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Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor protein downstream of the DNA sensor cyclic GMP-AMP synthase (cGAS) that is activated by cytosolic microbial and self-DNA during infections, and in the context of loss of cellular integrity, to instigate the production of type-I IFN and pro-inflammatory cytokines. To date, whether the cGAS-STING pathway is present in platelets and contributes to platelet functions is not defined. Using a combination of pharmacological and genetic approaches, we demonstrate here that megakaryocytes and platelets possess a functional cGAS-STING pathway. Our results suggest that in megakaryocytes, STING stimulation activates a type-I IFN response, and during thrombopoiesis, cGAS and STING are transferred to proplatelets. Finally, we show that both murine and human platelets contain cGAS and STING proteins, and the cGAS-STING pathway contributes to potentiation of platelet activation and aggregation. Taken together, these observations establish for the first time a novel role of the cGAS-STING DNA sensing axis in the megakaryocyte and platelet lineage., (© 2023 El-Mortada et al.)

During infections, neutrophil extracellular traps act like a meshwork of molecules that captures microbes. In contrast, during sterile inflammation the presence of NETs is usually associated with tissue damage and uncontrolled inflammation. In this context, DNA acts both as activator of NETs formation and immunogenic molecule fueling inflammation within the injured tissue microenvironment. Pattern recognition receptors that specifically bind to and get activated by DNA such as Toll-like receptor-9 (TLR9), cyclic GMP-AMP synthase (cGAS), Nod-like receptor protein 3 (NLRP3) and Absence in Melanoma-2 (AIM2) have been reported to play a role in NETs formation and detection. However, how these DNA sensors contribute to NETs-driven inflammation is not well understood. Whether these DNA sensors have unique roles or on the contrary they are mostly redundant is still elusive. In this review, we summarize the known contribution of the above DNA sensors to the formation and detection of NETs in the context of sterile inflammation. We also highlight scientific gaps needed to be addressed and propose future direction for therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aubé, Bidias and Pépin.)

Cytoplasmic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential component of antiviral responses. Upon synthesis, cGAMP binds to the stimulator of interferon (IFN) genes (STING) in infected and adjacent cells through intercellular transfer by connexins forming gap-junctions, eliciting a strong IFN-β-driven antiviral response. We demonstrate here that Genistein, a flavonoid compound naturally occurring in soy-based foods, inhibits cGAS-STING antiviral signaling at two levels. First, Genistein pretreatment of cGAMP-producing cells inhibited gap-junction intercellular communication, resulting in reduced STING responses in adjacent cells. In addition, Genistein directly blocked STING activation by the murine agonist DMXAA, by decreasing the interaction of STING with TBK1 and IKKε. As a result, Genistein attenuated STING signaling in human and mouse cells, dampening antiviral activity against Semliki Forest Virus infection. Collectively, our findings identify a previously unrecognized proviral activity of Genistein mediated via its inhibitory effects at two levels of cGAS-STING signaling. IMPORTANCE Several reports suggest that Genistein exhibits antiviral activities against DNA viruses. Our work uncovers a previously unrecognized proviral effect of Genistein, through inhibition of the cGAS-STING pathway at the level of cGAMP transfer and its sensing by STING. This suggests that the use of Genistein as an antiviral should be taken with caution as it may reduce the protective antiviral effects elicited by host STING activation.

Fanconi Anemia (FA) is a genome instability syndrome caused by mutations in one of the 23 repair genes of the Fanconi pathway. This heterogenous disease is usually characterized by congenital abnormalities, premature ageing and bone marrow failure. FA patients also show a high predisposition to hematological and solid cancers. The Fanconi pathway ensures the repair of interstrand crosslinks (ICLs) DNA damage. Defect in one of its proteins prevents functional DNA repair, leading to the accumulation of DNA breaks and genome instability. Accumulating evidence has documented a close relationship between genome instability and inflammation, including the production of type-I Interferon. In this context, type-I Interferon is produced upon activation of pattern recognition receptors by nucleic acids including by the cyclic GMP-AMP synthase (cGAS) that detects DNA. In mouse models of diseases displaying genome instability, type-I Interferon response is responsible for an important part of the pathological symptoms, including premature aging, short stature, and neurodegeneration. This is illustrated in mouse models of Ataxia-telangiectasia and Aicardi-Goutières Syndrome in which genetic depletion of either Interferon Receptor IFNAR, cGAS or STING relieves pathological symptoms. FA is also a genetic instability syndrome with symptoms such as premature aging and predisposition to cancer. In this review we will focus on the different molecular mechanisms potentially leading to type-I Interferon activation. A better understanding of the molecular mechanisms engaging type-I Interferon signaling in FA may ultimately lead to the discovery of new therapeutic targets to rescue the pathological inflammation and premature aging associated with Fanconi Anemia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Landelouci, Sinha and Pépin.)

Activation of the STING pathway upon genotoxic treatment of cancer cells has been shown to lead to anti-tumoral effects, mediated through the acute production of interferon (IFN)-β. Conversely, the pathway also correlates with the expression of NF-κB-driven pro-tumorigenic genes, but these associations are only poorly defined in the context of genotoxic treatment, and are thought to correlate with a chronic engagement of the pathway. We demonstrate here that half of the STING-expressing cancer cells from the NCI60 panel rapidly increased expression of pro-tumorigenic IL-6 upon genotoxic DNA damage, often independent of type-I IFN responses. While preferentially dependent on canonical STING, we demonstrate that genotoxic DNA damage induced by camptothecin (CPT) also drove IL-6 production through non-canonical STING signaling in selected cancer cells. Consequently, pharmacological inhibition of canonical STING failed to broadly inhibit IL-6 production induced by CPT, although this could be achieved through downstream ERK1/2 inhibition. Finally, prolonged inhibition of canonical STING signaling was associated with increased colony formation of MG-63 cells, highlighting the duality of STING signaling in also restraining the growth of selected cancer cells. Collectively, our findings demonstrate that genotoxic-induced DNA damage frequently leads to the rapid production of pro-tumorigenic IL-6 in cancer cells, independent of an IFN signature, through canonical and non-canonical STING activation; this underlines the complexity of STING engagement in human cancer cells, with frequent acute pro-tumorigenic activities induced by DNA damage. We propose that inhibition of ERK1/2 may help curb such pro-tumorigenic responses to DNA-damage, while preserving the anti-proliferative effects of the STING-interferon axis., Competing Interests: MG receives funding from Noxopharm Ltd. to study the activity of STING inhibitors in cancer. MG does not personally own any shares/equity in Noxopharm Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Al-Asmari, Rajapakse, Ullah, Pépin, Croft and Gantier.)

Amaryllidaceae plants are rich in alkaloids with biological properties. Pancratium trianthum is an Amaryllidaceae species widely used in African folk medicine to treat several diseases such as central nervous system disorders, tumors, and microbial infections, and it is used to heal wounds. The current investigation explored the biological properties of alkaloid extracts from bulbs of P. trianthum collected in the Senegalese flora. Alkaloid extracts were analyzed and identified by chromatography and mass spectrometry. Alkaloid extracts from P. trianthum displayed pleiotropic biological properties. Cytotoxic activity of the extracts was determined on hepatocarcinoma Huh7 cells and on acute monocytic leukemia THP-1 cells, while agar diffusion and microdilution assays were used to evaluate antibacterial activity. Antiviral activity was measured by infection of extract-treated cells with dengue virus (DENV GFP ) and human immunodeficiency virus-1 (HIV-1 GFP ) reporter vectors. Cytotoxicity and viral inhibition were the most striking of P. trianthum 's extract activities. Importantly, non-cytotoxic concentrations were highly effective in completely preventing DENV GFP replication and in reducing pseudotyped HIV-1 GFP infection levels. Our results show that P. trianthum is a rich source of molecules for the potential discovery of new treatments against various diseases. Herein, we provide scientific evidence to rationalize the traditional uses of P. trianthum for wound treatment as an anti-dermatosis and antiseptic agent.

Dengue fever, caused by dengue virus (DENV), is the most prevalent arthropod-borne viral disease and is endemic in many tropical and subtropical parts of the world, with an increasing incidence in temperate regions. The closely related flavivirus Zika virus (ZIKV) can be transmitted vertically in utero and causes congenital Zika syndrome and other birth defects. In adults, ZIKV is associated with Guillain-Barré syndrome. There are no approved antiviral therapies against either virus. Effective antiviral compounds are urgently needed. Amaryllidaceae alkaloids (AAs) are a specific class of nitrogen-containing compounds produced by plants of the Amaryllidaceae family with numerous biological activities. Recently, the AA lycorine was shown to present strong antiflaviviral properties. Previously, we demonstrated that Crinum jagus contained lycorine and several alkaloids of the cherylline, crinine, and galanthamine types with unknown antiviral potential. In this study, we explored their biological activities. We show that C. jagus crude alkaloid extract inhibited DENV infection. Among the purified AAs, cherylline efficiently inhibited both DENV (50% effective concentration [EC 50 ], 8.8 μM) and ZIKV replication (EC 50 , 20.3 μM) but had no effect on HIV-1 infection. Time-of-drug-addition and -removal experiments identified a postentry step as the one targeted by cherylline. Consistently, using subgenomic replicons and replication-defective genomes, we demonstrate that cherylline specifically hinders the viral RNA synthesis step but not viral translation. In conclusion, AAs are an underestimated source of antiflavivirus compounds, including the effective inhibitor cherylline, which could be optimized for new therapeutic approaches.

