Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

Plant-Derived, Nodule-Specific Cysteine-Rich Peptides as a Novel Source of Biopesticides for Controlling Citrus Greening Disease.

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.

Host Plant Adaptation Drives Changes in Diaphorina citri Proteome Regulation, Proteoform Expression, and Transmission of 'Candidatus Liberibacter asiaticus', the Citrus Greening Pathogen.

The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, 'Candidatus Liberibacter asiaticus' (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

Proteomic Polyphenism in Color Morphotypes of Diaphorina citri, Insect Vector of Citrus Greening Disease.

Diaphorina citri is a vector of "Candidatus Liberibacter asiaticus" (CLas), associated with citrus greening disease. D. citri exhibit at least two color morphotypes, blue and non-blue, the latter including gray and yellow morphs. Blue morphs have a greater capacity for long-distance flight and transmit CLas less efficiently as compared to non-blue morphs. Differences in physiology and immunity between color morphs of the insect vector may influence disease epidemiology and biological control strategies. We evaluated the effect of CLas infection on color morph and sex-specific proteomic profiles of D. citri. Immunity-associated proteins were more abundant in blue morphs as compared to non-blue morphs but were upregulated at a higher magnitude in response to CLas infection in non-blue insects. To test for differences in color morph immunity, we measured two phenotypes: (1) survival of D. citri when challenged with the entomopathogenic fungus Beauveria bassiana and (2) microbial load of the surface and internal microbial communities. Non-blue color morphs showed higher mortality at four doses of B. bassinana, but no differences in microbial load were observed. Thus, color morph polyphenism is associated with two distinct proteomic immunity phenotypes in D. citri that may impact transmission of CLas and resistance to B. bassiana under some conditions.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Candidatus Liberibacter asiaticus Minimally Alters Expression of Immunity and Metabolism Proteins in Hemolymph of Diaphorina citri, the Insect Vector of Huanglongbing.

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ' Candidatus Liberibacter asiaticus' ( CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology.

UNLABELLED: Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCE: The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy.

Ranking the harm of non-medically used prescription opioids in the UK.

A panel of nine experts applied multi-criteria decision analysis (MCDA) to determine the relative overall harm to users and harms to others of street heroin (injected and smoked) and eleven non-medically used prescription opioids. The experts assessed harm scores for each of the 13 opioids on each of 20 harm criteria, weighted the criteria and explored the resulting weighted harm scores for each opioid. Both forms of heroin scored very high: overall harm score of 99 for injected heroin and 72 for smoked heroin on a scale of 0-100. The main feature that distinguishes both forms of street heroin use is that their harm to others is more than five times that of the other eleven opioids. The overall harm score of fentanyl (including injection of fentanyl extracted from patches) and diamorphine (medically prescribed form of heroin) was 54 and 51, respectively, whereas that of orally used opioids ranged from 32 (pethidine) to 11 (codeine-containing pharmaceuticals). Injected street heroin, fentanyl and diamorphine emerged as most harmful to users, with the latter two very low in harm to others. Pethidine, methadone, morphine and oxycodone are also low in harm to others, while moderate in harm to users. We conclude that the overall harms of non-medically used prescription opioids are less than half that of injected street heroin. These data may give a basis for precautionary regulatory measures that should be considered if the rising trend in non-medical use of prescription opioids were to become evident in the UK.

Adaptation to nicotine feeding in Myzus persicae.

Lineages of the generalist hemipteran herbivore Myzus persicae (green peach aphid) that have expanded their host range to include tobacco often have elevated nicotine tolerance. The tobacco-adapted M. persicae lineage used in this study was able to reproduce on nicotine-containing artificial diets at concentrations that were 15-fold higher than those that were lethal to a non-adapted M. persicae lineage. Fecundity of the nicotine-tolerant M. persicae lineage was increased by 100 µM nicotine in artificial diet, suggesting that this otherwise toxic alkaloid can serve as a feeding stimulant at low concentrations. This lineage also was pre-adapted to growth on tobacco, exhibiting no drop in fecundity when it was moved onto tobacco from a different host plant. Although growth of the non-tobacco-adapted M. persicae lineage improved after three generations on tobacco, this higher reproductive rate was not associated with increased nicotine tolerance. Myzus persicae gene expression microarrays were used to identify transcripts that are up-regulated in response to nicotine in the tobacco-adapted lineage. Induced expression was found for CYP6CY3, which detoxifies nicotine in M. persicae, other genes encoding known classes of detoxifying enzymes, and genes encoding secreted M. persicae salivary proteins.

