Visualising substrate-fingermark interactions: Solid-state NMR spectroscopy of amino acid reagent development on cellulose substrates

© 2015 Elsevier Ireland Ltd. Most spectroscopic studies of the reaction products formed by ninhydrin, 1,2-indanedione-zinc (Ind-Zn) and 1,8-diazafluoren-9-one (DFO) when reacted with amino acids or latent fingermarks on paper substrates are focused on visible absorption or luminescence spectroscopy. In addition, structural elucidation studies are typically limited to solution-based mass spectrometry or liquid nuclear magnetic resonance (NMR) spectroscopy, which does not provide an accurate representation of the fingermark development process on common paper substrates. The research presented in this article demonstrates that solid-state carbon-13 magic angle spinning NMR (13C-MAS-NMR) is a technique that can not only be utilised for structural studies of fingermark enhancement reagents, but is a promising technique for characterising the effect of paper chemistry on fingermark deposition and enhancement. The latter opens up a research area that has been under-explored to date but has the potential to improve our understanding of how fingermark secretions and enhancement reagents interact with paper substrates

On the reactivity of zinc hydroxide acetate dihydrate in ethanol

Zinc hydroxide acetate dihydrate, Zn5(OH)8(CH 3CO2)2·2H2O, reacts in ethanol at room temperature to yield a mixture of zinc oxide and anhydrous zinc acetate. The process is driven by dehydration of the starting salt. Dehydration of Zn5(OH)8(CH3CO2) 2·2H2O also occurs when it is heated in air, but the product obtained in that case depends on the heating rate, environment and temperature. For example, when it is kept in a sealed silica capillary at 100 °C, Zn5(OH)8(CH3CO2) 2·1.5H2O is formed after 15 min, whereas treatment in the range 90-100°C in an open environment results in the formation of anhydrous zinc hydroxide acetate. Heating of any of these products further causes their decomposition to Zn(CH3CO2)2 and ZnO. The coordination bonding mode of the acetate groups in the anhydrous layered zinc hydroxide acetate prepared by reaction with ethanol was studied by using solid-state NMR spectroscopy. The presence of chelating, unidentate and bidentate bridging modes for the carbonyl carbon atom was revealed, but there was no evidence for the inclusion of ethanol in the resultant structure. Therefore, the reaction in ethanol offers a convenient strategy to prepare anhydrous zinc hydroxide acetate and/or zinc oxide, because it avoids the sensitivity of the thermally induced dehydroxlation process to time, temperature and environment. Decomposition of zinc hydroxide acetate dihydrate in ethanol can be exploited to generate ZnO nanoparticles at room temperature. The mechanism involves the removal of waters of hydration and the formation of Zn5(OH)8(CH3CO2)2 followed by the generation of Zn(CH3CO)2 and ZnO. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Towards Compound Identification of Synthetic Opioids in Non-targeted Screening Using Machine Learning Techniques.

The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a Naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH series or U series, with an accuracy of 89.5%. The model was most accurate for the fentanyl analogues, most likely due to their greater number in the training data. This classification model can provide guidance to an analyst when determining a suspected structure. A retention time prediction model was also trained for a wide array of synthetic opioids. This model utilised Gaussian Process Regression to predict the retention time of analytes based on multiple generated molecular features with 79.7% of the samples predicted within ± 0.1 min of their experimental retention time. Once the suspected structure of an unknown compound is determined, molecular features can be generated and input for the prediction model to compare with experimental retention time. The incorporation of machine learning prediction models into a compound identification workflow can assist putative identifications with greater confidence and ultimately save time and money in the purchase and/or production of superfluous certified reference materials

Collision-induced dissociation studies of synthetic opioids for non-targeted analysis

