Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the ...Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3′ single-stranded (3′-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3′-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.
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•Xrn1-sensitive Unstable Transcripts (XUTs) are 3′-extended isoforms of stable lncRNAs•Nonsense-Mediated Decay preferentially targets long XUTs with single-stranded 3′ end•Antisense XUTs form double-stranded RNA in vivo•Formation of double-stranded RNA protects XUTs from Nonsense-Mediated Decay
Wery et al. used single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA in vivo mapping to show that antisense Xrn1-sensitive Unstable Transcripts (XUTs) form double-stranded RNA in yeast and that 3′ single-stranded extension mediates XUTs degradation by the Nonsense-Mediated Decay (NMD) pathway, assisted by the Mtr4 and Dbp2 RNA helicases.
This study proposes a proteomic-based strategy for the identification of the origin species of glues used as binding media and adhesives in artworks. The methodology, based on FTICR high resolution ...mass spectrometry, was evaluated on glues from different animal origin (i.e., bovine, rabbit, and fish). The analysis of the peptide mixture resulting from the enzymatic hydrolysis of the proteins led to the identification of species-specific peptides. Up to 15 specific peptides were identified for the bovine species and three for the rabbit species and, in the case of sturgeon glue, three fish-specific peptides were found by sequence homology to the rainbow trout. Then, the method was applied to authenticate different rabbit skin glue samples, including a 100 year-old sample named “Colle à Doreurs” coming from the “Maison Totin-Frères”. For this sample, two specific peptides of rabbit collagen were identified. To evaluate the method in a complex matrix, model paints composed of lead white, linseed oil, and animal glue were prepared. Species-specific peptides were identified in each paint sample. Finally, a gilt sample from St Maximin church dating from the eighteenth century was analyzed, and 13 peptides specific to bovine collagens were identified starting from very low sample amount (50 μg).
This work provides the first identification of fish glue from a few micrograms of a 17(th) century artwork sample using an adapted proteomics approach. Fish glue has been widely used as a binder in ...various art objects such as paintings, manuscripts or polychrome objects however its authentication remains particularly challenging. The lack of information on fish species in genomic and proteomic databases represents a major drawback. A supplementary difficulty is provided by the historical sample features, i.e. a few micrograms of a 17(th) century polychrome object with a multilayered structure. SYPRO® Ruby staining was used as a screening technique to probe the presence of proteins in the sample cross-section. Results revealed the presence of several layers containing proteins among which a thin proteinaceous layer located between the silver leaf and the glaze. This thin layer is described as fish glue coating by historical sources but its composition has not been identified yet. The optimized methodology, based on high resolution mass spectrometry and adapted bioinformatic tools, was successfully applied to 50 μg of a polychromy sample and resulted in the identification of several collagen proteins. Extensive interpretation of data generated by tandem mass spectrometry allowed the identification of proteins from different biological origins. In particular, seven peptides specific to fish collagen proteins were identified for the first time proving the presence of fish glue in the sample and corroborating information found in historical texts dealing with the polychromy technique.
This manuscript deals with the identification of protein residues in amphorae, including particularly identification of protein species. The work described was performed on fishes, the anchovy ...(Engraulis encrasicolus) and bonito (Sarda sarda) species frequently found in the Mediterranean area. Based on proteomic techniques, the analytical strategy was adapted to analysis of protein residues from tiny ceramic fragments. The major difficulty was to extract proteins and limit their hydrolysis during the sample preparation; consequently, multiple soft extraction techniques were evaluated. The most valuable results were obtained using a solution containing high amounts of denaturing agents, urea and thiourea, reducing agent, dithiothreitol, and detergent, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate. The analysis using nano liquid chromatography-nano electrospray ionization double quadrupole time-of-flight mass spectrometry resulted in the identification of up to 200 proteins for the anchovy and bonito species, among which 73 peptides were found to be fish-specific. Because bonito and anchovy species are not documented and fully sequenced in genomic databases, the preliminary protein identification was realized via sequence homology to other fish sequenced species. Amino acid substitutions of peptides were assigned on the basis of the interpretation of tandem mass spectrometry spectra using de novo sequencing; these peptides, not reported up to now in databases, constitute species-specific markers. The method developed was finally applied to an archaeological sample replica impregnated with a mixture of fish tissue from both species; this experiment successfully led to the identification of 17 fish proteins, including 33 fish-specific peptides. This work shows that the analytical method developed has great potential for the identification of protein species in complex archaeological samples.
