Emergence, Diversity, and Control of New Psychoactive Substances: A Global Perspective.

The phenomenon of new psychoactive substances (NPS), which came to the attention of the wider international community at the beginning of the 2010s, has been unprecedented in terms of the sheer number of substances, their rate of emergence, chemical diversity, and range of pharmacological effects. In particular, the chemical diversity has been a challenge to promoting a better understanding of the NPS market - a fundamental requirement for effective policy decisions and interventions. This manuscript highlights the significant chemical diversity of NPS and describes an alternative, complementary, and pragmatic classification based on pharmacological effects, which aligns NPS to traditional controlled drugs and enhances understanding of the phenomenon. It further reviews actions taken at the international level to address the NPS issue, including changes in the scope of control of some NPS and the enhancement of the United Nations Early Warning Advisory on NPS to deal with the dynamics and evolution of the market.

Targeted overexpression of CRH receptor subtype 1 in central amygdala neurons: effect on alcohol-seeking behavior.

RATIONALE: The corticotropin-releasing hormone (CRH) system is a key mediator of stress-induced responses in alcohol-seeking behavior. Recent research has identified the central nucleus of the amygdala (CeA), a brain region involved in the regulation of fear and stress-induced responses that is especially rich in CRH-positive neurons, as a key player in mediating excessive alcohol seeking. However, detailed characterization of the specific influences that local neuronal populations exert in mediating alcohol responses is hampered by current limitations in pharmacological and immunohistochemical tools for targeting CRH receptor subtype 1 (CRHR1). OBJECTIVE: In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool. METHODS: Co-expression of CRHR1 in calcium-calmodulin-dependent kinase II (αCaMKII) neurons of the amygdala was demonstrated by double immunohistochemistry using a Crhr1-GFP reporter mouse line. A Cre-inducible Crhr1-expressing adeno-associated virus (AAV) was site-specifically injected into the CeA of αCaMKII-CreERT2 transgenic rats to analyze the role of CRHR1 in αCaMKII neurons on alcohol self-administration and reinstatement behavior. RESULTS: Forty-eight percent of CRHR1-containing cells showed co-expression of αCaMKII in the CeA. AAV-mediated gene transfer in αCaMKII neurons induced a 24-fold increase of Crhr1 mRNA in the CeA which had no effect on locomotor activity, alcohol self-administration, or cue-induced reinstatement. However, rats overexpressing Crhr1 in the CeA increased responding in the stress-induced reinstatement task with yohimbine serving as a pharmacological stressor. CONCLUSION: We demonstrate that CRHR1 overexpression in CeA-αCaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.

Environmental Selection, Dispersal, and Organism Interactions Shape Community Assembly in High-Throughput Enrichment Culturing.

A central goal of microbial ecology is to identify and quantify the forces that lead to observed population distributions and dynamics. However, these forces, which include environmental selection, dispersal, and organism interactions, are often difficult to assess in natural environments. Here, we present a method that links microbial community structures with selective and stochastic forces through highly replicated subsampling and enrichment of a single environmental inoculum. Specifically, groundwater from a well-studied natural aquifer was serially diluted and inoculated into nearly 1,000 aerobic and anaerobic nitrate-reducing cultures, and the final community structures were evaluated with 16S rRNA gene amplicon sequencing. We analyzed the frequency and abundance of individual operational taxonomic units (OTUs) to understand how probabilistic immigration, relative fitness differences, environmental factors, and organismal interactions contributed to divergent distributions of community structures. We further used a most probable number (MPN) method to estimate the natural condition-dependent cultivable abundance of each of the nearly 400 OTU cultivated in our study and infer the relative fitness of each. Additionally, we infer condition-specific organism interactions and discuss how this high-replicate culturing approach is essential in dissecting the interplay between overlapping ecological forces and taxon-specific attributes that underpin microbial community assembly.IMPORTANCE Through highly replicated culturing, in which inocula are subsampled from a single environmental sample, we empirically determine how selective forces, interspecific interactions, relative fitness, and probabilistic dispersal shape bacterial communities. These methods offer a novel approach to untangle not only interspecific interactions but also taxon-specific fitness differences that manifest across different cultivation conditions and lead to the selection and enrichment of specific organisms. Additionally, we provide a method for estimating the number of cultivable units of each OTU in the original sample through the MPN approach.

