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Rapid analysis of surrendered or seized drug samples provides important intelligence for health (e.g. treatment or harm reduction), and custodial services. Herein, three in-situ techniques, GC-MS, 1H NMR and FT-IR spectroscopy, with searchable libraries, are used to analyse 318 samples qualitatively, using technique specific library-based searches, obtained over the period 24th – 29th August 2019. 259 samples were identified as consisting of a single component, of which cocaine was the most prevalent (n = 158). Median match scores for all three techniques were ≥ 0.84 and showed agreement except for metformin (n = 1), oxandrolone (identified as vitamin K by IR (n = 4)), diazepam (identified as zolpidem by FT-IR (n = 2)) and 2-Br-4,5-DMPEA (n = 1), a structural isomer of 2C-B identified as a polymer of cellulose (cardboard) by FT-IR. 51 samples were found to consist of two or more components, of which 49 were adulterated cocaine samples (45 binary and 4 tertiary samples). GC-MS identified all components present in the 49 adulterated cocaine samples, whereas IR identified only cocaine in 88 % of cases (adulterant only = 12 %). The breakdown for 1H NMR spectroscopy was all components identified (51 %), cocaine only (33 %), adulterant only (10 %), cocaine and one adulterant (tertiary mixtures only, 6 %).

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (t G = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-[pent-4-en-1-yl]-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50, 11.5 nM and ADB-4en-PINACA (EC50, 11.6 nM) are similar to that of MDMB-4en-PINACA (EC50, 4.3 nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation. Funding Agencies|Linkopings Universitet; Dubai Police Force [2427681]; Leverhulme TrustLeverhulme Trust [RC-2015-01]; Scottish Prison Service [01865]; Linkoping University; VINNOVAVinnova [2019-03566]; European CommissionEuropean CommissionEuropean Commission Joint Research Centre [E! 113377]

[Image: see text] An automated approach to the collection of (1)H NMR (nuclear magnetic resonance) spectra using a benchtop NMR spectrometer and the subsequent analysis, processing, and elucidation of components present in seized drug samples are reported. An algorithm is developed to compare spectral data to a reference library of over 300 (1)H NMR spectra, ranking matches by a correlation-based score. A threshold for identification was set at 0.838, below which identification of the component present was deemed unreliable. Using this system, 432 samples were surveyed and validated against contemporaneously acquired GC–MS (gas chromatography–mass spectrometry) data. Following removal of samples which possessed no peaks in the GC–MS trace or in both the (1)H NMR spectrum and GC–MS trace, the remaining 416 samples matched in 93% of cases. Thirteen of these samples were binary mixtures. A partial match (one component not identified) was obtained for 6% of samples surveyed whilst only 1% of samples did not match at all.

Fluorinated fentanyl regioisomers have become increasingly common in forensic casework. This study reports the synthesis of eight fluorinated fentanyl derivatives as pure reference materials and their complete NMR, infrared and mass spectral characterisation. Presumptive colour tests and TLC results are presented as an initial screening method for these compounds. The fully validated GC–MS method (employing SIM mode) allows the quantification of the target analytes (LOD = 9 – 20 ng/mL, LOQ = 31 – 67 ng/mL), fentanyl, heroin, acetaminophen and caffeine, within 13 minutes. In most cases, the regioisomeric fluorofentanyls were resolved from each other except for the 3’- and 4’-fluorinated derivatives (5b and 5c). To achieve full separation, an orthogonal benchtop 19F NMR method allows the identification and quantification of target analogues (LOD = 74 – 400 μg/mL, LOQ = 290 – 1340 μg/mL). The 19F NMR method allows the detection of fluorinated fentanyl analogues at a low concentration (2.4% w/w) in heroin.