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most cells constitutively secrete mitochondrial DNA and proteins in extracellular vesicles (EVs). While EVs are small vesicles that transfer material between cells, Mitochondria-Derived Vesicles (MDVs) carry material specifically between mitochondria and other organelles. Mitochondrial content can enhance inflammation under pro-inflammatory conditions, though its role in the absence of inflammation remains elusive. Here, we demonstrate that cells actively prevent the packaging of pro-inflammatory, oxidized mitochondrial proteins that would act as damage-associated molecular patterns (DAMPs) into EVs. Importantly, we find that the distinction between material to be included into EVs and damaged mitochondrial content to be excluded is dependent on selective targeting to one of two distinct MDV pathways. We show that Optic Atrophy 1 (OPA1) and sorting nexin 9 (Snx9)-dependent MDVs are required to target mitochondrial proteins to EVs, while the Parkinson's disease-related protein Parkin blocks this process by directing damaged mitochondrial content to lysosomes. Our results provide insight into the interplay between mitochondrial quality control mechanisms and mitochondria-driven immune responses.

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2'-methoxyethyl (2'MOE) modifications. Through a screen of 192 2'OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2'OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Research works on operator monitoring underline the benefit of taking into consideration several signal modalities to improve accuracy for an objective mental state diagnosis. Heart rate (HR) is one of the most utilized systemic measures to assess cognitive workload (CW), whereas, respiration parameters are hardly utilized. This study aims at verifying the contribution of analyzing respiratory signals to extract features to evaluate driver's activity and CW variations in driving. Eighteen subjects participated in the study. The participants carried out two different cognitive tasks requiring different CW demands, a single task as well as a competing cognitive task realized while driving in a simulator. Our results confirm that both HR and breathing rate (BR) increase in driving and are sensitive to CW. However, HR and BR are differently modulated by the CW variations in driving. Specifically, HR is affected by both driving activity and CW, whereas, BR is suitable to evidence a variation of CW only when driving is not required. On the other hand, spectral features characterizing respiratory signal could be also used similarly to HR variability indices to detect high CW episodes. These results hint the use of respiration as an alternative to HR to monitor the driver mental state in autonomic vehicles in order to predict the available cognitive resources if the user has to take over the vehicle.

Murine models of Salmonella enterica serovar Typhimurium infection are one of the commonest tools to study host-pathogen interactions during bacterial infections. Critically, the outcome of S . Typhimurium infection is impacted by the genetic background of the mouse strain used, with macrophages from C57BL/6 and BALB/c mice lacking the capacity to control intracellular bacterial replication. For this reason, the use of congenic strains, which mix the genetic backgrounds of naturally protected mouse strains with those of susceptible strains, has the capacity to significantly alter results and interpretation of S . Typhimurium infection studies. Here, we describe how macrophage knockout cell lines generated by CRISPR/Cas9 gene editing can help determine the contribution of background contaminations in the phenotypes of primary macrophages from congenic mice, on the outcome of S . Typhimurium infection studies. Our own experience illustrates how the CRISPR/Cas9 technology can be used to complement pre-existing knockout models, and shows that there is great merit in performing concurrent studies with both genetic models, to exclude unanticipated side-effects on host-pathogen interactions.

Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) β stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with Salmonella Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation., (© 2018 Nejad et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

MicroRNA (miRNA) detection by reverse transcription (RT) quantitative real-time PCR (RT-qPCR) is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3'-end length variants ("isomiRs"), little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3'-end miRNA isoforms can exhibit 3'-length specific regulatory functions, underlining the need to develop strategies to differentiate 3'-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20-21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3'-isomiRs.

Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans., (Copyright © 2017 Pépin et al.)

Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as a Dicer-interacting protein candidate. Confocal immunofluorescence microscopy revealed the colocalization of Dicer and TWEAK proteins at the perinuclear region of HeLa cells. The Dicer-TWEAK protein interaction was confirmed by coimmunoprecipitation and found not likely to be mediated by RNA. TWEAK dose-dependently reduced pre-microRNA conversion into mature microRNA in Dicer activity assays using extracts of transfected human HEK 293 cells. TWEAK expression also impaired microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. These findings suggest a role for TWEAK-a pro-inflammatory cytokine-in regulating Dicer function and microRNA biogenesis, and its possible involvement in regulating gene expression during inflammatory processes and diseases.