Estimating the harms of nicotine-containing products using the MCDA approach.

BACKGROUND: An international expert panel convened by the Independent Scientific Committee on Drugs developed a multi-criteria decision analysis model of the relative importance of different types of harm related to the use of nicotine-containing products. METHOD: The group defined 12 products and 14 harm criteria. Seven criteria represented harms to the user, and the other seven indicated harms to others. The group scored all the products on each criterion for their average harm worldwide using a scale with 100 defined as the most harmful product on a given criterion, and a score of zero defined as no harm. The group also assessed relative weights for all the criteria to indicate their relative importance. FINDINGS: Weighted averages of the scores provided a single, overall score for each product. Cigarettes (overall weighted score of 100) emerged as the most harmful product, with small cigars in second place (overall weighted score of 64). After a substantial gap to the third-place product, pipes (scoring 21), all remaining products scored 15 points or less. INTERPRETATION: Cigarettes are the nicotine product causing by far the most harm to users and others in the world today. Attempts to switch to non-combusted sources of nicotine should be encouraged as the harms from these products are much lower.

Analysis of drugs seized from amnesty bins at two major United Kingdom summer music festivals using two portable gas chromatography-mass spectrometry (GC-MS) instruments.

Globally, the number of drug users and the proportion of the drug using population has increased from 210 million in 2009 to 269 million in 2019. Several studies suggest that music festival attendees are more likely to abuse illicit substances and have a high-risk profile. Consequently, it is crucial to develop robust field drug analysis methods that facilitate harm reduction and drug monitoring. The work presented in this report aimed at developing and validating qualitative analytical methods for 3,4-methylenedioxymethamphetamine, 4-bromo-2,5-dimethoxyphenethylamine (2C-B), ketamine and N-ethylpentylone on two portable gas chromatography-mass spectrometry (GC-MS) systems: Griffin G510 (Teledyne FLIR, West Lafayette, IN) and Torion T-9 (PerkinElmer, Shelton, CT). The diagnostic ability of the mobile GC-MS units was assessed on 200 samples in total, seized at two large summer music festivals in the United Kingdom. The method validation process included selectivity/specificity, limit of identification, carry-over, ruggedness/robustness, and inter- and intra-day precision (repeatability and reproducibility). The Griffin G510 demonstrated a limit of identification from 1 mg/mL for 2C-B to 0.063 mg/mL for ketamine and good ruggedness and precision results. The precision for 2C-B using the Torion T-9 was poorer than for the Griffin G510, but equivalent for the other compounds tested. Correct identifications (versus benchtop GC-MS) for the two festivals were 85%-86% and 74%-83% for the Griffin G510 and the Torion T-9, respectively. The two portable instruments were able to adequately cover current on-site drug-testing analytical gaps and proved to be a powerful addition to the on-site drug analysis techniques.

Assessment of a Single Quadrupole Mass Spectrometer Combined with an Atmospheric Solids Analysis Probe for the On-Site Identification of Amnesty Bin Drugs.

The surging number of people who abuse drugs has a great impact on healthcare and law enforcement systems. Amnesty bin drug analysis helps monitor the "street drug market" and tailor the harm reduction advice. Therefore, rapid and accurate drug analysis methods are crucial for on-site work. An analytical method for the rapid identification of five commonly detected drugs ((3,4-methylenedioxymethamphetamine (MDMA), cocaine, ketamine, 4-bromo-2,5-dimethoxyphenethylamine, and chloromethcathinone)) at various summer festivals in the U.K. was developed and validated employing a single quadrupole mass spectrometer combined with an atmospheric pressure solids analysis probe (ASAP-MS). The results were confirmed on a benchtop gas chromatography-mass spectrometry instrument and included all samples that challenged the conventional spectroscopic techniques routinely employed on-site. Although the selectivity/specificity step of the validation assessment of the MS system proved a challenge, it still produced 93% (N = 279) and 92.5% (N = 87) correct results when tested on- and off-site, respectively. A few "partly correct" results showed some discrepancies between the results, with the MS-only unit missing some low intensity active ingredients (N-ethylpentylone, MDMA) and cutting agents (caffeine, paracetamol, and benzocaine) or detecting some when not present. The incorrect results were mainly based on library coverage. The study proved that the ASAP-MS instrument can successfully complement the spectroscopic techniques used for qualitative drug analysis on- and off-site. Although the validation testing highlighted some areas for improvement concerning selectivity/specificity for structurally similar compounds, this method has the potential to be used in trend monitoring and harm reduction.

Analysis of illicit drugs in purchased and seized electronic cigarette liquids from the United Kingdom 2014-2021.

Increasing popularity and known shortfalls in the regulation of electronic cigarettes (ECs) emphasises the urgent need for closer content monitoring and for comprehensible information on their possible health effects. This study investigated components of EC liquids in samples submitted from 2014 to 2021 and discussed the trends driven by legislation changes. Samples originating from prisoners, teenagers and 'test purchases' of commercially available ECs were analysed by gas chromatography-mass spectrometry (GC-MS). For those containing delta-9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD), the content of these components was quantified by liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to show variation of these compounds in EC liquids; 112 EC liquids were included in this study. Nicotine was detected in 87 (78%) of the EC liquids analysed. Twenty-two, including samples from before and after introduction of the UK Psychoactive Substances Act (2016), contained one or more synthetic cannabinoid receptor agonist (SCRA). THC was detected in only 11 samples, whereas a single sample was found to contain CBD only. Six samples contained a mixture of THC and CBD. In all cases where information was available, the THC/CBD content was less than that stated on the product label. The data collected showed great variation in EC liquid content. Therefore, it is important that users are educated regarding risks associated with EC use. Additionally, substances now controlled under both the UK Misuse of Drugs Act and Psychoactive Substances Act were present. These substances each carry a potential risk to health, which is possibly exacerbated if multiple compounds are inhaled concomitantly.

Risk of caffeine toxicity associated with the use of 'legal highs' (novel psychoactive substances).

PURPOSE: Caffeine has desired stimulant effects similar to but weaker than both classical recreational drugs and novel psychoactive substances. This study was undertaken to determine the caffeine content of a sample of novel psychoactive substances, and we discuss the implications for the management of acute recreational drug toxicity. METHODS: Six novel psychoactive products ('legal highs') that were not declared to contain caffeine were purchased from different Internet suppliers; one additional product was supplied by the UK police force. Analysis of these products was undertaken using infrared spectroscopy (IR), gas chromatography with mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS) to identify the active ingredient(s) and measure the caffeine content of the product. RESULTS: All seven products, which weighed approximately 1 g each, contained only caffeine as the active pharmacological compound. There was significant variation in the percentage caffeine content (<2 to 96%), with four powders containing very significant caffeine contents of 87-96%. CONCLUSION: This study shows that individuals are at risk of significant caffeine toxicity related to the high caffeine content of some novel psychoactive substances. Clinicians, including clinical pharmacologists, need to be aware of this to ensure that the management of acute recreational drug toxicity is appropriate and that over-correction of any hypokalaemia does not occur.

Laser capture of tomato pericarp tissues for microscale carotenoid analysis by supercritical fluid chromatography.