© 2019 Klingberg, Cawley, Shimmon and Fu. The continual introduction of a large number of new psychoactive substances, along with the large turnover of these substances, necessitates the development of non-targeted detection strategies to keep pace with the ever-changing drug market. The production of certified reference materials often lags behind the introduction of new substances to the market, therefore these detection strategies need to be able to function without relying on reference materials or library spectra. Synthetic opioids have recently emerged as a drug class of particular concern due to the health issues caused by their incredibly high potency. A common method which has been used for non-targeted analysis in the past involves the identification of common product ions formed as a result of the fragmentation of the parent molecule. These common fragments can then potentially be used as markers to indicate the presence of a particular class of compounds within a sample. In this study, standards of a number of different synthetic opioids, including 14 fentanyl derivatives, 7 AH series opioids, 4U series opioids, 4W series opioids and MT-45, were subjected to collision-induced dissociation studies to determine how the compounds fragment. The spectra obtained from these studies included a number of diagnostic fragments common to the different opioid classes that, when used in combination, show potential for use as class predictors. By using simple data processing techniques, such as extracted ion chromatograms, these diagnostic product ions identified can be applied to a non-targeted screening workflow

Development and validation of a presumptive colour spot test method for the detection of piperazine analogues in seized illicit materials

The increasingly large quantities of potentially illicit samples received for confirmatory analysis highlights the importance and demand for preliminary testing procedures that are simple, rapid, selective, inexpensive and able to be used in the field. Colour testing fulfils the aforementioned requirements and is a technique frequently employed to achieve presumptive identification. Piperazine analogues (often marketed as 'legal ecstasy') are a group of psychoactive substances that have recently become established on the illicit drug market and are not effectively discriminated or identified by current colour testing methods. Herein, we report on the development and validation of a chemical spot test for piperazine analogues present in unknown seized materials using the spectrophotometric reagent, sodium 1,2-naphthoquinone-4-sulphonate (NQS). Primary testing revealed that NQS reacts almost instantly to form an intense, bright orange-red coloured complex with the representative piperazine 1-benzylpiperazine (BZP) at room temperature. The results of the test, assessed by colour development, were evaluated visually and variables affecting the coloured reaction were optimised. The colour test method was validated to meet requirements for use in preliminary screening, providing qualitative and reliable presumptive test results. Validation studies show that the characteristic colour change is unique to the piperazine class at room temperature, and is unaffected by the presence of common cutting agents, i.e. glucose and caffeine, in test samples of 5% purity, and other drugs such as N-methyl-3,4-methylenedioxyamphetamine (MDMA). The NQS reagent stability was found to be limited to storage in a refrigerated environment for no more than one week before results were affected. The operational limit of detection was found to be 40 μg. © 2013 The Royal Society of Chemistry

Deletion of Integron-Associated Gene Cassettes Impact on the Surface Properties of Vibrio rotiferianus DAT722

Background: The integron is a genetic recombination system that catalyses the acquisition of genes on mobilisable elements called gene cassettes. In Vibrio species, multiple acquired gene cassettes form a cassette array that can comprise 1-3% of the bacterial genome. Since 75% of these gene cassettes contain genes encoding proteins of uncharacterised function, how the integron has driven adaptation and evolution in Vibrio species remains largely unknown. A feature of cassette arrays is the presence of large indels. Using Vibrio rotiferianus DAT722 as a model organism, the aim of this study was to determine how large cassette deletions affect vibrio physiology with a view to improving understanding into how cassette arrays influence bacterial host adaptation and evolution. Methodology/Principal Findings: Biological assays and proteomic techniques were utilised to determine how artificially engineered deletions in the cassette array of V. rotiferianus DAT722 affected cell physiology. Multiple phenotypes were identified including changes to growth and expression of outer membrane porins/proteins and metabolic proteins. Furthermore, the deletions altered cell surface polysaccharide with Proton Nuclear Magnetic Resonance on whole cell polysaccharide identifying changes in the carbohydrate ring proton region indicating that gene cassette products may decorate host cell polysaccharide via the addition or removal of functional groups. Conclusions/Significance: From this study, it was concluded that deletion of gene cassettes had a subtle effect on bacterial metabolism but altered host surface polysaccharide. Deletion (and most likely rearrangement and acquisition) of gene cassettes may provide the bacterium with a mechanism to alter its surface properties, thus impacting on phenotypes such as biofilm formation. Biofilm formation was shown to be altered in one of the deletion mutants used in this study. Reworking surface properties may provide an advantage to the bacterium's interactions with organisms such as bacteriophage, protozoan grazers or crustaceans. © 2013 Rapa et al

Finding the proverbial needle: Non-targeted screening of synthetic opioids in equine plasma.