Toxicology in international drug control-Prioritizing the most harmful, persistent and prevalent substances.

The nature of the global drugs market has evolved rapidly and has become more complex with the emergence of new psychoactive substances (NPS), some of which have been associated with increased abuse, hospital emergency admissions and sometimes fatalities. NPS are characterized by geographic heterogeneity, with some only transient in nature and others not satisfying the criteria for harm required for international control. Consequently, a pragmatic response of the international community is to prioritize the most harmful, persistent and prevalent substances for action - an objective, which is hampered by the paucity of data on harms. The report describes a United Nations Office on Drugs and Crime (UNODC) initiative, in collaboration with the International Association of Forensic Toxicologists (TIAFT), to collect, analyze and share toxicology data at a global level to reinforce the ability of the international community in making informed decisions using a scientific evidence-based approach, in identifying the most harmful NPS.

CRF receptor type 2 neurons in the posterior bed nucleus of the stria terminalis critically contribute to stress recovery.

The bed nucleus of the stria terminalis (BNST) is critical in mediating states of anxiety, and its dysfunction has been linked to stress-related mental disease. Although the anxiety-related role of distinct subregions of the anterior BNST was recently reported, little is known about the contribution of the posterior BNST (pBNST) to the behavioral and neuroendocrine responses to stress. Previously, we observed abnormal expression of corticotropin-releasing factor receptor type 2 (CRFR2) to be associated with post-traumatic stress disorder (PTSD)-like symptoms. Here, we found that CRFR2-expressing neurons within the pBNST send dense inhibitory projections to other stress-related brain regions (for example, the locus coeruleus, medial amygdala and paraventricular nucleus), implicating a prominent role of these neurons in orchestrating the neuroendocrine, autonomic and behavioral response to stressful situations. Local CRFR2 activation by urocortin 3 depolarized the cells, increased the neuronal input resistance and increased firing of action potentials, indicating an enhanced excitability. Furthermore, we showed that CRFR2-expressing neurons within the pBNST are critically involved in the modulation of the behavioral and neuroendocrine response to stress. Optogenetic activation of CRFR2 neurons in the pBNST decreased anxiety, attenuated the neuroendocrine stress response, ameliorated stress-induced anxiety and impaired the fear memory for the stressful event. Moreover, activation following trauma exposure reduced the susceptibility for PTSD-like symptoms. Optogenetic inhibition of pBNST CRFR2 neurons yielded opposite effects. These data indicate the relevance of pBNST activity for adaptive stress recovery.

Newer insights into the role of miRNA a tiny genetic tool in psychiatric disorders: focus on post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a mental disorder occurring in about 2-9% of individuals after their exposure to life-threatening events, such as severe accidents, sexual abuse, combat or a natural catastrophe. Because PTSD patients are exposed to trauma, it is likely that epigenetic modifications have an important role in disease development and prognosis. For the past two decades, abnormal expression of the epigenetic regulators microRNAs (miRs) and miR-mediated gene regulation have been given importance in a variety of human diseases, such as cancer, heart disease and viral infection. Emerging evidence supports a role for miR dysregulation in psychiatric and neurological disorders, including schizophrenia, bipolar disorder, anxiety, major depressive disorder, autism spectrum disorder and Tourette's syndrome. Recently mounting of evidence supports the role of miR both in preclinical and clinical settings of psychiatric disorders. Abnormalities in miR expression can fine-tune the expression of multiple genes within a biological network, suggesting that miR dysregulation may underlie many of the molecular changes observed in PTSD pathogenesis. This provides strong evidence that miR not only has a critical role in PTSD pathogenesis, but can also open up new avenues for the development of diagnostic tools and therapeutic targets for the PTSD phenotype. In this review, we revisit some of the recent evidence associated with miR and PTSD in preclinical and clinical settings. We also discuss the possible clinical applications and future use of miRs in PTSD therapy.