Recreational MDMA use is a worldwide problem. Tablet dosage varies, thus entailing a requirement for quantitative analysis. The quantification of MDMA in tablets using benchtop 1H NMR spectroscopy via either linear regression (‘manual’ method) or partial least square regression (‘automated’ method) approaches are reported, without the need for an internal standard, and compared against contemporaneously obtained GC–MS data. Twenty samples were evaluated of which 15 were proven to contain MDMA, via qualitative NMR (hit score ≥ 0.97) and GC–MS (Rt = 5.6 min) analysis. Quantitative NMR analysis showed that the mean value of MDMA content was 42.6% w/w by the manual method and 45.9% w/w by the automated method. The mean value obtained from GC analysis was 44.0% w/w. A substantial proportion (n = 9) of the tablets tested possessed > 190 mg of MDMA (range 133–223 mg, average of all techniques’ calculations for each tablet). This value is higher than the reported average MDMA content of tablets by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), which was ca. 125 mg of MDMA per tablet in 2016.

The development of RNA sequencing (RNAseq) and corresponding emergence of public datasets have created new avenues of transcriptional marker search. The long non-coding RNAs (lncRNAs) constitute an emerging class of transcripts with a potential for high tissue specificity and function. Using a dedicated bioinformatics pipeline, we propose to construct a cell-specific catalogue of unannotated lncRNAs and to identify the strongest cell markers. This pipeline uses ab initio transcript identification, pseudoalignment and new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data.For an application model, we focused on Mesenchymal Stem Cells (MSCs), a type of adult multipotent stem-cells of diverse tissue origins. Frequently used in clinics, these cells lack extensive characterisation. Our pipeline was able to highlight different lncRNAs with high specificity for MSCs. In silico methodologies for functional prediction demonstrated that each candidate represents one specific state of MSCs biology. Together, these results suggest an approach that can be employed to harness lncRNA as cell marker, showing different candidates as potential actors in MSCs biology, while suggesting promising directions for future experimental investigations.

International audience; L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements.

The Plasmodium falciparum var gene family encodes ∼60 surface antigens by which parasites escape the host immune responses via clonal expression of var genes. However, the mechanism controlling this mutual exclusivity, associated with alterations in chromatin assembly, is not understood. Here, we determined how expression of the var gene family is regulated by two RecQ DNA helicase family members, PfRecQ1 and PfWRN, in P. falciparum . Through genetic manipulation, we found that the complete var repertoire was silenced on PfRecQ1 knockout, whereas their expression did not show noticeable changes when PfWRN was knocked out. More important, mutually exclusive expression of var genes could be rescued by complementation of PfRecQ1. In addition, knocking out either of these two helicase genes changed the perinuclear cluster distribution of subtelomeres and subtelomeric var genes. Whereas deletion of PfRecQ1 increased the heterochromatin mark trimethylated (H3K9me3) at the transcription start site (TSS) of the var gene upsC1 , that deletion had no effect on the global distribution of H3K9me3 over gene bodies, including those for the var genes. ChIP-seq assay showed that PfRecQ1 was enriched globally at the TSSs of all genes, whereas PfWRN-enriched regions occurred at the gene bodies of the var gene family, but not of other genes or at TSSs of all genes. On PfRecQ1 deletion, the upsC1 var gene moved from the active perinuclear transcription region to a silenced region of the upsC type. These findings imply that PfRecQ1, but not PfWRN, is essential for maintaining the clonal expression of var genes.

Background: High-throughput next generation sequencing (NGS) technologies enable the detection of biomarkers used for tumor classification, disease monitoring and cancer therapy. Whole-transcriptome analysis using RNA-seq is important, not only as a means of understanding the mechanisms responsible for complex diseases but also to efficiently identify novel genes/exons, splice isoforms, RNA editing, allele-specific mutations, differential gene expression and fusion-transcripts or chimeric RNA (chRNA). Methods: We used Crac, a tool that uses genomic locations and local coverage to classify biological events and directly infer splice and chimeric junctions within a single read. Crac’s algorithm extracts transcriptional chimeric events irrespective of annotation with a high sensitivity, and CracTools was used to aggregate, annotate and filter the chRNA reads. The selected chRNA candidates were validated by real time PCR and sequencing. In order to check the tumor specific expression of chRNA, we analyzed a publicly available dataset using a new tag search approach. Results: We present data related to acute myeloid leukemia (AML) RNA-seq analysis. We highlight novel biological cases of chRNA, in addition to previously well characterized leukemia chRNA. We have identified and validated 17 chRNAs among 3 AML patients: 10 from an AML patient with a translocation between chromosomes 15 and 17 (AML-t(15;17), 4 from patient with normal karyotype (AML-NK) 3 from a patient with chromosomal 16 inversion (AML-inv16). The new fusion transcripts can be classified into four groups according to the exon organization. Conclusions: All groups suggest complex but distinct synthesis mechanisms involving either collinear exons of different genes, non-collinear exons, or exons of different chromosomes. Finally, we check tumor-specific expression in a larger RNA-seq AML cohort and identify new AML biomarkers that could improve diagnosis and prognosis of AML.

International audience; AbstractThe third international conference on Transposable Elements (ICTE) was held 16–19 April 2016 in Saint Malo, France. Organized by the French Transposition Community (Research group of the CNRS: “Mobile genetic elements: from mechanism to populations, an integrative approach”) and the French Society of Genetics, the conference’s goal was to bring together researchers who study transposition in diverse organisms, using multiple experimental approaches. The meeting gathered 180 participants from all around the world. Most of them contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into six scientific sessions: “Taming mobile DNA: self and non-self recognition”; “Trans-generational inheritance”; “Mobile DNA genome structure and organization, from molecular mechanisms to applications”; “Remembrance of (retro)transposon past: mobile DNA in genome evolution”; and finally “The yin and the yang of mobile DNA in human health”.

International audience; The human LINE-1 retrotransposon has the ability to mobilize into a new genomic location through an intracellular replication cycle. Immunofluorescence and in situ hybridization experiments have been developed to detect subcellular localization of retrotransposition intermediates (i.e., ORF1p, ORF2p, and L1 mRNA). Currently, these protocols are also used to validate the interaction between retrotransposition complex components and potential cellular partners involved in L1 replication. Here, we describe in details methods for the identification of LINE-1 proteins and/or RNA in cells transfected with vectors expressing engineered human LINE-1 elements.

Supplementary Material : Supplementary tables S1–S3andfigure S1areavailableatMolecular Biology and Evolutiononline (http://www.mbe.oxfordjournals.org/).; International audience; Transposable elements comprise more than 45% of the human genome and long interspersed nuclear element 1 (LINE-1 or L1) is the only autonomous mobile element remaining active. Since its identification, it has been proposed that L1 contributes to the mobilization and amplification of other cellular RNAs and more recently, experimental demonstrations of this function has been described for many transcripts such as Alu, a nonautonomous mobile element, cellular mRNAs, or small noncoding RNAs. Detailed examination of the mobilization of various cellular RNAs revealed distinct pathways by which they could be recruited during retrotransposition; template choice or template switching. Here, by analyzing genomic structures and retrotransposition signatures associated with small nuclear RNA (snRNA) sequences, we identified distinct recruiting steps during the L1 retrotransposition cycle for the formation of snRNA-processed pseudogenes. Interestingly, some of the identified recruiting steps take place in the nucleus. Moreover, after comparison to other vertebrate genomes, we established that snRNA amplification by template switching is common to many LINE families from several LINE clades. Finally, we suggest that U6 snRNA copies can serve as markers of L1 retrotransposition dynamics in mammalian genomes.

Background Plant leucine-rich repeat receptor-like kinases (LRR-RLKs) are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs. Results We analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle. Conclusions In view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens.