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of microRNAs, which are known to regulate messenger RNA (mRNA) translation. In order to improve our understanding of the molecular context in which Dicer functions and how it is regulated in human cells, we sought to expand its protein interaction network by employing a yeast two-hybrid screening strategy. This approach led to the identification and characterization of cytoskeleton-linking endoplasmic reticulum (ER) membrane protein of 63 kDa (CLIMP-63) as a novel Dicer-interacting protein. CLIMP-63 interacts with Dicer to form a high molecular weight complex, which is electrostatic in nature, is not mediated by RNA and is catalytically active in pre-microRNA processing. CLIMP-63 is required for stabilizing Dicer protein and for optimal regulation of a reporter gene coupled to the 3' untranslated region of HMGA2 mRNA in human cells. Interacting with a portion of the luminal domain of CLIMP-63 and within minutes of its synthesis, our results suggest that Dicer transits through the ER, is glycosylated and can be secreted by cultured human cells with CLIMP-63. Our findings define CLIMP-63 as a novel protein interactor and regulator of Dicer function, involved in maintaining Dicer protein levels in human cells.

The authors present 3 cases that demonstrate a return to DFC following periods of inactivity. The offences occurred in Paris and its suburbs and in each of the cases there were two distinct periods of activity by the offenders with 2, 8 and 22 victims attributed to each of the perpetrators. To 20mg of decontaminated and cut hair, 100 pg/mg of clonazepam-d4 was added as internal standard. Hair specimens were extracted with CH(2)Cl(2)/ether after incubation overnight at 56 degrees C in pH 7.6 buffer. Extractions were performed on blood and urine using Toxi-tube A with 5 ng/mL of clonazepam-d4. The residues were analyzed by LC-ESI-MS/MS. Calibration curves in blood and urine (0.5-500 ng/mL) were prepared by spiking aliquots of blank fluids (r(2)>0.9816 for all drugs). LOD in body fluids ranged 0.5-10 ng/mL. Calibration curves in hair (0.5-100 pg/mg) were prepared by spiking aliquots of blank hair (r(2)>0.9877 for all drugs). LOD in hair ranged 0.5-5 pg/mg. Case #1: Two young women were raped with an interval of approximately 1 year between the incidents. Lorazepam (present, <2 pg/mg) was detected in hair obtained from the first victim, and zolpidem (19 pg/mg) in hair of the second one. The offender was in jail between the two offences. Case #2: The offender approached a total of 8 men and women who were aged over 50 years. The offender was in jail between the two series of respectively 3 and 5 victims. Zopiclone was detected in victims' hair (n=7) at concentrations 13-42 pg/mg. Case #3: The offender stole thousands of Euros using credit cards obtained from 22 different wealthy victims. He employed a cocktail of up to 6 drugs made up of: flunitrazepam, clonazepam, doxylamine, cyamemazine, zolpidem and lorazepam. Drugs were detected in all victims' hair (n=18) at concentrations in the range 1-81 pg/mg for all drugs. Between the two series (of respectively 4 and 16 victims) the offender spent 6 months in jail, and then police spent 6 months looking for him while he was under judiciary control prior to his judgment. Segmental hair analysis permits retrospective information on drug exposure and should be considered in the investigation of drug-facilitated crimes not only to prove single exposure but also when there has been any appreciable delay in samples being obtained for analysis. Indeed, in 56% cases reported in this paper, due to the long time that elapsed between offences and the opportunity to obtain samples for analysis hair analysis was considered the only viable matrix to investigate the possibility of drug involvement in the crimes. Our experience demonstrates that the incidence of re-offending in DFC after a period of inactivity (often due to imprisonment) may be of concern, notably in big cities., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Tabernanthe iboga belongs to the Apocynaceae family. In this study, we report the case of a 37-year-old black male working as a security agent in Paris and found dead naked on the beach in Gabon after consumption of iboga. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken. Biological fluids were liquid-liquid extracted with saturated NH4Cl pH 9.5 and methylene chloride/isopropanol (95/5, v/v) in presence of clonazepam-d(4), used as internal standard. After decontamination with dichloromethane, hair was cut into small pieces then sonicated for 2h in saturated NH4Cl pH 9.5 before extraction by methylene chloride/isopropanol (95/5, v/v). After evaporation the residues were reconstituted in methanol/ACN/formate buffer pH 3, from which 10 microL were injected into an ODB Uptisphere C(18) column (150 mm x 2.1mm, 5 microm) and eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 200 microL/min. A Quantum Ultra triple-quadrupole mass spectrometer was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESI). For each compound, detection was related to three daughter ions (ibogaine: m/z 311.4-->122.1, 174.1 and 188.1; noribogaine: m/z 297.4-->122.1, 159.1 and 160.1; clonazepam-d(4): m/z 319.9-->218.1, 245.1 and 274.1). Ibogaine and noribogaine were detected in all autopsy samples. Hair segmentation was not possible as hair was very short and frizzy. Concentrations of 1.2 and 2.5 ng/mg, respectively were detected. Neither other licit or illicit drugs nor alcohol were found. The presence of ibogaine and noribogaine in all autopsy samples was consistent with the recent absorption of Tabernanthe iboga, which was assumed to be responsible of the drowning fatality. The history of exposure, regarding hair analysis, is discussed. LC-MS/MS appears to be the best method for analyzing complex and poorly volatile alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured.