'Some patients are more equal than others': Patient-centred care differential in two-tier inpatient ward hospitals in Ghana.

OBJECTIVE: To examine differences in patient-centred care among private and public inpatients in public hospitals and whether satisfaction with patient-centred care differ between the patient groups. METHOD: Cross-sectional data collected from inpatients in private wards (n=300) and public wards (n=520) in Ghana, using a structured questionnaire modelled on four dimensions of patient-centred care: respect and dignity, emotional support, interpersonal relations and information sharing. RESULTS: Patient-centred care differed significantly among private and public patients (p<0.001), with an effect size ranging from medium to large. Private patients rated patient-centred care higher than public patients in all the items of the four dimensions. Satisfaction with patient-centred care discriminated between the patient groups. Satisfaction was significantly high for private patients who are aged 50+ (p<0.001), had high education (p<0.05) and high income (p<0.001) compared to the same category of public patients. CONCLUSION: Physicians behaviour is stereotyping and less favourable to public patients, suggesting inequitable access to patient-centred care for inpatients from high and low socioeconomic backgrounds. PRACTICE IMPLICATIONS: Hospitals with private and public wards should be compelled to properly coordinate and regulate the activities of physicians to avoid fragmented care for inpatients.

Sex differences in NMDA GluN1 plasticity in rostral ventrolateral medulla neurons containing corticotropin-releasing factor type 1 receptor following slow-pressor angiotensin II hypertension.

There are profound, yet incompletely understood, sex differences in the neurogenic regulation of blood pressure. Both corticotropin signaling and glutamate receptor plasticity, which differ between males and females, are known to play important roles in the neural regulation of blood pressure. However, the relationship between hypertension and glutamate plasticity in corticotropin-releasing factor (CRF)-receptive neurons in brain cardiovascular regulatory areas, including the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN), is not understood. In the present study, we used dual-label immuno-electron microscopy to analyze sex differences in slow-pressor angiotensin II (AngII) hypertension with respect to the subcellular distribution of the obligatory NMDA glutamate receptor subunit 1 (GluN1) subunit of the N-methyl-D-aspartate receptor (NMDAR) in the RVLM and PVN. Studies were conducted in mice expressing the enhanced green fluorescence protein (EGFP) under the control of the CRF type 1 receptor (CRF1) promoter (i.e., CRF1-EGFP reporter mice). By light microscopy, GluN1-immunoreactivity (ir) was found in CRF1-EGFP neurons of the RVLM and PVN. Moreover, in both regions tyrosine hydroxylase (TH) was found in CRF1-EGFP neurons. In response to AngII, male mice showed an elevation in blood pressure that was associated with an increase in the proportion of GluN1 on presumably functional areas of the plasma membrane (PM) in CRF1-EGFP dendritic profiles in the RVLM. In female mice, AngII was neither associated with an increase in blood pressure nor an increase in PM GluN1 in the RVLM. Unlike the RVLM, AngII-mediated hypertension had no effect on GluN1 localization in CRF1-EGFP dendrites in the PVN of either male or female mice. These studies provide an anatomical mechanism for sex-differences in the convergent modulation of RVLM catecholaminergic neurons by CRF and glutamate. Moreover, these results suggest that sexual dimorphism in AngII-induced hypertension is reflected by NMDA receptor trafficking in presumptive sympathoexcitatory neurons in the RVLM.

The p.Arg86Gln change in GARP2 (glutamic acid-rich protein-2) is a common West African-related polymorphism.

