Phospho-RNA sequencing with circAID-p-seq.

Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3' end. Unfortunately, current library preparation protocols rely only on a 3' hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3' phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3'-phospho RNA molecules.

One-shot analysis of translated mammalian lncRNAs with AHARIBO.

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.

SMN-primed ribosomes modulate the translation of transcripts related to spinal muscular atrophy.

The contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains unclear. Here, we demonstrate that survival motor neuron (SMN) protein-the loss of which causes the neuromuscular disease spinal muscular atrophy (SMA)-binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs that are enriched for translational enhancer sequences in the 5' untranslated region (UTR) and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins that are involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs encoding proteins that are relevant to SMA pathogenesis.

A long noncoding RNA acts as a post-transcriptional regulator of heat shock protein (HSP70) synthesis in the cold hardy Diamesa tonsa under heat shock.

Cold stenothermal insects living in glacier-fed streams are stressed by temperature variations resulting from glacial retreat during global warming. The molecular aspects of insect response to environmental stresses remain largely unexplored. The aim of this study was to expand our knowledge of how a cold stenothermal organism controls gene expression at the transcriptional, translational, and protein level under warming conditions. Using the chironomid Diamesa tonsa as target species and a combination of RACE, qPCR, polysomal profiling, western blotting, and bioinformatics techniques, we discovered a new molecular pathway leading to previously overlooked adaptive strategies to stress. We obtained and characterized the complete cDNA sequences of three heat shock inducible 70 (hsp70) and two members of heat-shock cognate 70 (hsc70). Strikingly, we showed that a novel pseudo-hsp70 gene encoding a putative long noncoding RNA (lncRNA) which is transcribed during thermal stress, acting as a ribosome sponge to provide post-transcriptional control of HSP70 protein levels. The expression of the pseudo-hsp70 gene and its function suggest the existence of a new and unexpected mechanism to cope with thermal stress: lowering the pace of protein production to save energy and optimize resources for recovery.

How do cardiologists select patients for dual antiplatelet therapy continuation beyond 1 year after a myocardial infarction? Insights from the EYESHOT Post-MI Study.

BACKGROUND: Current guidelines suggest to consider dual antiplatelet therapy (DAPT) continuation for longer than 12 months in selected patients with myocardial infarction (MI). HYPOTHESIS: We sought to assess the criteria used by cardiologists in daily practice to select patients with a history of MI eligible for DAPT continuation beyond 1 year. METHODS: We analyzed data from the EYESHOT Post-MI, a prospective, observational, nationwide study aimed to evaluate the management of patients presenting to cardiologists 1 to 3 years from the last MI event. RESULTS: Out of the 1633 post-MI patients enrolled in the study between March and December 2017, 557 (34.1%) were on DAPT at the time of enrolment, and 450 (27.6%) were prescribed DAPT after cardiologist assessment. At multivariate analyses, a percutaneous coronary intervention (PCI) with multiple stents and the presence of peripheral artery disease (PAD) resulted as independent predictors of DAPT continuation, while atrial fibrillation was the only independent predictor of DAPT interruption for patients both at the second and the third year from MI at enrolment and the time of discharge/end of the visit. CONCLUSIONS: Risk scores recommended by current guidelines for guiding decisions on DAPT duration are underused and misused in clinical practice. A PCI with multiple stents and a history of PAD resulted as the clinical variables more frequently associated with DAPT continuation beyond 1 year from the index MI.

Active Ribosome Profiling with RiboLace.

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

riboWaltz: Optimization of ribosome P-site positioning in ribosome profiling data.

Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine the trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of ribosome profiling data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies. riboWaltz was implemented in R and is available as an R package at https://github.com/LabTranslationalArchitectomics/RiboWaltz.

In Vivo Translatome Profiling in Spinal Muscular Atrophy Reveals a Role for SMN Protein in Ribosome Biology.

Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration.

DNA damage and translational response during detoxification from copper exposure in a wild population of Chironomus riparius.