Synthetic opioids are a class of compounds that are of particular concern due to their high potency and potential health impacts. With the relentless emergence of new synthetic opioid derivatives, non-targeted screening strategies are required that do not rely on the use of library spectra or reference materials. In this study, product ion searching, and Kendrick mass defect analysis were investigated for non-targeted screening of synthetic opioids. The estimated screening cut-offs for these techniques ranged between 0.05 and 0.1 ng/mL. These techniques were designed to not be reliant on a particular vendor's software, meaning that they can be applied to existing drug screening protocols, without requiring the development and validation of new analytical procedures. The efficacy of the developed techniques was tested through blind trials, with spiked samples inserted amongst authentic plasma samples, which demonstrated the usefulness of these methods for high-throughput screening. The use of a non-targeted screening workflow that contains complementary techniques can increase the likelihood of detecting compounds of interest within a sample, as well as the confidence in detections that are made

A Comparison of Two Ovine Lumbar Intervertebral Disc Injury Models for the Evaluation and Development of Novel Regenerative Therapies

© The Author(s) 2018. Study Design: Large animal research. Objective: Lumbar discectomy is the most commonly performed spinal surgical procedure. We investigated 2 large animal models of lumbar discectomy in order to study the regenerative capacity of mesenchymal stem cells following disc injury. Methods: Twelve adult ewes underwent baseline 3-T magnetic resonance imaging (MRI) followed by lumbar intervertebral disc injury by either drill bit (n = 6) or annulotomy and partial nucleotomy (APN) (n = 6). Necropsies were performed 6 months later. Lumbar spines underwent 3-T and 9.4-T MRI prior to histological, morphological and biochemical analysis. Results: Drill bit-injured (DBI) and APN-injured discs demonstrated increased Pfirrmann grades relative to uninjured controls (P <.005), with no difference between the 2 models. Disc height index loss was greater in the APN group compared with the DBI group (P <.005). Gross morphology injury scores were higher in APN than DBI discs (P <.05) and both were higher than controls (P <.005). Proteoglycan was reduced in the discs of both injury models relative to controls (P <.005), but lower in the APN group (P <.05). Total collagen of the APN group disc regions was higher than DBI and control discs (P <.05). Histology revealed more matrix degeneration, vascular infiltration, and granulation in the APN model. Conclusion: Although both models produced disc degeneration, the APN model better replicated the pathobiology of human discs postdiscectomy. We therefore concluded that the APN model was a more appropriate model for the investigation of the regenerative capacity of mesenchymal stem cells administered postdiscectomy

Universal detection of foot and mouth disease virus based on the conserved VP0 protein

Background: Foot and mouth disease virus (FMDV), a member of the picornaviridae that causes vesicular disease in ungulates, has seven serotypes and a large number of strains, making universal detection challenging. The mature virion is made up of 4 structural proteins, virus protein (VP) 1 – VP4, VP1-VP3 of which form the outer surface of the particle and VP4 largely contained within. Prior to mature virion formation VP2 and VP4 occur together as VP0, a structural component of the pre-capsid which, as a result of containing the internal VP4 sequence, is relatively conserved among all strains and serotypes. Detection of VP0 might therefore represent a universal virus marker. Methods: FMDV virus protein 0 (VP0) was expressed in bacteria as a SUMO fusion protein and the SUMO carrier removed by site specific proteolysis. Rabbit polyvalent sera were generated to the isolated VP0 protein and their reactivity characterised by a number of immunoassays and by epitope mapping on peptide arrays. Results: The specific VP0 serum recognised a variety of FMDV serotypes, as virus and as virus-like-particles, by a variety of assay formats. Epitope mapping showed the predominant epitopes to occur within the unstructured but highly conserved region of the sequence shared among many serotypes. When immunogold stained VLPs were assessed by TEM analysis they revealed exposure of epitopes on the surface of some particles, consistent with particle breathing hitherto reported for some other picornaviruses but not for FMDV. Conclusion: A polyvalent serum based on the VP0 protein of FMDV represents a broadly reactive reagent capable of detection of many if not all FMDV isolates. The suggestion of particle breathing obtained with this serum suggests a reconsideration of the FMDV entry mechanism

Color spot test as a presumptive tool for the rapid detection of synthetic cathinones

© 2018 Journal of Visualized Experiments. Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing