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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

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated 1 H NMR analysis and compared to gas chromatography-mass spectrometry (GC-MS) data. Two seized drug samples, SS1 and SS2, were identified to contain 4-fluoroamphetamine by 1 H NMR (match score median = 0.9933) and GC-MS (RRt = 5.42-5.43 min). The amount of 4-fluoroamphetamine present was 42.8%-43.4% w/w and 48.7%-49.2% w/w for SS1 and SS2, respectively, from quantitative 19 F NMR analysis, which is in agreement with the amount determined by GC-MS (39.9%-41.4% w/w and 49.0%-49.3% w/w). The total time for the qualitative 1 H NMR and quantitative 19 F NMR analysis is ~10 min. This contrasts to ~40 min for the GC-MS method. The NMR method also benefits from minimal sample preparation. Thus, benchtop NMR affords rapid, and discriminatory, analysis of the drug present in a seized sample.

Fentanyl, also known as ‘jackpot’, is a synthetic opiate that is 50–100 times more potent than morphine. Clandestine laboratories produce analogues of fentanyl, known as fentalogues to circumvent legislation regarding its production. Three pyridyl fentalogues were synthesized and then hyperpolarized by signal amplification by reversible exchange (SABRE) to appraise the forensic potential of the technique. A maximum enhancement of ‐168‐fold at 1.4 T was recorded for the ortho pyridyl 1H nuclei. Studies of the activation parameters for the three fentalogues revealed that the ratio of ligand loss trans to hydride and hydride loss in the complex [Ir(IMes)(L)3(H)2]+ (IMes=1,3‐bis(2,4,6‐trimethylphenyl)imidazole‐2‐ylidene) ranged from 0.52 to 1.83. The fentalogue possessing the ratio closest to unity produced the largest enhancement subsequent to performing SABRE at earth's magnetic field. It was possible to hyperpolarize a pyridyl fentalogue selectively from a matrix that consisted largely of heroin (97 : 3 heroin:fentalogue) to validate the use of SABRE as a forensic tool., No more hiding for pyridyl fentalogues! The hyperpolarization technique SABRE (signal amplification by reversible exchange) is utilised for the successful enhancement of 1H NMR signals of pyridyl functionalised fentalogues. We further demonstrate the successful selective polarization of one the fentalogues against a strong background‐signal of heroin.

Two-pore domain K

Background The development of RNA sequencing (RNAseq) and the 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. Therefore, we tested the biomarker potential of lncRNAs on Mesenchymal Stem Cells (MSCs), a complex type of adult multipotent stem cells of diverse tissue origins, that is frequently used in clinics but which is lacking extensive characterization. Results We developed a dedicated bioinformatics pipeline for the purpose of building a cell-specific catalogue of unannotated lncRNAs. The pipeline performs ab initio transcript identification, pseudoalignment and uses new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data. We next applied it on MSCs, and our pipeline was able to highlight novel lncRNAs with high cell specificity. Furthermore, with original and efficient approaches for functional prediction, we demonstrated that each candidate represents one specific state of MSCs biology. Conclusions We showed that our approach can be employed to harness lncRNAs as cell markers. More specifically, our results suggest different candidates as potential actors in MSCs biology and propose promising directions for future experimental investigations.

The huge body of publicly available RNA-sequencing (RNA-seq) libraries is a treasure of functional information allowing to quantify the expression of known or novel transcripts in tissues. However, transcript quantification commonly relies on alignment methods requiring a lot of computational resources and processing time, which does not scale easily to large datasets. K-mer decomposition constitutes a new way to process RNA-seq data for the identification of transcriptional signatures, as k-mers can be used to quantify accurately gene expression in a less resource-consuming way. We present the Kmerator Suite, a set of three tools designed to extract specific k-mer signatures, quantify these k-mers into RNA-seq datasets and quickly visualize large dataset characteristics. The core tool, Kmerator, produces specific k-mers for 97% of human genes, enabling the measure of gene expression with high accuracy in simulated datasets. KmerExploR, a direct application of Kmerator, uses a set of predictor gene-specific k-mers to infer metadata including library protocol, sample features or contaminations from RNA-seq datasets. KmerExploR results are visualized through a user-friendly interface. Moreover, we demonstrate that the Kmerator Suite can be used for advanced queries targeting known or new biomarkers such as mutations, gene fusions or long non-coding RNAs for human health applications.