BACKGROUND/AIMS: The aim of the present study is to probe the potential association between previously-reported GARP2 mutations and retinitis pigmentosa (RP) using Scottish RP patients and controls. METHODS: Exons 4, 5 and 8 in DNA from blood or buccal samples (130 autosomal recessive and simplex RP patients, 31 controls) were amplified and analysed for single-strand conformational polymorphism by capillary electrophoresis (CE-SSCP) and confirmed by sequencing. RESULTS: The p.Arg86Gln mutation in exon 4 was found in just one patient (out of 130), and in 10 of the 31 unaffected subjects. All of these occurrences were in people of West African origin (patient and controls). Two polymorphisms in exon 5, p.His100Arg and p.Gly109Gly, and a c.534+20A>G change in the intronic region flanking the 3' end of exon 8 were also found not to be associated with RP. CONCLUSIONS: The Scottish population examined here had no mutations in the GARP2 exons surveyed that could be associated with RP. The p.Arg86Gln mutation actually appears to be a polymorphism common in ethnic West Africans and not associated with RP. This change may provide a useful marker for West African ancestry.

Chromatographic retention behaviour, modelling and separation optimisation of the quaternary ammonium salt isometamidium chloride and related compounds on a range of reversed-phase liquid chromatographic stationary phases.

This paper describes the reversed-phase liquid chromatographic behaviour of the trypanocidal quaternary ammonium salt isometamidium chloride and its related compounds on a range of liquid chromatographic phases possessing alkyl and phenyl ligands on the same inert silica. In a parallel study with various extended polar selectivity phases which possessed different hydrophobic/silanophilic (hydrogen bonding) activity ratios, the chromatographic retention/selectivities of the quaternary ammonium salts was shown to be due to a co-operative mechanism between hydrophobic and silanophilic interactions. The highly aromatic and planar isometamidium compounds were found to be substantially retained on stationary phases containing aromatic functionality via strong π-π interactions. The chemometric approach of principal component analysis was used to characterise the chromatographic behaviour of the isometamidium compounds on the differing phases and to help identify the dominant retention mechanism(s). Two-dimensional (temperature/gradient) retention modelling was employed to develop and optimise a rapid liquid chromatography method for the separation of the six quaternary ammonium salts within 2.5 min which would be suitable for bioanalysis using liquid chromatography-mass spectrometry. This is the first reported systematic study of the relationship between stationary phase chemistries and retention/selectivity for a group of quaternary ammonium salts.

The effect of anticoagulants on the distribution of chromium VI in blood fractions.

Metal-on-metal resurfacing arthroplasty is associated with elevated circulating levels of cobalt and chromium ions. To establish the long-term safety of metal-on-metal resurfacing arthroplasty, it has been recommended that during clinical follow-up of these patients, the levels of these metal ions in blood be monitored. In this article, we provide information on the distribution of chromium VI ions (the predominant form of chromium released by cobalt-chrome alloys in vivo and in vitro) in blood fractions. Chromium VI is predominantly partitioned into red blood cells compared with plasma (analysis of variance, P < .05). The extent of accumulation in red blood cells is influenced by the anticoagulant used to collect the blood, with EDTA giving a lower partitioning into red cells compared with sodium citrate and sodium heparin.

Spatial distribution of diarrhoea and microbial quality of domestic water during an outbreak of diarrhoea in the Tshikuwi community in Venda, South Africa.

Total microbial quality assessment and geographical information system were used for evaluating the quality of water and the spatial distribution of diarrhoea cases in Tshikuwi, a rural community in South Africa, during an outbreak of diarrhoea. The water-abstraction points included two groundwater storage tanks, namely Tank 1 and Tank 2 and the Khandanama river. Indicator microbial counts for total coliforms, faecal coliforms, enterococci, and heterotrophic bacteria exceeded the limit for no risk as stipulated by the South African water-quality guidelines for domestic use for Tank 1 and the Khandanama river. Vibrio, Salmonella, and Shigella species were prevalent in the Khandanama river. The spatial distribution of diarrhoea cases showed a hot-spot of diarrhoea cases close to Tank 1 and the Khandanama river. Results of chi-square analysis showed that the proportion of infection from each water source was different or that infection depends on the type of water source (alpha = 0.05). The demonstrated spatial clustering of diarrhoea cases might have been influenced by the poor microbial quality of water used from Tank 1 and the Khandanama river. The results further highlight the urgent need of water-treatment facilities and monitoring of water quality in rural communities of South Africa.

