The analysis and detection of new psychoactive substances in biological matrices

New psychoactive substances (NPSs) have appeared on the recreational drug market at an unprecedented rate in recent years. Many are not new drugs but failed products of the pharmaceutical industry. The speed and variety of drugs entering the market poses a new complex challenge for the forensic toxicology community. The detection of these substances in biological matrices can be difficult as the exact compounds of interest may not be known. Many NPS are sold under the same brand name and therefore users themselves may not know what substances they have ingested. The majority of analytical methods for the detection of NPSs tend to focus on a specific class of compounds rather than a wide variety. In response to this, a robust and sensitive method was developed for the analysis of various NPS by solid phase extraction (SPE) with gas chromatography mass spectrometry (GCMS). Sample preparation and derivatisation were optimised testing a range of SPE cartridges and derivatising agents, as well as derivatisation incubation time and temperature. The final gas chromatography mass spectrometry method was validated in accordance with SWGTOX 2013 guidelines over a wide concentration range for both blood and urine for 23 and 25 analytes respectively. This included the validation of 8 NBOMe compounds in blood and 10 NBOMe compounds in urine. This GC-MS method was then applied to 8 authentic samples with concentrations compared to those originally identified by NMS laboratories. The rapid influx of NPSs has resulted in the re-analysis of samples and thus, the stability of these substances is crucial information. The stability of mephedrone was investigated, examining the effect that storage temperatures and preservatives had on analyte stability daily for 1 week and then weekly for 10 weeks. Several laboratories identified NPSs use through the cross-reactivity of these substances with existing screening protocols such as ELISA. The application of Immunalysis ketamine, methamphetamine and amphetamine ELISA kits for the detection of NPS was evaluated. The aim of this work was to determine if any cross-reactivity from NPS substances was observed, and to determine whether these existing kits would identify NPS use within biological samples. The cross- reactivity of methoxetamine, 3-MeO-PCE and 3-MeO-PCP for different commercially point of care test (POCT) was also assessed for urine. One of the newest groups of compounds to appear on the NPS market is the NBOMe series. These drugs pose a serious threat to public health due to their high potency, with fatalities already reported in the literature. These compounds are falsely marketed as LSD which increases the chance of adverse effects due to the potency differences between these 2 substances. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was validated in accordance with SWGTOX 2013 guidelines for the detection for 25B, 25C and 25I-NBOMe in urine and hair. Long-Evans rats were administered 25B-, 25C- and 25I-NBOMe at doses ranging from 30-300 µg/kg over a period of 10 days. Tail flick tests were then carried out on the rats in order to determine whether any analgesic effects were observed as a result of dosing. Rats were also shaved prior to their first dose and reshaved after the 10-day period. Hair was separated by colour (black and white) and analysed using the validated LC-MS/MS method, assessing the impact hair colour has on the incorporation of these drugs. Urine was collected from the rats, analysed using the validated LC-MS/MS method and screened for potential metabolites using both LC-MS/MS and quadrupole time of flight (QToF) instrumentation.

The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

The impact and control of malignant catarrhal fever in Tanzania

Malignant Catarrhal Fever (MCF), an often-lethal infectious disease, presents as a variable complex of lesions in susceptible ungulate species. The disease is caused by a -herpesvirus following transmission from an inapparent carrier host. Two major epidemiological forms exist: wildebeest-associated MCF (WA-MCF), in which the virus is transmitted to susceptible species by wildebeest calves less than approximately four months of age, and sheepassociated MCF (SA-MCF) in which the virus is spread by sheep (primarily adolescents). Due to the lack of an in-vitro propagation system for the causative agent of the more economically significant SA-MCF, and with the expectation that cross-protective immunity may be provided, vaccine development has focused on the more easily propagated alcelaphine herpesvirus-1 (AlHV-1) that causes WA-MCF. In 2008 a direct viral challenge trial showed that a novel vaccine, employing an attenuated AlHV-1 (atAlHV-1) `C5000 virus strain, protected British Friesian-Holstein (FH) cattle against an intranasal challenge with virulent AlHV-1 `C5000 virus. For cattle keeping people living near wildebeest calving areas in sub-Saharan Africa an effective vaccine would have value as it would release them from the costly annual disease avoidance strategy of having to move their herds away from the oncoming wildebeest. On the other hand, an effective vaccine will release herd owners from the need to avoid MCF, allowing them to graze their cattle alongside wildebeest on the highly nutritious pastures of the calving areas. As such conservationists have raised concerns that the development of a vaccine might lead to detrimental grazing competition. The principle objective of this study was to test the novel vaccine on Tanzanian shorthorn zebu cross cattle (SZC).We did this firstly using a natural challenge field trial (Chapter Two) which demonstrated that immunisation with the atAlHV-1 vaccine was well tolerated and induced an oro-nasopharyngeal AlHV-1-specific and -neutralising antibody response. This resulted in an immunity in SZC cattle that was partially protective and reduced naturally transmitted infection by 56%. We also demonstrated that non-fatal infections occurred with a much higher frequency than previously thought. Because the calculated efficacy of the vaccine was less than that seen in British FH cattle we wanted to determine whether host factors, particular to SZC cattle, had impacted the outcomes of the field trial. To do this we repeated the 2008 direct viral challenge trial using SZC cattle (Chapter Four). During this trial we also investigated whether the recombinant bacterial flagellin monomer (FliC), when used as an adjuvant, might improve the vaccine’s efficacy. The findings from this trial indicated that direct challenge with pathogenic AlHV-1 is effective at inducing MCF in SZC cattle and that FliC is not an appropriate adjuvant for this vaccine. Furthermore, with less control group cattle dying of MCF than expected we speculate that SZC cattle may have a degree of resistance to MCF that affords them protection from infection and developing fatal disease. In Chapter Three we investigated aspects of the epidemiology of MCF, specifically whether wildebeest placenta, long implicated by Maasai cattle owners as a source of MCF, might play a role in viral transmission. Additionally, through comparative sequence analysis, at two specific genes (A9.5 and ORF50) of wild-type and atAlHV-1, we investigated whether the `C5000 strain, the source of which was taken from Africa more than 40 years ago, was appropriate for vaccine development. The detection of AlHV-1 virus in approximately 50% of placentae indicated that infection can occur in-utero and that this tissue might play a role in disease transmission. And, despite describing three new alleles of the A9.5 gene (supporting previous evidence that this gene is polymorphic and encodes a secretory protein with interleukin-4 as the major homologue), the observation that the most frequently detected haplotypes, in both wild-type and attenuated AlHV-1, were identical suggests that AlHV-1 has a slow molecular clock and that the attenuated strain was appropriate for vaccine development. In Chapter Five we present the first quantitative assessment of the annual MCF avoidance costs that Maasai pastoralists incur. In particular we estimated that as a result of MCF avoidance 64% of the total daily milk yield during the MCF season was not available to be used by the 81% of the family unit remaining at the permanent boma. This represents an upper-bound loss of approximately 8% of a household0s annual income. Despite these considerable losses we concluded that, given an incidence of fatal MCF in cattle living in wildebeest calving areas of 5% to 10%, if herd owners were to stop trying to avoid MCF by allowing their cattle to graze alongside wildebeest, any gains made through increased availability of milk, improved body condition and reduced energy demands would be offset by an increase in MCF-incidence. With the development of an effective vaccine, however, this alternative strategy might become optimal. The overall conclusion we draw therefore is that, despite the substantial costs incurred each year avoiding MCF, the partial protection afforded by the novel vaccine strategy is not sufficient to warrant a wholesale change in disease avoidance strategy. Nonetheless, even the partial protection provided by this vaccine could be of value to protect animals that cannot be moved, for example where some of the herd remain at the boma to provide milk or where land-use changes make traditional disease avoidance difficult. Furthermore, the vaccine may offer a feasible solution to some of the current land-use challenges and conflicts, providing a degree of protection to valuable livestock where avoidance strategies are not possible, but with less risk of precipitating the potentially damaging environmental consequences, such as overgrazing of highly nutritious seasonal pastures, that might result if herd owners decide they no longer need to avoid wildebeest.