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.

Poisoning by colchicine may occur following ingestion of this alkaloid used for the treatment of acute gouty arthritis. The authors report two fatalities and describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) triple-quadrupole method for the determination of colchicine in autopsy samples. One milliliter of heart blood, femoral blood, urine, bile, gastric, and vitreous each were extracted with saturated NH4Cl at pH 9.6 and dichloromethane/5% isopropanol. Separation was achieved on a C18-Xterra column with a mobile phase consisting of 2 mM ammonium formate buffer (pH 3)/acetonitrile in a gradient mode. Four product ions of the protonated molecule were monitored. The method was fully validated in whole blood (1 mL) and was linear in the range of 0.5-50 ng/mL (r2>0.99). The limit of detection was 0.1 ng/mL (50 times S/N), and the limit of quantitation was 0.5 ng/mL with RSDs<11.8% intraday (n=6), <18.7% interday (n=18), and accuracy<3% (n=18). Case #1: a 33-year-old nurse committed suicide by the ingestion of 80 colchicine 1-mg tablets. She died 61 h later after resuscitation procedures. Colchicine was found in heart blood at 5.2 ng/mL, femoral blood at 17.4 ng/mL, urine at 19.4 ng/mL, bile at 42.8 ng/mL, gastric at 348 ng/mL, and vitreous at 3 ng/mL. Case #2: a 57-year-old man with gout was found dead at home. Colchicine was found in heart blood at 22.8 ng/mL, femoral blood at 21.9 ng/mL, lung blood at 45.2 ng/mL, urine at 148.5 ng/mL, bile at 1818.5 ng/mL, gastric at 219.8 ng/mL, and vitreous at 0.5 ng/mL. These results were consistent with death. Because of its good sensitivity, this LC-ESI-MS-MS triple-quadrupole method is suitable for the determination of colchicine not only in fatalities but also for pharmacokinetic studies.

In recent years, reports of drug-facilitated crimes (DFC) have been increasing. The drugs involved are sometimes difficult to detect, because of their low dosages and the long time ellapsed between alleged DFC and blood and urine sampling. In order to detect benzodiazepines and benzodiazepine-like hypnotics, we developed an approach for hair analysis by liquid chromatography-tandem mass spectrometry using a triple stage quadrupole with an electrospray ionization (LC-ESI-MS/MS). Separation was performed on an Uptisphere ODB C18 column using a gradient of 2mM formate buffer and acetonitrile. For the 23 compounds studied, detection limits are lower than 2 pg/mg, but a specific extraction procedure is needed for 7-amino metabolites. Over a 1-year period within the city limits of Paris and three suburbs, we tested blood and urine from victims of sexual assaults, robbery and battery in which psychoactive substances were suspected of being involved. Hair was collected 4-8 weeks after the alleged DFC. Over the 128 cases studied, results of simultaneous analysis of blood, urine, and hair allowed us to conclude that 23 cases were real DFC. In 18 cases, no conclusion was possible since no hair was sampled and/or results were negative. In 56 cases, victims were previously using narcotics, cannabis, and/or a prescribed drug, according to the compounds detected in hair strands. Thirty-one cases were not DFC cases. This study indicates that the prevalence of zolpidem and clonazepam is high, followed by bromazepam, nordazepam, and midazolam. Others benzodiazepines and analogs are rare. LC-ESI-MS/MS is a good tool for toxicological investigations of DFC. Testing blood, urine, and hair by this technique may reveal drug presence, even if it was administered at a single therapeutic dose. That may be helpful to prosecute perpretators or to exclude a drug-facilitated crime.