Over recent years there has been a progressive increase in the adulteration of common illicit street drugs, such as heroin and cocaine, with fentanyl and its derivatives (fentalogues) being the cause of over doses ending with fatal repercussions. Consequently, there is a need for the development of sensitive, selective and reliable analytical protocols for their separation and quantification. Herein, we report for the first time, a combination of high-performance liquid chromatography with a dual-diode array and electrochemical (amperometric) detector achieved for the simultaneous detection and quantification of heroin (HRN), fentanyl and ten fentalogues; the amperometric detection is achieved using a commercially available impinging jet flow-cell that incorporates in-house screen-printed graphite macroelectrodes (SPEs). Both protocols are analytically compared and contrasted in terms of their experimental parameters and chromatographic conditions with the separation and quantification being optimized, with these protocols demonstrating a high sensitivity and reproducibility. The proposed methods were successfully applied for the analysis of the investigated drugs of abuse, in the presence of common adulterants (e.g. caffeine, paracetamol and benzocaine), co-formulated excipients (starch, lactose, aerosil 200, etc.) and simultaneously within seized street samples.

Supercritical Fluid Chromatography (SFC-UV) employing a carbon dioxide (CO 2) and 10 mM ammonium acetate in MeOH-water (95:5 v/v) gradient provides a rapid analysis (t G

Mirfentanil, a fentanyl derivative that is a μ-opioid partial agonist, is hyperpolarised via Signal Amplification By Reversible Exchange (SABRE), a para-hydrogen-based technique. [Ir(IMes)(COD)Cl] (IMes=1,3-bis(2,4,6-trimethylphenyl)imidazole-2-ylidene, COD=cyclooctadiene) was employed as the polarisation transfer catalyst. Following polarisation transfer at 6.5 mT, the pyrazine-protons were enhanced by 78-fold (polarisation, P=0.04 %). The complex [Ir(IMes)(H) (mirfentanil) (MeOH)] is proposed to form based on the observation of two hydrides at δ −22.9 (trans to mirfentanil) and −24.7 (trans to methanol). In a mixture of mirfentanil and heroin, the former could be detected using SABRE at concentrations less than 1 % w/w. At the lowest concentration analyzed, the amount of mirfentanil present was 0.18 mg (812 μM) and produced a signal enhancement of −867-fold (P=0.42 %). following polarisation transfer at 6.5 mT. 2 2 +

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated

The huge body of publicly available RNA-sequencing (RNA-seq) libraries is a treasure of functional information allowing to quantify the expression of known or novel transcripts in tissues. However, transcript quantification commonly relies on alignment methods requiring a lot of computational resources and processing time, which does not scale easily to large datasets. K-mer decomposition constitutes a new way to process RNA-seq data for the identification of transcriptional signatures, as k-mers can be used to quantify accurately gene expression in a less resource-consuming way. We present the Kmerator Suite, a set of three tools designed to extract specific k-mer signatures, quantify these k-mers into RNA-seq datasets and quickly visualize large dataset characteristics. The core tool, Kmerator, produces specific k-mers for 97% of human genes, enabling the measure of gene expression with high accuracy in simulated datasets. KmerExploR, a direct application of Kmerator, uses a set of predictor gene-specific k-mers to infer metadata including library protocol, sample features or contaminations from RNA-seq datasets. KmerExploR results are visualized through a user-friendly interface. Moreover, we demonstrate that the Kmerator Suite can be used for advanced queries targeting known or new biomarkers such as mutations, gene fusions or long non-coding RNAs for human health applications.