L’analyse d’échantillons du Patrimoine Culturel est primordiale pour des questions de compréhension de technique, conservation et restauration. Cependant, ces échantillons sont rares et précieux et ...l’analyse doit être effectuée sur une faible quantité de matière, ce qui nécessite le développement et l’optimisation de méthodologies analytiques appropriées. L’objectif de ce travail de thèse a été de développer des méthodes analytiques dans le but d’étudier les protéines dans des échantillons du Patrimoine Culturel.Les changements structuraux et les modifications chimiques des protéines des liants de peinture, provoqués par interaction avec les autres composés ou par le vieillissement ont été mis en évidence grâce à l’utilisation de la micro-spectrométrie Raman et de l’analyse protéomique. Les travaux ont ensuite ciblés un type de substance protéique abondamment utilisé en tant qu’adhésif et liant de peinture : la colle animale. Une méthodologie, basée sur l’analyse par spectrométrie de masse à haute résolution et permettant l’identification de l’espèce d’origine des colles animales est présentée. Elle a été appliquée avec succès sur un échantillon de dorure du 18ème siècle révélant ainsi la nature de la colle animale utilisée pour la préparation de la couche d’apprêt et pour la fixation des feuilles d’or. Enfin, une problématique liée à l’identification du contenu des amphores, qui est à l’heure actuelle un véritable challenge à cause des conditions de conservation des objets archéologiques a été abordée. Le développement d’une méthode d’analyse protéomique permettant d’identifier les résidus de protéines, piégés dans les tessons d’amphores archéologiques est exposée.
The analysis of Cultural Heritage samples brings information on the understanding of a technique and is essential for conservation and restoration issues. However, as these samples are scarce and precious little amount is available for the analysis, which requires the development and the optimization of appropriate analytical methods. The purpose of this thesis was to develop analytical methods for the study of proteins in Cultural Heritage samples. Structural changes and chemical modifications of proteins in paint binder, due to the presence of other components or due to ageing have been investigated by using micro-Raman spectrometry and proteomics analysis. The work was then focused on a particular kind of proteinaceous substance frequently used as painting binder and adhesives: the animal glue. A method, based on high resolution mass spectrometry and allowing the identification of the origin species of the glues on the basis of species-specific peptides is presented. It was successfully applied on a gilt sample dating from the 18th century. Bovine glue was identified in the plaster support and as adhesive for the gold leaf. The development of analytical tools for the analysis of archeological samples is also a challenging task owing to the conditions in which such objects have been stored. A proteomics-based method for the identification of protein residues in archaeological ceramics is presented. Here, the work was focused on the identification of fish-specific peptides because analysis of fish amphorae was expected.
L'analyse d'échantillons du Patrimoine Culturel est primordiale pour des questions de compréhension de technique, de conservation et de restauration. Cependant, ces échantillons sont rares et ...précieux et l'analyse doit être effectuée sur une faible quantité de matière, ce qui nécessite le développement et l'optimisation de méthodes analytiques appropriées. L'objectif de ce travail de thèse a donc été de développer des méthodes analytiques dans le but d'étudier les protéines dans des échantillons du Patrimoine Culturel. Les changements structuraux et les modifications chimiques des protéines des liants de peinture, provoqués par interaction avec les autres composés ou par le vieillissement ont été mis en évidence grâce à l'utilisation de la micro-spectrométrie Raman et de l'analyse protéomique. Les travaux ont ensuite ciblés un type de substance protéique abondamment utilisé en tant qu'adhésif et liant de peinture : la colle animale. Une méthodologie, basée sur l'analyse par spectrométrie de masse à haute résolution et permettant l'identification de l'espèce d'origine des colles animales est présentée. Elle a été appliquée avec succès sur un échantillon de dorure du 18ème siècle révélant ainsi la nature de la colle animale utilisée pour la préparation de la couche d'apprêt et pour la fixation des feuilles d'or. Enfin, une problématique liée à l'identification du contenu des amphores, qui est à l'heure actuelle un véritable challenge à cause des conditions de conservation des objets archéologiques, a été abordée. Le développement d'une méthode d'analyse protéomique permettant d'identifier les résidus de protéines piégés dans les tessons d'amphores archéologiques est exposée.