The number of reports on drug facilitated crimes is increasing these last years. Apart from ethanol and cannabis, benzodiazepines (BZD) and analogs are the most common drugs reported to be used probably due to their amnesic and sedative properties. We have developed a rapid and sensitive method using LC-MS/MS triple stage quadrupole (TSQ) for the determination of single exposure to bromazepam (Lexomil, 6 mg) and clonazepam (Rivotril, 2 mg) in urine and hair of healthy volunteers. Chromatography was carried out on a Uptisphere ODB 5 microm, 2.1 mm x 150 mm column (Interchim) with a gradient of acetonitrile and formate 2 mM buffer, pH 3. Urine was extracted with Toxitube A (Varian) and allowed the detection of bromazepam, 3-hydroxy-bromazepam, clonazepam and 7-Aminoclonazepam for more than 6 days. Head hair, collected 1 month after the exposure, was treated by incubation with Soerensen buffer pH 7.6, followed by liquid-liquid extraction with dichloromethane for common BZD. A specific pre-treatment for amino-BZD, with an incubation of 15 min at 95 degrees C in 0.1 N NaOH before liquid-liquid extraction with dichloromethane, gave better recoveries and repeatability. After single exposure, bromazepam was present in powdered hair at 28 pg/mg and 7-Aminoclonazepam at 22 pg/mg in the first 1-cm segment, while no clonazepam was detectable. This method was applied in two forensic cases. It allowed us to determine bromazepam in urine 3 days after the alleged offense and in cut head hair at a concentration of 6.7 pg/mg only in the 2-cm proximal segment. The other case showed the presence of clonazepam and 7-Aminoclonazepam in urine a few hours after the offense and the presence of 7-Aminoclonazepam at about 3.2 pg/mg in axillary hair 4 months later.

The purported lowering of sex opposition, coupled with a possible abrupt unconsciousness-inducing effect and ease of administration in spiked drinks have resulted in the use of hypnotics in cases of drug-facilitated offense. Among these compounds, lorazepam possesses amnesic properties and can impair an individual rapidly. The chances to detect this substance increase if the most sensitive methods are used and if the biological fluid which allows the longest possible detection time is available. In order to document the window of detection of lorazepam, we have orally administered 2.5 mg of the drug to three volunteers and collected oral fluid (n = l) over 8 h, urine (n = 2) over 144 h and hair (n = 3) 4 weeks after exposure. Lorazepam was analyzed by LC-MS/MS after alkalinisation (to pH 8.4 with phosphate buffer) and extraction by dichloromethane/diethyl ether in presence of diazepam-d5, used as internal standard. Reversed-phase separation on a XTerra C18 column was achieved in 12 min, under gradient conditions. Molecular ions (m/z 321 and 290 for lorazepam and the IS, respectively) were selected in Ql and the corresponding daughter ions (m/z 303 and 275 for lorazepam and m/z 154 and 198 for the IS) were detected in Q3 after collision with argon. Urine tested positive for lorazepam over 144 h (2-4 ng/ml), with a peak detected after 24 h exposure (411-880 ng/ml). Oral fluid tested positive for lorazepam over 8 h (0.7 ng/ml). Despite a limit of quantitation at 1 pg/mg, we were unable to detect a single lorazepam dose in hair, contrarily to most other benzodiazepines that are detectable. Therefore, in case of drug-facilitated crimes involving lorazepam, urine appears as the best specimen to document exposure, particularly if LC-MS/MS is used.

We have developed a GC-MS-MS assay for GHB in human hair. Five milligrams of washed hair were hydrolyzed by 1M or 0.01M NaOH before a liquid-liquid extraction with ethyl acetate under acidic conditions. GHB-d(6) was used as the internal standard. TMS derivatives were formed before injection. TBDMS derivatives were used in cases of strong chromatographic interferences or in a confirmatory procedure. Analysis of basal levels of GHB in 61 drug-free donors gave the following results: the mean measured concentration for blond hair was 0.60 ng/mg (n = 12), SD = 0.19 ng/mg, and extreme figures were in the range 0.35-0.95 ng/mg. For brown hair, the mean measured concentration was 0.90 ng/mg (n = 30), SD = 0.42 ng/mg, and extreme figures 0.41-1.86 ng/mg. For black hair, the mean measured concentration was 0.90 ng/mg (n = 19), SD = 0.37 ng/mg, and extreme figures 0.32-1.54 ng/mg, showing no significant differences depending on hair color. Analysis of basal levels of GHB of 12 or more specimens in segmented hair showed a mean concentration of 1.22 ng/mg (0.31-8.4 ng/mg) and a relative standard deviation for each individual ranging from 6.75% to 37.98%. GHB was administered to a healthy 53-year-old white male (light brown hair) at oral dosages of 30, 45, and 60 mg/kg. Beard hair was collected just before administration and 24 h after (and each day for one week for the last dose), and a 7.5-cm scalp hair lock was collected 7 days after the last dose. A rise in GHB concentration was observed in beard hair for the 45 and 60 mg/kg dosages with a maximum at 24 h, whereas no change was observed for the 30 mg/kg dosage. Scalp hair was segmented into 3-mm long segments. The three proximal last segments showed significantly (0.0005 < p < 0.005) different concentrations of GHB (1.22, 1.27, and 1.66 ng/mg, respectively) when compared with the basal physiological level of GHB in this same person (mean = 0.62 ng/mg, SD = 0.15 ng/mg). A case of daily GHB abuse during bodybuilding allowed us to determine a concentration of GHB of 14 ng/mg, in a 2-cm long segment (black hair). A case of rape under the influence of GHB was documented through hair analysis (black hair) and positive analysis of the glass she used. Sampled 7 days after the sexual assault, the three last 3-mm long proximal segments tested for GHB exhibited concentrations of 3.1-5.3 and 4.3 ng/mg, respectively, whereas the mean physiological level determined in this woman was 0.71 ng/mg, SD = 0.17 ng/mg. The authors advise a two-step hair sampling as evidence of GHB consumption: the first sample at the time of exposure to show the contamination by sweat of the proximal segment in case of recent administration with a significant rise of hair level at the root, and the second after at least 3 or 4 weeks to avoid this contamination and determine the levels incorporated in the hair matrix before, during, and after the exposure.