Copper is one of the predominant components of pesticides employed in agriculture and known to be highly toxic once it reaches aquatic organisms. The impact of sublethal concentrations of this metal on wild insects is not yet completely understood. Studies addressing alterations in different levels of gene expression are still lacking. We previously demonstrated that in a wild population of Chironomus riparius, HSP and CYP families of genes were up-regulated at the transcriptional level after copper exposure. Here, we analyse the impact of copper at the genomic, translational and protein functional level, obtaining a comprehensive picture of the molecular reply to this metal. We studied genotoxicity in C. riparius larvae by Comet Assay, the translational response by polysomal profiling and the detoxification capacity by the CYP450 enzymes activity. Fourth-instar larvae from a mountain stream polluted by agricultural land run-off (NE-Italy) were exposed for 3 h copper concentrations ≤ LC50. We report DNA damage induced by copper, even at sublethal levels, as demonstrated by significant increases in all the comet parameters at concentrations ≥1 mg L-1. By estimating the transcript-specific translational efficiency, we observe a specific up-regulation of CYP4G. Furthermore, the enzymatic activity of CYP450 enzymes is increased at all sublethal copper concentrations, confirming the role of this protein family in the detoxification processes. Surprisingly, the HSP transcripts are up-regulated at the transcriptional level, but these changes are buffered at the translational level suggesting the existence of still unknown post-transcriptional controls that may be connected to survival processes.

Antiplatelet therapy and outcome in patients undergoing surgery following coronary stenting: Results of the surgery after stenting registry.

OBJECTIVES: The aim of the present study was to define the feasibility and clinical impact of complying with national consensus recommendations on perioperative management of antiplatelet therapy in patients with coronary stents undergoing cardiac and noncardiac surgery. BACKGROUND: There are limited evidence-based recommendations on the perioperative management of antiplatelet therapy in stented patients undergoing surgery. METHODS: The recommendations provided by the national consensus document were applied in a multicenter, prospective registry of consecutive patients with prior coronary stenting undergoing any type of surgery at 19 hospitals in Italy. The primary end-point was in-hospital net adverse clinical events (NACE) represented by the composite of all-cause death, myocardial infarction, probable/definite stent thrombosis and Bleeding Academic Research Consortium (BARC) grade ≥3 bleeding. Patients were followed for 30 days. RESULTS: A total of 1,082 patients were enrolled. Adherence to consensus recommendations occurred in 85% of the cases. Perioperative aspirin and dual antiplatelet therapy were maintained in 69.7 and 10.5% of the cases, respectively. In-hospital NACE rate was 12.7%, being significantly higher in patients undergoing cardiac surgery (36.3% vs. 7.3%, P < 0.01), mainly due to BARC 3 bleeding events (32.3%). At 30 days, MACE rates were similar (3.5% vs. 3.5%, P = NS) in patients undergoing cardiac and noncardiac surgery, whereas BARC ≥3 bleeding events were significantly higher with cardiac surgery (36.3% vs. 5.6%, P < 0.01). CONCLUSIONS: The results of this registry demonstrate the safety and feasibility of applying a national consensus document on the perioperative management of antiplatelet therapy in stented patients undergoing surgery. © 2016 Wiley Periodicals, Inc.

Thermal survival limits of young and mature larvae of a cold stenothermal chironomid from the Alps (Diamesinae: Pseudodiamesa branickii [Nowicki, 1873]).

The threats posed by climate change make it important to expand knowledge concerning cold and heat tolerance in stenothermal species from habitats potentially threatened by temperature changes. Thermal limits and basal metabolism variations were investigated in Pseudodiamesa branickii (Diptera: Chironomidae) under thermal stress between -20 and 37 °C. Supercooling point (SCP), lower (LLTs) and upper lethal temperatures (ULTs), and oxygen consumption rate were measured in overwintering young (1st and 2nd instar) and mature (3rd and 4th instar) larvae from an Alpine glacier-fed stream. Both young and mature larvae were freezing tolerant (SCPs = -7.1 °C and -6.4 °C, respectively; LLT100 -20 °C) and thermotolerant (ULT50 = 31.7 ± 0.4, 32.5 ± 0.3, respectively). However, ontogenetic differences in acute tolerance were observed. The LLT50 calculated for the young larvae (= -7.4 °C) was almost equal to their SCP (= -7.1 °C) and the overlapping of the proportion of mortality curve with the CPIF curve highlighted that the young larvae are borderline between freezing tolerance and freezing avoidance. Furthermore, a lower ULT100 in the young larvae (of ca. 1 °C), suggests that they are less thermotolerant than mature larvae. Finally, young larvae exhibit a higher oxygen consumption rate (mgO2 /gAFDM/h) at any temperature tested and are overall less resistant to oxygen depletion compared to mature larvae at ≥10 °C. These findings suggest that mature larvae enter into a dormant state by lowering their basal metabolism until environmental conditions improve in order to save energy for life cycle completion during stressful conditions.

Peering at Brain Polysomes with Atomic Force Microscopy.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

Transcriptional profiling induced by pesticides employed in organic agriculture in a wild population of Chironomus riparius under laboratory conditions.

