Atypical postmortem redistribution in chronic methadone consumers.

Available literature demonstrates that methadone is prone to moderate postmortem redistribution, but subject to high interindividual variability in the central to peripheral blood concentration ratios (C/P). In this case series, 10 cases of chronic methadone users displaying C/P < 1 (range 0.26-0.82) are described. Femoral, cardiac and ante-mortem blood concentrations of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) are reported for all cases, as well as sex, age, case history, results of the pathological investigation, other toxicological findings and cause and manner of death. EDDP blood concentrations, similar in both central and peripheral blood, as well as antemortem blood concentration results in Case 4, demonstrate that this atypical C/P < 1 finding is attributable to postmortem changes and not analytical or pre-analytical artifacts. Case 4 is a particularly instructive example, with femoral blood concentration (966 ng/mL) approximately twice as high as cardiac blood (499 ng/mL) and ante-mortem blood (418 ng/mL, collected 38 min prior to death)-clearly demonstrating that cardiac blood methadone concentration is more representative of the antemortem blood concentration in this case. In Case 4 and four others, toxicological interpretation based on femoral blood concentration alone would have been misleading. Based on these results and evidence from the literature, it is hypothesized that methadone bioaccumulates in the tissues of chronic users and redistributes from thigh tissues into femoral blood, increasing the concentration postmortem. This case series highlights how femoral blood is not always preserved from postmortem changes and that the analysis of multiple blood sources is necessary to avoid a misleading toxicological interpretation-particularly for cases of chronic methadone users.

On-site drug detection coasters: An inadequate tool to screen for GHB and ketamine in beverages.

With drug facilitated sexual assault (DFSA) being alleged in 15-20 % of sexual assault cases, drink spiking is a serious concern for several people, casting doubts over the expected safety at events in public spaces. On-site drug testing material is often touted as a solution, allowing attendees to test their drinks for the presence of certain so-called "date-rape drugs". In this manuscript, we aim to evaluate the efficiency of such a coaster device, manufactured by Drink Safe Technologies (Tallahassee, Florida, United States) and sold by Alco Prevention Canada (Laval, Québec, Canada), in detecting drink spiking by GHB and ketamine. From the onset, several generic arguments call into question the practicality of the test: limitations set by the manufacturer on drinks that can be tested, cost, waiting time, interpretation in suboptimal lighting and elevated limits of detection (LODs) compared to a standard recreational or impairing dose. More importantly, the test simply isn't effective at detecting the targeted drugs. The GHB test reagent was identified as bromocresol green using surface-enhanced Raman spectroscopy (SERS). Therefore, it does not detect GHB, but any matrix with a pH higher than 5.5. The ketamine test reagent was identified as cobalt thiocyanate, a non-specific chemical commonly used in colorimetric drug testing. Performance tests were carried with more than 22 drug-free and drug-spiked (≥125 % of the LOD) matrices, including solvent solutions (water, methanol), fixed pH solutions, and an array of popular drinks (including wine, beer, cocktails and spirits). While specificity in drug-free drinks was 100 % for both GHB and ketamine, provided that the manufacturer's limitations on drinks were respected, sensitivity in drug spiked drinks (at 150 % of the LOD) was 0 % for ketamine and between 31 % and 69 % for GHB, depending on whether one classifies inconclusive results as negatives or positives. We conclude that these coasters are an inadequate tool to screen for GHB and ketamine in beverages.

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

Direct detection of alternative open reading frames translation products in human significantly expands the proteome.

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

An out-of-frame overlapping reading frame in the ataxin-1 coding sequence encodes a novel ataxin-1 interacting protein.

Spinocerebellar ataxia type 1 is an autosomal dominant cerebellar ataxia associated with the expansion of a polyglutamine tract within the ataxin-1 (ATXN1) protein. Recent studies suggest that understanding the normal function of ATXN1 in cellular processes is essential to decipher the pathogenesis mechanisms in spinocerebellar ataxia type 1. We found an alternative translation initiation ATG codon in the +3 reading frame of human ATXN1 starting 30 nucleotides downstream of the initiation codon for ATXN1 and ending at nucleotide 587. This novel overlapping open reading frame (ORF) encodes a 21-kDa polypeptide termed Alt-ATXN1 (Alternative ATXN1) with a completely different amino acid sequence from ATXN1. We introduced a hemagglutinin tag in-frame with Alt-ATXN1 in ATXN1 cDNA and showed in cell culture the co-expression of both ATXN1 and Alt-ATXN1. Remarkably, Alt-ATXN1 colocalized and interacted with ATXN1 in nuclear inclusions. In contrast, in the absence of ATXN1 expression, Alt-ATXN1 displays a homogenous nucleoplasmic distribution. Alt-ATXN1 interacts with poly(A)(+) RNA, and its nuclear localization is dependent on RNA transcription. Polyclonal antibodies raised against Alt-ATXN1 confirmed the expression of Alt-ATXN1 in human cerebellum expressing ATXN1. These results demonstrate that human ATXN1 gene is a dual coding sequence and that ATXN1 interacts with and controls the subcellular distribution of Alt-ATXN1.

PrP(C) homodimerization stimulates the production of PrPC cleaved fragments PrPN1 and PrPC1.

An endoproteolytic cleavage termed α-cleavage between residues 111/112 is a characteristic feature of the cellular prion protein (PrP(C)). This cleavage generates a soluble N-terminal fragment (PrPN1) and a glycosylphosphatidylinositol-anchored C-terminal fragment (PrPC1). Independent studies demonstrate that modulating PrP(C) α-cleavage represents a potential therapeutic strategy in prion diseases. The regulation of PrP(C) α-cleavage is unclear. The only known domain that is essential for the α-cleavage to occur is a hydrophobic domain (HD). Importantly, the HD is also essential for the formation of PrP(C) homodimers. To explore the role of PrP(C) homodimerization on the α-cleavage, we used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) and termed Fv-PrP is incubated in the presence of a dimerizer AP20187 ligand. We show that homodimerization leads to a considerable increase of PrP(C) α-cleavage in cultured cells and release of PrPN1 and PrPC1. Interestingly, enforced homodimerization increased PrP(C) levels at the plasma membrane, and preventing PrP(C) trafficking to the cell surface inhibited dimerization-induced α-cleavage. These observations were confirmed in primary hippocampal neurons from transgenic mice expressing Fv-PrP. The proteases responsible for the α-cleavage are still elusive, and in contrast to initial studies we confirm more recent investigations that neither ADAM10 nor ADAM17 are involved. Importantly, PrPN1 produced after PrP(C) homodimerization protects against toxic amyloid-ß (Aß) oligomers. Thus, our results show that PrP(C) homodimerization is an important regulator of PrP(C) α-cleavage and may represent a potential therapeutic avenue against Aß toxicity in Alzheimer's disease.

A novel ribozyme-based prophylaxis inhibits influenza A virus replication and protects from severe disease.

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses.

The peach latent mosaic viroid replication initiation site is located at a universal position that appears to be defined by a conserved sequence.

Viroids replicate through a rolling circle mechanism that is exclusively RNA dependent. In this study, we initially revisited the determination of the replication initiation sites of peach latent mosaic viroid (PLMVd). A universal initiation site for each of the PLMVd polarities (position A50/C51 and U284 for the plus and minus strands, respectively) that is shared by a relatively wide repertoire of viroid variants was identified, in agreement with a previous report based on a different methodology. Subsequently, an in vitro selection procedure based on a model rolling circle replication assay was developed. This latter experiment led to the identification of a highly conserved CAGACG box which is reminiscent of the sequence found in the vicinity of the PLMVd initiation sites. The conserved sequence contributes to delineating the initiation site and provides an explanation for the presence of a specific universal initiation site on the PLMVd molecule.

Cloning, purification, and enzymatic properties of dipeptidyl peptidase IV from the swine pathogen Streptococcus suis.

In this study, the dipeptidyl peptidase IV (DPP IV) of the swine pathogen Streptococcus suis was cloned, overexpressed in Escherichia coli, and characterized. The coding region comprises 2,268 nucleotides containing an open reading frame that codes for a 755-amino-acid protein with a calculated molecular mass of 85 kDa. The amino acid sequence contained the sequence Gly-X-Ser-X-X-Gly, which is a consensus motif flanking the active-site serine shared by serine proteases. The recombinant DPP IV showed a high affinity for the synthetic peptide glycine-proline-p-nitroanilide and was strongly inhibited by Hg2+ and diprotin A.