We report one fatal case of asphyxia caused by leaves of common ivy. Macroscopic examination of the corpse during the autopsy disclosed an incredible quantity of leaves of Hedera helix in the mouth and throat of the decedent. In order to rule out the possibility of poisoning by the toxic saponins contained in the plant, we have developed an efficient LC-EI/MS-MS assay of hederacoside C, alppha-hederin, and hederagenin in biological fluids and plant material. Sample cleanup involved solid-phase extraction of the toxins on C18 cartridges followed by LC analysis under reversed-phase conditions in the gradient elution mode. Solute identification was performed using full scan MS-MS spectrum of the analytes. Oleandrine was used as internal standard. Under these conditions, saponins in powdered dried leaves of Hedera helix were measured at a concentration of 21.83 mg/g for hederacoside C, 0.41 mg/g for alpha-hederin and 0.02 mg/g for hederagenin. No toxin was detected in cardiac blood, femoral blood, or urine of the deceased, but hederacoside C was quantitated at 857 ng/mL in the gastric juice. These findings led us to conclude that the man committed suicide and that the death was caused by suffocation by leaves of common ivy.

In an effort to improve laboratories performing hair analysis in forensic cases, the French Society of Analytical Toxicology (S.F.T.A.) has implemented a proficiency testing program since 1992. Actually about 10 laboratories are participating. Each survey is dedicated to one analyte or one pharmacological class: opiates (6-monoacetylmorphine, morphine and codeine), cocaine and benzoylecgonine, tetrahydrocannabinol, buprenorphine and norbuprenorphine, beta-blockers (metoprolol, atenolol), beta 2-agonists (salbutamol, clenbuterol). Animal hair was tested for clenbuterol. Prior to sending, hair samples were reduced to a powdered form, well mixed to ensure homogeneity, and then tested by GC/MS or HPLC/MS. Results confirm those obtained in a preliminary study on opiates and cocaine analysis in hair: a common analytical procedure has to be used by all the participants, including hydrolysis of hair. It is essential to work on authentic drug-positive hair samples and not on spiked samples. Participation at this program is free of charge and considered as an educational tool. Comparison of the results with those of other laboratories in Europe and USA shows that the analytical methods used during this program are in accordance with all the other procedures.

In France during a famous bicycle race, the newspapers documented the degree in which doping seemed to be supervised in some teams by managers and doctors. Use of anabolic steroids and other substances was officially banned in the mid-seventies by sports authorities. This policy has been enforced through urine testing before competition. It is well known, however, that a latency period is all that is necessary to defeat these tests. Nevertheless, hair analysis could be a promising tool when testing for periods that are not accessible to urinalysis any more. We have developed different sensitive methods for testing hair for amphetamines, anabolic steroids and their esters and corticosteroids. For amphetamines, 50 mg of hair were digested with 1 M NaOH, extracted with ethyl acetate, derivatized with TFA and analyzed by gas chromatography positive chemical-ionization mass spectrometry. For corticosteroids, 50 mg of powdered hair were treated with methanol in an ultrasonic bath and subsequently purified using a C18 solid phase extraction column. Analysis was realized by high performance liquid chromatography coupled to electrospray-ionization tandem mass spectrometry. For anabolic steroids and their esters, 100 mg of powdered hair were treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid phase extraction on aminopropyl and silica cartridges. Residue was derivatized with MSTFA prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. Thirty cyclists were sampled and tested both in hair and in urine. Amphetamine was detected 10 times in hair (out of 19 analyses) compared to 6 times in urine (out of 30 analyses). Corticosteroids were detected 5 times in hair (methylprednisolone 1 case, triamcinolone acetonide 3 cases and hydrocortisone acetate 1 case) in hair (out of 12 analyses) compared to 12 times (triamcinolone acetonide 10 cases and betamethasone 2 cases) in urine (out of 30 analyses). Anabolic steroids were detected twice (nandrolone 1 case, and testosterone undecanoate 1 case) in hair (out of 25 analyses) compared to none in urine (out of 30 analyses).