Plant organs and tissues are comprised of an array of cell types often superimposed on a gradient of developmental stages. As a result, the ability to analyze and understand the synthesis, metabolism, and accumulation of plant biomolecules requires improved methods for cell- and tissue-specific analysis. Tomato (Solanum lycopersicum) is the world's most valuable fruit crop and is an important source of health-promoting dietary compounds, including carotenoids. Furthermore, tomato possesses unique genetic activities at the cell and tissue levels, making it an ideal system for tissue- and cell-type analysis of important biochemicals. A sample preparation workflow was developed for cell-type-specific carotenoid analysis in tomato fruit samples. Protocols for hyperspectral imaging of tomato fruit samples, cryoembedding and sectioning of pericarp tissue, laser microdissection of specific cell types, metabolite extraction using cell wall digestion enzymes and pressure cycling, and carotenoid quantification by supercritical fluid chromatography were optimized and integrated into a working protocol. The workflow was applied to quantify carotenoids in the cuticle and noncuticle component of the tomato pericarp during fruit development from the initial ripening to full ripe stages. Carotenoids were extracted and quantified from cell volumes less than 10nL. This workflow for cell-type-specific metabolite extraction and quantification can be adapted for the analysis of diverse metabolites, cell types, and organisms.

3,4-Methylenedioxymethamphetamine quantification via benchtop 1H qNMR spectroscopy: Method validation and its application to ecstasy tablets collected at music festivals.

We describe a method validation for the quantification of 3,4-methylenedioxymethamphetamine (MDMA) in tablets based on the United Nations Office on Drugs and Crime (UNODC) guideline for quantitative Nuclear Magnetic Resonance analysis (qNMR). qNMR experiments were carried out on a 60 MHz benchtop NMR spectrometer employing ethylene carbonate as an internal calibrant. A series of 'ecstasy' tablets seized at music events were quantified and the results discussed regarding their within-batch variation and yearly median dose. The method showed good specificity and selectivity, with linearity, precision, accuracy, and recovery well within the UNODC recommended criteria. The limit of detection and quantification are 0.33 mg/mL and 0.10 mg/mL respectively, proving the method works well on small amounts of MDMA. Overall, the lowest amount of MDMA free base detected in this study was 9.35 mg in a piperazine mix, while the highest dosed tablet contained 237.55 mg MDMA free base, with a 9.1% decrease in median amount compared to the pre-pandemic data (2019), but still higher than the data collected in a previous study (105 mg median amount of MDMA free base in 2018). The within-batch variation was insignificant for one of the seizures but showed greater variation for the other, which confirmed that the MDMA content of a single tablet may not reflect that of the whole batch. This dynamic upward change in tablet dosage highlights the importance of ongoing trend monitoring and specific prevention intervention to counteract the negative consequences associated with MDMA use. Benchtop NMR has been successfully employed in quality control, material science and more recently, drug analysis. The present study demonstrates its beneficial application in forensic science overcoming the limitations of currently available instruments and techniques employed in harm reduction and field testing.

Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis.

A microfluidic device capable of rapidly analyzing cells in a high-throughput manner using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an electron multiplying charge coupled device camera with high frame rate (>100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities.

Variability in the 3,4-methylenedioxymethamphetamine content of 'ecstasy' tablets in the UK.

BACKGROUND: Toxicity, such as hyperpyrexia, associated with the use of 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') appears to be related to serum MDMA concentrations. However, there does not appear to be a similar association with the number of tablets ingested, suggesting variation in the tablet content of MDMA. Although work has shown this variation in other areas of the world, no studies have reported on the variation of MDMA content in UK ecstasy tablets. METHODS: Ecstasy tablets seized from individuals attending nightclubs were analysed qualitatively to determine if they contained MDMA and quantitatively to determine the MDMA content per tablet. RESULTS: The mean amount of MDMA hydrochloride in 101 seized ecstasy tablets was 58.7±22.9 mg per tablet, with a range of 20 mg to 131 mg per tablet. The majority (96.0%) of tablets contained less than 100 mg MDMA per tablet. There appeared to be a bimodal distribution of MDMA content at approximately 20-40 mg per tablet and 60-80 mg per tablet. CONCLUSION: There is variability in the MDMA content of ecstasy tablets in the UK. This variability could potentially put users at increased risk of acute harm due to inadvertent excess ingestion of MDMA, as they are unaware of the differences in the MDMA content. Repeat sampling and quantification of MDMA content of ecstasy tablets in the UK will allow better education of users about the potential harms associated with the variability in the MDMA content. In addition, it will provide information to allow the monitoring of changes in not only the MDMA content, but also other adulterants, in ecstasy tablets.