Long Interspersed Element-1 (LINE-1 or L1) sequences comprise ∼17% of human DNA and ongoing L1 retrotransposition continues to impact genome evolution. The L1-encoded proteins also can mobilize other cellular RNAs (e.g., Alu retrotransposons, SVA retrotransposons, and U6 snRNAs), which comprise ∼13% of human DNA. Here, we demonstrate that the trans-mediated mobilization of non-L1 RNAs can occur by either template choice or template-switching mechanisms. Remarkably, these mechanisms are not mutually exclusive, as both processes can operate sequentially on the same RNA template. Finally, we provide evidence that efficient U6 snRNA retrotransposition requires both ORF1p and ORF2p, providing indirect evidence for the action of ORF1p in U6 snRNA retrotransposition. Thus, we propose that the LINE-1-encoded reverse transcriptase can mediate the retrotransposition of non-L1 RNAs by distinct mechanisms.

The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comprise 28% of the human genome. They share the same L1-encoded endonuclease for insertion, which recognizes an A+T-rich sequence. Under a simple model of insertion distribution, this nucleotide preference would lead to the prediction that the populations of both elements would be biased towards A+T-rich regions. Genomic L1 elements do show an A+T-rich bias. In contrast, Alu is biased towards G+C-rich regions when compared to the genome average. Several analyses have demonstrated that relatively recent insertions of both elements show less G+C content bias relative to older elements. We have analyzed the repetitive element and G+C composition of more than 100 pre-insertion loci derived from de novo L1 insertions in cultured human cancer cells, which should represent an evolutionarily unbiased set of insertions. An A+T-rich bias is observed in the 50 bp flanking the endonuclease target site, consistent with the known target site for the L1 endonuclease. The L1, Alu, and G+C content of 20 kb of the de novo pre-insertion loci shows a different set of biases than that observed for fixed L1s in the human genome. In contrast to the insertion sites of genomic L1s, the de novo L1 pre-insertion loci are relatively L1-poor, Alu-rich and G+C neutral. Finally, a statistically significant cluster of de novo L1 insertions was localized in the vicinity of the c-myc gene. These results suggest that the initial insertion preference of L1, while A+T-rich in the initial vicinity of the break site, can be influenced by the broader content of the flanking genomic region and have implications for understanding the dynamics of L1 and Alu distributions in the human genome.

Mobile elements such as transposons and retrotransposons constitute a substantial fraction of eukaryotic genomes. Such elements represent ~45% of the human genome. Long INterspersed Element-1 (LINE-1 or L1) constitutes about 17% of human DNA and is the only known autonomously active human retrotransposon. L1s are bi-cistronic elements that encode two proteins, ORF1p and ORF2p, which are essential for retrotransposition. These proteins associate with the RNA from which they were translated to form an L1 ribonucleoprotein particle (L1-RNP), which is a hypothesized retrotransposition intermediate. The insertion of a new L1 copy occurs by a mechanism known as target-site primed reverse transcription (TPRT), which is performed by the endonuclease and reverse-transcriptase activities of ORF2p and possibly other cellular factors. About 80 to 100 L1s per human genome retain the ability to retrotranspose by this mechanism, and therefore the ongoing mobility of L1 elements contin! ues to impact the evolution of our genome. Here, we developed new molecular tools to detect retrotransposition-competent L1 RNA and proteins through an epitope-tagging strategy. We used a biochemical approach to identify the 'basal' retrotransposition complex containing L1 RNA, epitope-tagged ORF1p and epitope-tagged ORF2p. We also tested retrotransposition-deficient L1s for their ability to form RNPs and to encode an ORF2p-specific reverse transcriptase activity. Using immunocytochemistry coupled with FISH, we observed a cytoplasmic accumulation of L1 RNPs in perinuclear foci in association with stress granules (SG). Interestingly, we observed that the SG protein markers consistently surrounded the L1 RNP foci. Point mutations in the RNA binding domain of ORF1p decreased the ability of the L1 proteins to form cytoplasmic aggregates, and confirmed the role of this domain in L1 RNP formation. Thus, these results further our understanding of the L1 RNP complex and the general mechanism of L1 retrotransposition.