We have described a rapid and simple solid-phase extraction on C18 cartridges of moclobemide suitable for the analysis of post-mortem whole blood and urine. The methods used for identification were GC-MS and HPLC-PDA. Quantification was performed by the HPLC-PDA technique with detection at 238 nm. The limit of detection was 0.012 microgram/ml in blood. A between-day precision study gave relative standard deviations which were always less than 4.7% over the entire range of calibration (0.2 to 20.0 micrograms/ml). The method was applied in a case of moclobemide overdose due to a deliberate ingestion of 4.5 g of the drug. A second case concerned a polyintoxication including moclobemide as one of the main toxins. The post-mortem whole blood concentrations were 15.5 and 13.8 micrograms/ml respectively. Determination of the drug in other biological specimens is also reported.

We describe a simple method for the urinary identification and blood quantitation of meprobamate suitable for any toxicological laboratory. After urinary screening using GC-MS technology, quantitation is performed by GC-MS in the selected-ion monitoring mode. Isolation of the drug is achieved by solid phase extraction on a C-18 cartridge. A specific elution is obtained by three volumes of acetone:triethylamine (99:1 v/v). Lidocaine is used as internal standard. RSDs (%) of the within-day and between-day precision studies are always less than 7.2 on the entire range of calibration. Linearity is inspected using an analysis of variance ANOVA. Homogeneity of the variances is tested using Hartley's test. Weighted linear regression is then computed. Limits of detection and quantification are given by an analysis of the blanks. The present method was applied in our laboratory over a period of 1 year. Meprobamate appeared as a drug which still has a significant frequency (5.5%) and is the most frequently involved in fatal pharmaceutical overdoses (15.3%). Post mortem concentrations ranged from 41 to 397 mg/l (mean = 182) and are compared to those of the literature.

We have developed a new solid-phase extraction (SPE) on C18 cartridges which allows a very simple protocol of manipulation and a single elution of opiates and cocainics from human hair samples. The method involved decontamination in a phosphate buffer and dichloromethane, pulverization in a ball mill, addition of deuterated internal standards, heated acid hydrolysis and SPE. Quantitation utilized gas chromatography and mass spectrometry. Between days precise study gave relative standard deviations always inferior to 8.9% for each compound at 4 ng/mg (except methylecgonine ester = 15.7%). Accuracy was tested using a t-statistic versus a reference material from the NIST. Limits of detection were calculated from an analysis of the blanks which contained between 0.12 and 0.28 ng/mg for each drug. The method was applied in forensic cases for 1 year of toxicological activity. Among the 108 analyses performed, 30 were positive for cocaine and 33 for opiates. Concentrations were in the range 0.9-242.0 ng/mg (cocaine), 0.3-71.3 ng/mg (benzoylecgonine), 0.0-9.8 ng/mg (methylecgonine ester), 0.0-2.9 ng/mg (cocaethylene), 0.1-11.5 ng/mg (codeine), 0.4-44.6 ng/mg (morphine) and 0.7-131.2 ng/mg (6-acetylmorphine). Ratios of the metabolites to parent drugs were proposed to avoid risk of external contamination.

We have presented the results concerning 135 judicial expert opinion over a 3-year period. We have compared the measured levels in hair of 6-acetylmorphine (6-AM) and of cocaine with the habitual use declared by the consumers. This allows us to propose three levels (low, medium, high) of consumption in relation to the level of the 6-AM marker found in hair for the consumption of heroin, and the level of cocaine as a marker for the cocaine intake.

We have described a simple, precise and sensitive assay of tetrachloroethylene in whole blood and tissues, suitable both for emergency cases and forensic medicine. The method employs gas chromatography and electron capture detection. The case report concerns a fatal exposure of a child to tetrachloroethylene. Concentrations of the chemical in different fluids and tissues were determined and compared to two other previously published fatalities.