Rapid screening procedure based on headspace solid-phase microextraction and gas chromatography-mass spectrometry for the detection of many recreational drugs in hair

An increasing number of synthetic drugs are appearing on the illicit market and on the scene of drug use by youngsters. Official figures are underestimated. In addition, immunochemical tests are blind to many of these drugs and appropriate analytical procedures for routine clinical and epidemiological purposes are lacking. Therefore, the perceived increasing abuse of recreational drugs has not been proved yet. In a previous paper, we proposed a procedure for the preliminary screening of several recreational substances in hair and other biological matrices. Unfortunately, this procedure cannot apply to cocaine. Consequently, we performed a new headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of cocaine, amphetamine (A), methamphetamine (MA), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE), N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), ketamine, and methadone in human hair. Hair was washed with water and acetone in an ultrasonic bath. A short acid extraction with 1M hydrochloric acid was needed; the fiber was exposed to a 5 min absorption at 90 degrees C and thermal desorption was performed at 250 degrees C for 3 min. The procedure was simple, rapid, required small quantities of sample and no derivatization. Good linearity was obtained over the 0.1-20.0 ng/mg range for the target compounds. Sensitivity was good enough: limits of detection (LOD) were 0.7 ng/mg of hair for the majority of substances. The intra-day precision ranged between 7 and 20%. This paper deals with the analytical performance of this procedure and its preliminary application to hair samples obtained on a voluntary basis from 183 young people (138 males and 45 females) in the Rome area.     

Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats

Background

Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored.

Methods

Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies.

Results

There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling.

Conclusions

The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT- rats.     

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l^?1 6-benzyladenine (BA) and 0.1 mg l^?1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l^?1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.     

Influence of carbohydrate source on xanthone content in root cultures of Gentiana dinarica Beck

The effects of different types and concentrations of sugars on root growth and xanthone production in root culture of Gentiana dinarica were investigated. The results showed that sucrose, glucose and fructose all supported root growth, and sucrose was superior in terms of growth index, dry mass and fresh/dry mass ratio then fructose or glucose at the same concentrations. However, considering equimolar concentration of sugars, their contribution to the root growth was similar. The HPLC analysis of roots indicated the presence of xanthone compounds, and the contents of norswertianin-1-O-primeveroside (1), norswertianin-1-O-glucoside (2), gentioside (3) and norswertianin (4) were evaluated. In all samples, norswertianin-1-O-primeveroside (1) was present in highest concentration, followed by norswertianin-1-O-glucoside (2), whereas gentioside (3) and norswertianin (4) were present in lower amounts. The production of xanthones was affected by both type and concentration of sugar. In general, roots growing in media supplemented with sucrose contained higher levels of xanthones. The amounts of xanthone primeveroses (1) and (3) increased with the increase of concentrations of all types of sugars, whereas higher sugar concentrations resulted in reduction of the contents of norswertianin-1-O-glucoside (2) and aglycone norswertianin (4). The roots were also evaluated regarding the content of total phenolics and higher accumulation of total phenolic compounds was observed in roots grown in fructose-containing medium. Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and high correlation between total phenolic content and antiradical activity was observed (r = ?0.83).     

Critical Role of TLR4 in Human Metapneumovirus Mediated Innate Immune Responses and Disease Pathogenesis

Human metapneumovirus (hMPV) is one of the main causes of acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including viruses. We have recently shown that hMPV-induced cytokine, chemokine and type I interferon secretion in dendritic cells occurs via TLR4, however, its role in hMPV-induced disease is unknown. In this study, wild-type(WT) and TLR4-deficient mice (TLR4^−/−) were infected with hMPV and examined for clinical disease parameters, such as body weight loss and airway obstruction, viral clearance, lung inflammation, dendritic cell maturation, T-cell proliferation and antibody production. Our results demonstrate that absence of TLR4 in hMPV-infected mice significantly reduced the inflammatory response as well as disease severity, shown by reduced body weight loss and airway obstruction and hyperresponsiveness (AHR), compared to WT mice. Levels of cytokines and chemokines were also significantly lower in the TLR4^−/− mice. Accordingly, recruitment of inflammatory cells in the BAL, lungs, as well as in lymph nodes, was significantly reduced in the TLR4^−/− mice, however, viral replication and clearance, as well as T-cell proliferation and neutralizing antibody production, were not affected. Our findings indicate that TLR4 is important for the activation of the innate immune response to hMPV, however it does play a role in disease pathogenesis, as lack of TLR4 expression is associated with reduced clinical manifestations of hMPV disease, without affecting viral protection.     

Xanthone compounds in shoot cultures of <Emphasis Type="Italic">Gentianella bulgarica</Emphasis>

Shoot cultures of Gentianella bulgarica established from seedling epicotyls were maintained on MS medium supplemented with BA 0.2 mg l⁻¹ + NAA 0.1 mg l⁻¹. Cultures were prone to precocious flowering requiring the use of small shoot buds for multiplication purposes. The contents of three xanthone compounds identified as DGL, BGL, and DMB, in different plant material were determined by HPLC. The analysis revealed that the production of xanthones was affected by different concentrations of BA in medium. Shoot cultures grown at higher BA concentrations contained more DGL than material grown in nature. The concentrations of other two xanthones were lower in shoot cultures than in plants from nature. The radical scavenging activity of plant extracts and xanthones was investigated by DPPH test. Samples from plants grown in nature showed the highest activity (IC₅₀ = 0.26 mg ml⁻¹), while the extracts of shoot cultures grown in media with higher concentrations of BA showed moderate activities (IC₅₀ from 1.6 to 4.4 mg ml⁻¹).     

Molecular Typing of MRSA and of Clinical Staphylococcus aureus Isolates from Ia?i,Romania

Romania is one of the countries with the highest prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the world. To obtain data on affiliation of MRSA to strains and clonal complexes and on the population of methicillin susceptible S. aureus (MSSA), clinical isolates from bloodstream infections, skin and soft tissue infections as well as from screening swabs were collected at hospitals in Ia?i, a city in the North-Eastern part of Romania. Isolates were characterised by microarray hybridisation. Nearly half of all isolates (47%), and about one third (34%) of bloodstream isolates were MRSA. The prevalence of the Panton-Valentine leukocidin (PVL) was also high (31% among MRSA, 14% among MSSA). The most common MRSA strain was a PVL-negative CC1-MRSA-IV that might have emerged locally, as a related MSSA was also common. PVL-positive CC8-MRSA-IV (“USA300”) and PVL-negative ST239-like MRSA-III were also frequently found while other MRSA strains were only sporadically detected. Among MSSA, PVL-positive CC121 as well as PVL-negative CC1, CC22 and CC45 predominated. Although this study provides only a snapshot of S. aureus/MRSA epidemiology in Romania, it confirms the high burden of MRSA and PVL on Romanian healthcare settings.     

Phosphorylation of murine caspase-9 by the protein kinase casein kinase 2 regulates its cleavage by caspase-8

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in tumor necrosis factor (TNF)-alpha-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of Ser(348) on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of Ser(348) abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-alpha-treated caspase-9 knock-out mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, whereas phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9, rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.