1,2-Dihaloalkenes readily undergo simultaneous or sequential difunctionalization through transition-metal-catalyzed reactions, which makes them attractive building blocks for complex unsaturated motifs. This review summarizes recent applications of such transformations in C–C and C–heteroatom bond forming processes. The facile synthesis of stereodefined alkene derivatives, as well as aromatic and heteroatomic­ compounds, from 1,2-dihaloalkenes is thus outlined.1 Introduction2 Synthesis of 1,2-Dihaloalkenes3 C–C Bond Forming Reactions4 C–Heteroatom Bond Forming Reactions5 Conclusion

Synthetic cannabinoid receptor agonists (SCRAs) are a continually evolving family of illicit drugs, with novel analogues frequently being detected. This paper reports the detection of a new N-hexyl-1H-indazole derivative, ADB-HINACA, within a herbal sample seized by law enforcement for the first time in the United Kingdom. The identity of the compound was confirmed via the synthesis of a pure ADB-HINACA reference standard and direct spectral comparison by 1H NMR and GC-EI-MS analysis. A full analytical profiling of ADB-HINACA by nuclear magnetic resonance (NMR), attenuated total reflection Fourier-transform infrared (FTIR) spectroscopy and gas chromatography-electron ionisation-mass spectrometry (GC-EI-MS) is reported and shows good concordance between the seized sample and the reference standard. A validated GC-EI-MS method for the routine quantification of the cannabinoid in seized samples (LOD: 1.7 μg/mL, LOQ: 5.8 μg/mL) was also developed and using this method, the seized herbal sample was determined to contain 4.9% w/w ADB-HINACA.

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.

The emergence of a wide variety of fentanyl analogues has become a problem for the identification of seized drug samples. While chemical databases are largely reactive to the emergence of new analogues, efforts should focus on the development of predictive models which can discern how new analogues differ from the parent drug. Principal component analysis (PCA) was performed on mass spectral data from 54 fentanyl analogues. Hierarchical clustering was used to group these analogues into meaningful classes. The model was able to classify 67 analogues not previously included in the model with high accuracy, based on the nature and position of the chemical modification.

The ability of cyanoacrylates (CA) to develop fingerprints was first discovered in 1977. Since then, the cyanoacrylate fuming method has reached widespread use, becoming one of the primary techniqu...

Trace emerging contaminants (ECs) occur in both waste and surface waters that are rich in particulates that have been found to sorb several organic contaminants. An analytical method based on off-line solid-phase extraction (SPE) followed by liquid chromatography-mass spectrometry (LC-MS) analysis was developed for the detection and quantification of 31 ECs from surface water, wastewater, suspended particulate matter (SPM) as well as sediments. Lyophilized sediments and air-dried SPM were subjected to ultrasonic extraction. Water samples and extracts were then concentrated and cleaned-up by off-line SPE. Quantification was realized using a Q Exactive mass spectrometer in both full scan (FS) and MS2 modes. These two modes were optimized and compared to determine which one was the most suitable for each matrix studied. Yeast estrogen screen assay (YES-assay) adapted from the direct measurement of estrogenic activity without sample extraction was tested on filtered wastewater samples. An endocrine disrupting effect was detected in all effluent samples analyzed with estradiol equivalent concentrations ranging from 4.4 to 720 ng eq E2 L−1 for the WWTP-1 and 6.5–42 ng eq E2 L−1 for the WWTP-2. The analytical methods were also applied on six samples of surface water, the corresponding SPM, the sediments and thirty-nine effluent samples from two wastewater treatment plants (WWTPs) sampled over a period of five months (February to June 2014).

Though microbial transformations are the primary mechanism of contaminant attenuation in wetlands, much remains to be known about microbial communities in urban wetlands. In this study, the microbial communities from urban wetlands with different runoff regimes (i.e., a contaminated remnant wetland, a constructed wetland, and a remnant wetland) were assessed for their capacity to attenuate and tolerate typical urban runoff pollutants. Results from denaturing gradient gel electrophoresis of 16S rRNA genes showed relatively high similarity in community composition among the wetlands. Community-level physiological profiles had similar results but exhibited within-site variation in both the contaminated remnant and remnant wetlands. All wetland communities were less tolerant to copper than 2,4-dichlorophenoxyacetic acid; however, the contaminated remnant wetland had the highest tolerance. All study wetlands had a limited capacity to biodegrade model chlorinated aromatic compounds (e.g., 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate). Though having different input regimes and contaminant exposure histories, the study wetlands were generally similar with respect to microbial community diversity and function. Additionally, the generally low capacity for these wetlands to biodegrade mobile chlorinated organic contaminants offers preliminary insight into the limited ecosystem services these wetlands may provide in urban environments.

