen Isotyps (Maus IgG1). Die alpha/beta-T-Zell-Depletion wurde mit Hilfe einer FACS-Analyse im peripheren Blut überprüft, wobei nur Tiere mit 0-5% "gegateter" (für R73 positiv) Zellen als alpha/beta-T-Zell-depletiert angesehen wurden, alle anderen Tiere wurden von der Analyse ausgeschlossen. Drei Wochen nach der T-Zell-Depletion wurden die Tiere entweder gonadektomiert (GX) bzw. scheinoperiert (Sham-GX), wobei alle Tiere zwei Monate post-GX acht und drei Tage vor der Tötung eine Fluorochrom-Doppelmarkierung (20mg/kg KGW Calcein; s.c.) erhielten. In Versuch II wurde die Rolle von alpha/beta-T-Lymphozyten für die Skelettwirkung von CsA, im Hinblick auf die klinische Anwendung von Cyclosporin A (CsA) bei Posttransplantations-Patienten untersucht. Dazu wurden 32 weibliche, 6 Monate alte und 32 männliche, 9 Monate alte Fischer-344-Ratten vorgesehen. Analog zu Versuch I wurde die Hälfte der Tiere entweder TX oder Sham-TX. Zwei Wochen nach der Operation erhielten die TX Ratten eine dreimalige R73 Gabe (20 mg/kg KGW; i.p.) im Abstand von drei Tagen. Die Sham-TX Tiere erhielten 20 mg/kg KGW eines unspezifischen murinen monoklonalen Antikörpers des gleichen Isotyps (Maus IgG1). Drei Wochen nach der T-Zell-Depletion wurden die Tiere für zwei Monate mit CsA (5 mg/kg KGW; s.c.) oder Vehikel (mittelkettige Triglyceride; s.c.) getötet. Mit Hilfe des Tiermodells konnte klar erarbeitet werden, dass die alpha/beta-T-Zell-Depletion per se keinen Effekt auf den Knochenturnover hat. Die Östrogen- oder Androgenmangel induzierte Osteopenie der Ratten wurden nicht durch eine Depletion von alpha/beta-T-Zellen moduliert. Ebenso bleibt die Skelettwirkung von CsA durch die alpha/beta-T-Zell-Depletion unbeeinflusst. Die, in der Literatur beschriebene, Geschlechtsspezifität der Skelettwirkung von CsA ist konsistent mit unserer In-vitro Untersuchung, konnte jedoch nicht bei unserer In-vivo Untersuchung gezeigt werden. Das neu etablierte Tiermodell, mit dem eine dauerhafte T-Zell-Depletion bei normalen adulten Ratten durch eine Kombination aus TX und T-Zell-Depletion mit Hilfe des R73 Antikörpers erreicht werden kann, ist auch für weitere osteoimmunologische Fragestellungen interessant

Highly Efficient siRNA Delivery from Core-Shell Mesoporous Silica Nanoparticles with Multifunctional Polymer Caps

A new general route for siRNA delivery is presented combining porous core-shell silica nanocarriers with a modularly designed multifunctional block copolymer. Specifically, the internal storage and release of siRNA from mesoporous silica nanoparticles (MSN) with orthogonal core-shell surface chemistry was investigated as a function of pore-size, pore morphology, surface properties and pH. Very high siRNA loading capacities of up to 380 microg/mg MSN were obtained with charge-matched amino-functionalized mesoporous cores, and release profiles show up to 80% siRNA elution after 24 h. We demonstrate that adsorption and desorption of siRNA is mainly driven by electrostatic interactions, which allow for high loading capacities even in medium-sized mesopores with pore diameters down to 4 nm in a stellate pore morphology. The negatively charged MSN shell enabled the association with a block copolymer containing positively charged artificial amino acids and oleic acid blocks, which acts simultaneously as capping function and endosomal release agent. The potential of this multifunctional delivery platform is demonstrated by highly effective cell transfection and siRNA delivery into KB-cells. A luciferase reporter gene knock-down of up to 90% was possible using extremely low cell exposures with only 2.5 microg MSN containing 32 pM siRNA per 100 microL well.Comment: Artikel including supporting informatio

Differential effects of purified low molecular weight Poly(I:C) in the maternal immune activation model depend on the laboratory environment.

The Poly (I:C) (polyriboinosinic-polyribocytidilic acid) paradigm of maternal immune activation (MIA) is most widely used as experimental model for the evaluation of the effects of gestational infection on the brain and behavior of the progeny. We have previously reported significant batch-to-batch variability in the effects of Poly (I:C), purchased from the same supplier (Sigma-Aldrich), on maternal and fetal immune responses and found these differences to be dependent on the relative amount of synthetic double-stranded RNA fragments in the high versus low molecular weight (LMW) range contained in the compound. We here resorted to Poly (I:C) purified for LMW dsRNA fragments to establish a MIA paradigm with increased reproducibility and enhanced standardization in an effort to refine the MIA paradigm and characterize its effect on offspring behavior. We found that the parallel application of LMW Poly (I:C) in two different MIA-experienced laboratories (Vienna and Zurich) yielded differential outcomes in terms of maternal immune responses and behavioral phenotypes in the offspring generation. In both experimental sites, administration of LMW Poly (I:C) induced a significant sickness response and cytokine induction in the pregnant dam and fetal brains, while the expected deficit in sociability as one main behavioral outcome parameter in the MIA progeny, was only present in the Zurich, but not the Vienna cohort. We conclude that although using Poly (I:C) purified for a defined molecular weight range reduces batch-to-batch variability, it does not make the MIA model more reliable and robust. The differential response in behavioral phenotypes of the MIA offspring between the two laboratories illustrates the highly complex interaction between prenatal and postnatal milieus - including the laboratory environment - that determine offspring phenotypic outcomes after MIA. Consequently, establishing a new MIA protocol or implementing the MIA model firstly under new or changed environmental conditions must include the assessment of offspring behavior to ensure solid and reproducible experimental outcomes