Postprocedural aortic regurgitation occurs in 10 to 20% of patients undergoing transcatheter aortic-valve replacement (TAVR) for aortic stenosis. We hypothesized that assessment of defects in ...high-molecular-weight (HMW) multimers of von Willebrand factor or point-of-care assessment of hemostasis could be used to monitor aortic regurgitation during TAVR.
We enrolled 183 patients undergoing TAVR. Patients with aortic regurgitation after the initial implantation, as identified by means of transesophageal echocardiography, underwent additional balloon dilation to correct aortic regurgitation. HMW multimers and the closure time with adenosine diphosphate (CT-ADP), a point-of-care measure of hemostasis, were assessed at baseline and 5 minutes after each step of the procedure. Mortality was evaluated at 1 year. A second cohort (201 patients) was studied to validate the use of CT-ADP in order to identify patients with aortic regurgitation.
After the initial implantation, HMW multimers normalized in patients without aortic regurgitation (137 patients). Among the 46 patients with aortic regurgitation, normalization occurred in 20 patients in whom additional balloon dilation was successful but did not occur in the 26 patients with persistent aortic regurgitation. A similar sequence of changes was observed with CT-ADP. A CT-ADP value of more than 180 seconds had sensitivity, specificity, and negative predictive value of 92.3%, 92.4%, and 98.6%, respectively, for aortic regurgitation, with similar results in the validation cohort. Multivariable analyses showed that the values for HMW multimers and CT-ADP at the end of TAVR were each associated with mortality at 1 year.
The presence of HMW-multimer defects and a high value for a point-of-care hemostatic test, the CT-ADP, were each predictive of the presence of aortic regurgitation after TAVR and were associated with higher mortality 1 year after the procedure. (Funded by Lille 2 University and others; ClinicalTrials.gov number, NCT02628509.).
Background
Recent studies have demonstrated that aldosterone levels measured in patients with heart failure or acute myocardial infarction (MI) are associated with long-term mortality, but the ...association with aldosterone levels in patients with coronary artery disease (CAD) outside these specific settings remains unknown. In addition, no clear mechanism has been elucidated to explain these observations. The present study was designed to evaluate the relationship between the level of aldosterone and the risk of death and acute ischaemic events in CAD patients with a preserved left ventricular (LV) function and no acute MI.
Methods and results
In 799 consecutive CAD patients referred for elective coronary angioplasty measurements were obtained before the procedure for: aldosterone (median = 25 pg/mL), brain natriuretic peptide (BNP) (median = 35 pg/mL), hsC-reactive protein (median = 4.17 mg/L), and left ventricular ejection fraction (mean = 58%). Patients with acute MI or coronary syndrome (ACS) who required urgent revascularization were not included in the study. The primary endpoint, cardiovascular death, occurred in 41 patients during a median follow-up period of 14.9 months. Secondary endpoints-total mortality, acute ischaemic events (acute MI or ischaemic stroke), and the composite of death and acute ischaemic events-were observed in 52, 54, and 94 patients, respectively. Plasma aldosterone was found to be related to BMI, hypertension and NYHA class, and inversely related to age, creatinine clearance, and use of beta-blockers. Multivariate Cox model analysis demonstrated that aldosterone was independently associated with cardiovascular mortality (P = 0.001), total mortality (P = 0.001), acute ischaemic events (P = 0.01), and the composite of death and acute ischaemic events (P = 0.004). Reclassification analysis, using integrated discrimination improvement (IDI) and net reclassification improvement (NRI), demonstrated incremental predictive value of aldosterone (P < 0.0001).
Conclusion
Our results demonstrate that, in patients with CAD but without heart failure or acute MI, the level of aldosterone is strongly and independently associated with mortality and the occurrence of acute ischaemic events.
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