Cet article présente une vue d’ensemble des outils et des méthodes de travail du compositeur José Evangelista. Après avoir discuté de la relation qu’entretient le compositeur avec le piano et l’ordinateur en tant qu’outils compositionnels, on examine le rôle des notes textuelles et des esquisses dans ses habitudes de travail. À l’aide d’exemples tirés des esquisses d’Alap et Gat (1998) et de Viola Song (2002), on découvre une méthode de travail où notes textuelles et esquisses musicales s’enchevêtrent et se fertilisent mutuellement. On constate également que le travail assidu du compositeur sur les procédés hétérophoniques a permis à sa méthode de travail de se développer de façon continue, sans grandes ruptures, au cours des 25 dernières années. On conclut de cet examen que, chez Evangelista, méthode de travail et esthétique constituent un tout organique à l’intérieur duquel la subjectivité du compositeur s’exprime sans entraves., This article presents an overview of the tools and working methods of composer José Evangelista. After discussing his relationship with the piano and the computer as compositional tools, we examine the role of textual notes and drafts in his working habits. On the basis of examples drawn from the drafts of Alap et Gat (1998) and Viola Song (2002), we discover a working method in which written text and musical drafts cohabitate and fertilize each other. We also note that the composer’s assiduous work on heterophonic processes allowed for a continuous evolution of his working method over the last 25 years. We conclude this examination by stating that in Evangelista’s work, working method and aesthetics constitute an organic whole within which the composer’s subjectivity expresses itself freely.

This chapter emphasises on the studies that have focused on understanding the mechanism of L1 retrotransposition, which were conducted since the publication of Mobile DNA in 1989. In addition, when appropriate the similarities and differences between the retrotransposition mechanisms of long interspersed nuclear elements (LINE-1s or L1s), closely related L1-like elements, and more distantly related non-LTR retrotransposons, are discussed. The majority of Cin4 elements are variably 5’ truncated, rearranged, or mutated. The basic structural features of these nonautonomous retrotransposons are introduced in the chapter. The cultured cell assay also has yielded unexpected information about L1 retrotransposition. First, in cultured cells, 5 to 10% of new L1 retrotransposition events occurs into the introns of actively transcribed genes. Second, because L1s can be considered processed pseudogenes, the L1 pA signal lacks conserved elements that normally reside downstream of the poly(A) addition site in canonical RNA polymerase II pA signals. Finally, because most L1s are 5’ truncated, it is possible that many transduction events are not detected because they completely lack L1 sequences. However, biochemical data argue that ORF1 binds particular A-rich sequences in L1 RNA with relatively high affinity and that ORF1p is more abundant than ORF2p. We just are beginning to realize the consequences of L1 retrotransposition on the human genome. Clearly, L1 is a mutagen. Moreover, because of the abundance of L1s, it is likely that L1s provide scaffolds for illegitimate recombination, which may contribute to the genome instability seen in many tumors.

B1 elements are an abundant class of short interspersed elements (SINEs) in the mouse genome and mobilize by a process known as retrotransposition. Here, we report the characterization of a mutagenic B1 insertion into exon 4 of the Atcay gene, which was previously shown to be responsible for the jittery mouse. Mutations in the human ortholog of this gene, ATCAY, are responsible for Cayman ataxia. The B1 insertion is B150-bp long, ends in a 45–50-bp polyadenylic acid (poly A) tail, is flanked by a perfect 13-bp target-site duplication, and is inserted into a sequence that resembles a LINE-1 endonuclease consensus cleavage site. Computational analysis indicates that the mutagenic insertion is most closely related to elements of the B1-C subfamily, and we have identified two possible progenitor B1 sequences on mouse chromosome 19. Together, these data demonstrate that B1 retrotransposition is ongoing in the mouse genome and is consistent with the hypothesis that the reverse transcriptase and endonuclease encoded by LINE-1 elements mediate B1 mobility. Hum Mutat 24:9–13, 2004. r 2004 Wiley-Liss, Inc.

Le retrotransposon LINE-1 (Ll) represente environ 17 % du genome humain. Du fait de son grand nombre de copies, il est implique dans des remaniements genomiques associes a des evenements de recombinaison homologue entre sites heterologues. De plus, meme si la vaste majorite des elements Ll sont inactifs, certains sont encore capables de se mobiliser par retrotransposition. Ll est donc un agent mutagene par insertion. De plus, des travaux ont aussi montre que les retrotransposons actifs etaient impliques dans la mobilisation d'autres sequences pour produire des retro-pseudogenes ou amplifier d’autres sequences repetees. Finalement, des etudes recentes ont montre que l’element Ll pourrait etre associe a de nouveaux rearrangements genomiques produits lors de l'insertion, tels que des deletions genomiques de grande taille. En conclusion, Ll peut etre considere comme un facteur important qui a affecte et modele le genome humain par l'intermediaire de plusieurs mecanismes.

Long interspersed elements (LINE-1s) are abundant retrotransposons in mammalian genomes that probably retrotranspose by target site-primed reverse transcription (TPRT). During TPRT, the LINE-1 endonuclease cleaves genomic DNA, freeing a 3' hydroxyl that serves as a primer for reverse transcription of LINE-1 RNA by LINE-1 reverse transcriptase. The nascent LINE-1 cDNA joins to genomic DNA, generating LINE-1 structural hallmarks such as frequent 5' truncations, a 3' poly(A)+ tail and variable-length target site duplications (TSDs). Here we describe a pathway for LINE-1 retrotransposition in Chinese hamster ovary (CHO) cells that acts independently of endonuclease but is dependent upon reverse transcriptase. We show that endonuclease-independent LINE-1 retrotransposition occurs at near-wildtype levels in two mutant cell lines that are deficient in nonhomologous end-joining (NHEJ). Analysis of the pre- and post-integration sites revealed that endonuclease-independent retrotransposition results in unusual structures because the LINE-1s integrate at atypical target sequences, are truncated predominantly at their 3' ends and lack TSDs. Moreover, two of nine endonuclease-independent retrotranspositions contained cDNA fragments at their 3' ends that are probably derived from the reverse transcription of endogenous mRNA. Thus, our results suggest that LINE-1s can integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

S1Bn is a plant short interspersed element (SINE) whose amplification probably involves the reverse transcription of an RNA intermediate. In this report, we identified and characterized S1Bn transcripts from different Brassica napus tissues. Despite the presence of a consensus internal POL III promoter in a large number of genomic S1Bn elements, we observed that S1Bn transcripts are rare in B. napus cells. The use of two very sensitive methods (RT-PCR and RACE PCR) allowed the characterization of 102 independent S1Bn cDNA clones from three different tissues (shoot, root and callus). From this analysis, we conclude that the majority of S1Bn transcripts probably result from a small number of cotranscriptional events where an S1Bn element is transcribed due to its presence in a POL II transcriptional unit. Specific POL III RNA transcripts, initiating at the first 5' nucleotide of the DNA element, are also present in the tested tissues and possibly result from the transcriptional activity of as few as three genomic elements. Two of these transcripts could represent master transcripts responsible for the amplification of S1Bn subfamilies. We also observed that the population of specific POL III transcripts varies among the three tested tissues and that some transcripts appear completely tissue-specific.

LINE-1 (L1) is the most represented sequence of the human genome (17% of the total genomic mass). Moreover, it has been proposed for many years and demonstrated more recently that L1 has contributed to the mobilization of pseudogenes, small non-coding RNAs, such as tRNAs or snRNAs, and SINEs. In fact, it is estimated that L1 is responsible for at least 30% of our genome. The mobilization of non-L1 RNAs can occur in different ways and at different steps of the retrotransposition cycle. Here, by looking at U6 snRNA sequences mobilized by L1, we have observed an ancient repeat sequence derived from U6, present in all primate genomes. We were able to trace its origin in Euarchota genomes, most likely during the divergence of the four orders; Scandentia, Dermoptera, Plesiadapiform (extinct) and Primates.

Comment la Bretagne devait-elle commémorer le centenaire de la loi de la Séparation des Églises et de l'État ? En réunissant pour un colloque commun, sous l'égide du Comité Renan de Tréguier et en collaboration avec l'Institut culturel de Bretagne, les principaux chercheurs des universités de Rennes, Brest et Nantes. En imposant d'elle-même une spécificité où la langue bretonne, la question scolaire, la force de la religion et des traditions jouent un rôle essentiel. En creusant cette spécificité par l'inscription de l'événement dans la longue durée d'un avant et d'un après. En éclairant les travaux des historiens à la lumière de la littérature et des arts. En choisissant pour centre de la manifestation Tréguier, le berceau de Renan : Tréguier où l'inauguration tumultueuse de son monument sous la présidence d'Émile Combes fait date. Et la puissante pensée même de Renan amenait naturellement à l'internationalisation du débat. C'est pourquoi ce colloque fut à son tour un événement. C'est pourquoi aussi ces actes sont à considérer comme lieu de mémoire dont l'actualité fait toujours un lieu de leçon.

Long interspersed element 1 (LINE-1 or L1) is an abundant retrotransposon that comprises ∼17% of human DNA (43, 69). Most L1s are retrotransposition defective because they are 5′ truncated, contain internal rearrangements, or harbor mutations within their open reading frames (25, 43). However, the average human genome is estimated to contain ∼80 to 100 retrotransposition-competent L1s (RC-L1s), and approximately 10% of these elements are classified as highly active or “hot” (6, 63). Human RC-L1s are ∼6.0 kb and contain a 5′ untranslated region (UTR), two nonoverlapping open reading frames (ORF1 and ORF2), and a 3′ UTR that ends in a poly(A) tail (Fig. ​(Fig.1A)1A) (13, 53, 66). ORF1 encodes a 40-kDa nucleic acid binding protein (30, 31, 33), whereas ORF2 has the potential to encode a 150-kDa protein with demonstrated endonuclease (L1 EN) and reverse transcriptase (L1 RT) activities (15, 19, 22, 51). ORF2p also contains a cysteine-rich domain (CX3CX7HX4C) of unknown function (17, 54). Both proteins are required for retrotransposition in cis (54), which most probably occurs by a mechanism termed “target site primed reverse transcription” (TPRT) (19, 47, 54, 72). However, how L1 integration is completed remains a mystery. FIG. 1. Simple sequence alterations at the 5′ genomic DNA/L1 junction. A. Rationale of the assay. The 3′ UTR of a human RC-L1 was tagged with a reporter cassette designed to detect retrotransposition events. Open rectangles indicate L1 ORF1 and ... We recently developed a plasmid-based rescue system that allows the recovery of L1 insertions in cultured human HeLa cells with minimal influence from selective pressures that occur during genome evolution. We found that L1 retrotransposition is associated with various forms of genetic instability and that the nascent L1 cDNA can undergo recombination with endogenous L1 elements, resulting in the formation of chimeric L1s. Consistent findings by Symer et al., using a colon cell line (HCT116) with an essentially normal karyotype, have led to the hypothesis that L1 retrotransposition can lead to various types of genomic instability (21, 72). Here, we describe the analysis of 100 L1 retrotransposition events in HeLa cells derived from four previously characterized RC-L1s (L1.2A, LRE-2, L1.3, and L1RP). Consistent with previous studies, we have found that retrotransposition is associated with the generation of intrachromosomal deletions, the creation of chimeric L1 elements, and the addition of non-L1 nucleotides at the 5′ insertion junction (21, 56, 72). In addition, we have observed novel rearrangements, including the mobilization of U6 small uracil-rich nuclear RNA (U6 snRNA) to a new genomic location, the formation of intrachromosomal duplications, intra-L1 rearrangements, and the generation of a possible interchromosomal translocation. Finally, we have determined that the L1 RT can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases. Together, these data indicate that the resolution of L1 retrotransposition intermediates in transformed human cell lines can lead to a variety of genomic rearrangements and lead us to propose that host processes act to restrict L1 retrotransposition during integration, limiting the number of full-length L1s in the genome.

LINE-1 (L1) retrotransposition continues to impact the human genome, yet little is known about how L1 integrates into DNA. Here, we developed a plasmid-based rescue system and have used it to recover 37 new L1 retrotransposition events from cultured human cells. Sequencing of the insertions revealed the usual L1 structural hallmarks; however, in four instances, retrotransposition generated large target site deletions. Remarkably, three of those resulted in the formation of chimeric L1s, containing the 5′ end of an endogenous L1 fused precisely to our engineered L1. Thus, our data demonstrate multiple pathways for L1 integration in cultured cells, and show that L1 is not simply an insertional mutagen, but that its retrotransposition can result in significant deletions of genomic sequence.

We characterized short interspersed elements (SINEs), of the CORE-suprafamily in egg-laying (monotremes), pouched (marsupials) and placental mammals. Five families of these repeats distinguished by the presence of distinct LINE-related 3′-segments shared tRNA-like promoter and the central core region. The putative active elements were reconstructed from the alignment of genomic repeats representing molecular fossils of sequences that amplified in the past and since then underwent multiple mutations. Their mode of proliferation by retroposition was indicated by the presence of: (1) internal RNA PolIII promoter; (2) simple sequence repeated tail; (3) direct repeats; and (4) subfamilies recording the evolution of elements. The copy number of CORE-SINEs in placental genomes was estimated at about 300,000; they were highly divergent and apparently ceased to amplify before radiation of these lineages. On the other hand, among almost half a million fossil elements present in marsupials and monotremes, the youngest subfamilies could still be retropositionally active. CORE-SINEs terminate in sequence repeats of a few nucleotides similar to their 3′-segment LINE-homologues, CR1, L2 and Bov-B. These three LINE elements fall into clades distinct from that of L1 elements which, similar to their co-amplifying SINEs, end in a poly(A) tail. We propose a model in which new CORE-families, with distinct 3′-segments, are created at the RNA level due to template switching between LINE and CORE-RNA during reverse transcription. The proposed mechanism suggests that such an adaptation to the changing amplification machinery facilitated the survival and prosperity of CORE-elements over long evolutionary periods in different lineages.

A 65-bp “core” sequence is dispersed in hundreds of thousands copies in the human genome. This sequence was found to constitute the central segment of a group of short interspersed elements (SINEs), referred to as mammalian-wide interspersed repeats, that proliferated before the radiation of placental mammals. Here, we propose that the core identifies an ancient tRNA-like SINE element, which survived in different lineages such as mammals, reptiles, birds, and fish, as well as mollusks, presumably for >550 million years. This element gave rise to a number of sequence families (CORE-SINEs), including mammalian-wide interspersed repeats, whose distinct 3′ ends are shared with different families of long interspersed elements (LINEs). The evolutionary success of the generic CORE-SINE element can be related to the recruitment of the internal promoter from highly transcribed host RNA as well as to its capacity to adapt to changing retropositional opportunities by sequence exchange with actively amplifying LINEs. It reinforces the notion that the very existence of SINEs depends on the cohabitation with both LINEs and the host genome.

The S1 element is a plant SINE (Short INterspersed Element) that was first described and studied in Brassica napus and is widely distributed among Cruciferae, especially in species of the Brassiceae tribe. We propose that S1 amplification in Cruciferae could represent a good eukaryotic model to study retroposition. This is based on the fact that S1 elements share clear structural and evolutionary characteristics with mammalian SINEs but are present in a much lower copy number (500 loci by haploid genome for the S1 element in B. napus compared to 700,000 loci by haploid genome for the Alu element in human). This low copy number allows the characterization of a large portion of SINEs from a given plant species. This can lead to a more precise understanding of the evolutionary history of SINE amplification and can more easily allow an evaluation of the impact of retroposition on the evolution of that species. It can also lead more rapidly to the characterization of genomic elements active in transcription and retroposition so that the cellular control of these elements can be addressed. Finally, we show that the study of S1 insertion sites can reveal information on the RNA reverse transcription and integration step of the retroposition process.