Optimisation of the Read-out Electronics of Muon Drift-Tube Chambers for Very High Background Rates at HL-LHC and Future Colliders

In the ATLAS Muon Spectrometer, Monitored Drift Tube (MDT) chambers and sMDT chambers with half of the tube diameter of the MDTs are used for precision muon track reconstruction. The sMDT chambers are designed for operation at high counting rates due to neutron and gamma background irradiation expected for the HL-LHC and future hadron colliders. The existing MDT read-out electronics uses bipolar signal shaping which causes an undershoot of opposite polarity and same charge after a signal pulse. At high counting rates and short electronics dead time used for the sMDTs, signal pulses pile up on the undershoot of preceding background pulses leading to a reduction of the signal amplitude and a jitter in the drift time measurement and, therefore, to a degradation of drift tube efficiency and spatial resolution. In order to further increase the rate capability of sMDT tubes, baseline restoration can be used in the read-out electronics to suppress the pile-up effects. A discrete bipolar shaping circuit with baseline restoration has been developed and used for reading out sMDT tubes under irradiation with a 24 MBq 90Sr source. The measurements results show a substantial improvement of the performance of the sMDT tubes at high counting rates

Effectiveness of Dietary Interventions in the Treatment of Endometriosis: a Systematic Review

A patients’ increasing interest in dietary modifications as a possible complementary or alternative treatment of endometriosis is observed. Unfortunately, the therapeutic potential of dietary interventions is unclear and to date no guidelines to assist physicians on this topic exist. The aim of this study, therefore, was to systematically review the existing studies on the effect of dietary interventions on endometriosis. An electronic-based search was performed in MEDLINE and COCHRANE. We included human and animal studies that evaluated a dietary intervention on endometriosis-associated symptoms or other health outcomes. Studies were identified and coded using standard criteria, and the risk of bias was assessed with established tools relevant to the study design. We identified nine human and 12 animal studies. Out of the nine human studies, two were randomized controlled trials, two controlled studies, four uncontrolled before-after studies, and one qualitative study. All of them assessed a different dietary intervention, which could be classified in one of the following principle models: supplementation with selected dietary components, exclusion of selected dietary components, and complete diet modification. Most of the studies reported a positive effect on endometriosis; they were however characterized by moderate or high-risk bias possibly due to the challenges of conducting dietary intervention trials. According to the available level of evidence, we suggest an evidence-based clinical approach for physicians to use during consultations with their patients. Further well-designed randomized controlled trials are needed to accurately determine the short-term and long-term effectiveness and safety of different dietary interventions

Factors Associated With Lameness in Tie Stall Housed Dairy Cows in South Germany

Lameness remains a major concern for animal welfare and productivity in modern dairy production. Even though a trend toward loose housing systems exists and the public expects livestock to be kept under conditions where freedom of movement and the expression of natural behavior are ensured, restrictive housing systems continue to be the predominant type of housing in some regions. Factors associated with lameness were evaluated by application of multiple logistic regression modeling on data of 1,006 dairy cows from 56 tie stall farms in Bavaria, South Germany. In this population, approximately every fourth cow was lame (24.44% of scored animals). The mean farm level prevalence of lameness was 23.28%. In total, 22 factors were analyzed regarding their association with lameness. A low Body Condition Score (BCS) (OR 1.54 [95%-CI 1.05-2.25]) as well as increasing parity (OR 1.41 [95%-CI 1.29-1.54]) entailed greater odds of lameness. Moreover, higher milk yield (OR 0.98 [95%-CI 0.96-1.00]) and organic farming (OR 0.48 [95%-0.25-0.92]) appeared to be protectively associated with lameness. Cows with hock injuries (OR 2.57 [95%-CI 1.41-4.67]) or with swellings of the ribs (OR 2.55 [95%-CI 1.53-4.23]) had higher odds of lameness. A similar association was observed for the contamination of the lower legs with distinct plaques of manure (OR 1.88 [95%-CI 1.14-3.10]). As a central aspect of tie stall housing, the length of the stalls was associated with lameness; with stalls of medium [(>158-171 cm) (OR 2.15 [95%-CI 1.29-3.58]) and short (171 cm). These results can help both gaining knowledge on relevant factors associated with lameness as well as approaching the problem of dairy cow lameness in tie stall operations