Metabolism of two new benzodiazepine-type anti-leishmanial agents in rat hepatocytes and hepatic microsomes and their interaction with glutathione in macrophages.

OBJECTIVES: To measure the metabolism and toxicity of 7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. METHODS: The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. KEY FINDINGS: BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 microM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. CONCLUSIONS: The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.

Comparative chronic in vitro toxicity of hexavalent chromium to osteoblasts and monocytes.

There is increasing concern regarding the long-term biological effects of exposure to low concentrations of metal ions, particularly chromium, in patients following implantation of metal-on-metal hip prostheses. To investigate this, we have evaluated the chronic in vitro response to clinically relevant concentrations of chromium VI in osteoblasts and monocytes over a 4-week period in continuous culture. The cell viability, intracellular reduced glutathione content, glutathione reductase activity and expression, and expression of glutamate cysteine ligase were monitored in both cell types. Monocytes were more susceptible to the toxicity of the metal, and at the end of 4 weeks exposure to 0.5 microM Cr VI, the protein content of cultures had declined to 23.4% +/- 1.0% of control cultures. Both cell types demonstrated temporal increases in reduced glutathione levels, glutathione reductase activity, and glutamate cysteine ligase expression. Only osteoblasts showed a transcriptional increase in reductase expression. Data suggest that both cell types mount an adaptive response to chronic exposure to Cr VI, but this is more potent in osteoblasts, and results in the relative resilience of this cell type to the effects of Cr VI on cell viability.

Simultaneous analysis of basic, acidic and neutral compounds on an endcapped octadecylsilane silica-based monolith by pressure-assisted capillary electrochromatography.

This simultaneous separation of basic, acidic and neutral analytes by pressure-assisted CEC (pCEC) using a hybrid (tetramethoxysilane and methyltrimethoxysilane) silica-based monolith, chemically modified with octadecyldimethylchlorosilane followed by endcapping with hexamethyldisilazane is described. The endcapping resulted in near Gaussian peaks for highly basic analytes such as nortriptyline without a significant loss in the EOF. The migration behaviour of analytes on this phase could be rationalised based on hydrophobicity, electrophoretic mobility and ion-exchange interactions. The high porosity of the monolith allowed manipulation of the linear velocity of mobile phases by the addition of varying amounts of pressure at the inlet to reduce analysis times and overcome the reversed migration of anionic species towards the detection window in cathodic EOF mode. The concomitant programmed application of pressure (2-4 bar) and voltage (27 kV) facilitated the simultaneous separation of four cationic, four neutral and two anionic compounds in 6 min with efficiencies ranging from 41 000 to 94 000, 57 000 to 77 000 and 180 000 to 210 000 theoretical plates/metre, respectively. The % RSD values of migration times and efficiencies in pCEC mode were less than 3.6 and 7.9%, respectively (n = 5).

CE hydrogen deuterium exchange-MS in peptide analysis.

CE and hydrogen-deuterium (H/D) exchange MS are useful tools in the analysis and characterisation of peptides. This study reports the facile coupling of these tools in the H/D exchange CE-MS analysis of model and pharmaceutically important peptides, using a sheath flow interface. The peptides varied in mass from 556 (leucine enkephalin) to 1620 Da (bombesin), and in charge state from 0.33 (leucine enkephalin) to 3.0 (substance P). The application of a BGE composed of ammonium formate buffer (25 mM, pD 3.5 in D(2)O (>98% D atom)), a sheath liquid composed of formic acid (0.25% v/v in D(2)O) and ACN (30:70 v/v), and dissolving the samples in a mixture of ACN/D(2)O (50:50 v/v) facilitates complete H/D exchange. Because of complete H/D exchange the ESI mass spectra produced are easy to interpret and comparable to those obtained from LC-MS analysis. The CE-H/D-MS approach has the advantage of requiring lower volumes of deuterated solvents. The b- and y-series fragments produced by using in-source decomposition correspond to those predicted. With the peptides studied, the complete exchange H/D exchange observed with both the molecular and fragment ions helps to confirm both amino acid composition and sequence.

Identification of the benzodiazepines as a new class of antileishmanial agent.

The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have identified the benzodiazepines as a structural class for antileishmanial hit optimisation, and demonstrated that their in vitro activity is comparable with the clinically used drug, sodium stibogluconate, and that the compounds are not toxic to macrophages.

Capillary electrochromatography of therapeutic peptides on mixed-mode butylmethacrylate monoliths.

In this study, a porous mixed-mode n-alkyl methacrylate-based monolith has been used in the separation of therapeutic peptides. While the sulfonic acid (SCX) moiety derived from 2-acrylamido-2-methyl-1-propanesulfonic acid supports the generation of a stable electroosmotic flow (EOF) at both acidic and basic pH values, the butyl ligands provide the nonpolar sites for chromatographic resolution. The performance of the monolith was evaluated regarding the influence of pH on chromatographic resolution of peptides. The suitability of the butylmethacrylate/SCX monolith for the analysis of therapeutic peptides containing basic centres, for example arginine, at moderately high pH 9.5 and the stability to repeat injections of a mixture of peptides was demonstrated. Separations with efficiencies as high as 5.0 x 10(5) plates/m were obtained and the migration behaviour of the peptides at both low (2.8) and high (9.5) pH values could be rationalised based on their charge, molecular mass/shape and relative hydrophobicities.

Formation and protein binding of the acyl glucuronide of a leukotriene B4 antagonist (SB-209247): relation to species differences in hepatotoxicity.

SB-209247 [(E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic acid], an anti-inflammatory leukotriene B4 receptor antagonist, was associated in beagle dogs but not male rats with an inflammatory hepatopathy. It also produced a concentration-dependent (10-1000 microM) but equal leakage of enzymes from dog and rat precision-cut liver slices. The hepatic metabolism of SB-209247 was investigated with reference to the formation of reactive acyl glucuronides. [14C]SB-209247 (100 micromol/kg) administered i.v. to anesthetized male rats was eliminated by biliary excretion of the acyl glucuronides of the drug and its sulfoxide. After 5 h, 1.03 +/- 0.14% (mean +/- S.E.M., n = 4) of the dose was bound irreversibly to liver tissue. The sulfoxide glucuronide underwent pH-dependent rearrangement in bile more rapidly than did the SB-209247 conjugate. [14C]SB-209247 was metabolized by sulfoxidation and glucuronidation in rat and dog hepatocytes, and approximately 1 to 2% of [14C]SB-209247 (100 microM) became irreversibly bound to cellular material. [14C]SB-209247 sulfoxide and glucuronide were the only metabolites produced by dog, rat, and human liver microsomes in the presence of NADPH and UDP-glucuronic acid (UDPGA), respectively. V(max) values for [14C]SB-209247 glucuronidation by dog, rat, and human microsomes were 2.6 +/- 0.1, 1.2 +/- 0.1, and 0.4 +/- 0.0 nmol/min/mg protein, respectively. Hepatic microsomes from all three species catalyzed UDPGA-dependent but not NADPH-dependent irreversible binding of [14C]SB-209247 (100-250 microM) to microsomal protein. Although a reactive acyl glucuronide was formed by microsomes from every species, the binding did not differ between species. Therefore, neither the acute cellular injury nor glucuronidation-driven irreversible protein binding in vitro is predictive of the drug-induced hepatopathy.