Copper (Cu) and azadirachtin (AZA-A+B) are pesticides allowed in organic agriculture whose environmental risk and toxicity for aquatic wildlife is only partially known. Reverse Transcription Polymerase Chain Reaction was used to assess the molecular effect of acute and short-term exposure (3, 24h) of Cu (0.01, 0.05, 1, 10, 25mgl(-1)) and AZA-A+B (0.2, 0.3, 0.4, 0.5, 1mgl(-1)) on the expression of five candidate genes (hsp70, hsc70, hsp40, hsp10 and cyP450) in a non-target species, Chironomus riparius. Fourth-instar larvae were collected from a mountain stream polluted by agricultural land run-off. All genes were responsive to both pesticides but each gene had a specific response to the different experimental concentrations and exposure times. A few similarities in transcriptional profiling were observed, such as a linear concentration-dependent response of hsp70 after 24h of exposure (at ≥1mgl(-1) of Cu and ≥0.2mgl(-1) of AZA-A+B) and an up-regulation regardless of the concentration of hsc70 after 24h of exposure (at ≥0mgl(-1) of Cu and ≥0.2mgl(-1) of AZA-A+B and the up-regulation of hsp70 after 3h of exposure at ~LC50 (Cu-LC50=26.1±2.5mgl(-1), AZA-A+B-LC50=1.1±0.2mgl(-1)). According to the results, hsp40, hsp10 and cyP450 may be defined as pesticide-dependent (i.e., hsp40 and hsp10 seemed to responded mainly to AZA-A+B and cyP450 to Cu), while hsc70 as time-dependent regardless of the pesticide (i.e., hsc70 responded only after 24h of treatment with Cu and AZA-A+B). This study gives new insights on the potential role of the C. riparius's hsps and cyP450 genes as sensitive biomarkers for freshwater monitoring.

Global translation variations in host cells upon attack of lytic and sublytic Staphylococcus aureus α-haemolysin.

Genome-wide analyses of translation can provide major contributions in our understanding of the complex interplay between virulent factors and host cells. So far, the activation of host translational control mechanisms by bacterial toxins, owing to specific recruitment of mRNAs, RNA-binding proteins (RBPs) and ncRNAs (non-coding RNAs), are far from being understood. In the present study, we characterize for the first time the changes experienced by the translational control system of host cells in response to the well-known Staphylococcus aureus α-haemolysin (AHL) under both sublytic and lytic conditions. By comparing variations occurring in the cellular transcriptome and translatome, we give evidence that global gene expression is primarily rewired at the translational level, with the contribution of the RBP ELAVL1 (HuR) in the sublytic response. These results reveal the importance of translational control during host-pathogen interaction, opening new approaches for AHL-induced diseases.

Studying translational control in non-model stressed organisms by polysomal profiling.

In stressed organisms, strategic proteins are selectively translated even if the global process of protein synthesis is compromised. The determination of protein concentrations in tissues of non-model organisms (thus with limited genomic information) is challenging due to the absence of specific antibodies. Moreover, estimating protein levels quantifying transcriptional responses may be misleading, because translational control mechanisms uncouple protein and mRNAs abundances. Translational control is increasingly recognized as a hub where regulation of gene expression converges to shape proteomes, but it is almost completely overlooked in molecular ecology studies. An interesting approach to study translation and its control mechanisms is the analysis of variations of gene-specific translational efficiencies by quantifying mRNAs associated to ribosomes. In this paper, we propose a robust and streamlined pipeline for purifying ribosome-associated mRNAs and calculating global and gene-specific translation efficiencies from non-model insect's species. This method might found applications in molecular ecology to study responses to environmental stressors in non-model organisms.

Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes.

Translation is increasingly recognized as a central control layer of gene expression in eukaryotic cells. The overall organization of mRNA and ribosomes within polysomes, as well as the possible role of this organization in translation are poorly understood. Here we show that polysomes are primarily formed by three distinct classes of ribosome assemblies. We observe that these assemblies can be connected by naked RNA regions of the transcript. We show that the relative proportions of the three classes of ribosome assemblies reflect, and probably dictate, the level of translational activity. These results reveal the existence of recurrent supra-ribosomal building blocks forming polysomes and suggest the presence of unexplored translational controls embedded in the polysome structure.

Thermal stress induces HSP70 proteins synthesis in larvae of the cold stream non-biting midge Diamesa cinerella Meigen.

Laboratory experiments on the cold stenothermal midge Diamesa cinerella (Diptera, Chironomidae) were performed to study the relationship between increasing temperature and heat shock proteins (HSP70) expression at translational level (Western blotting). Thermotolerance of IV instar larvae collected in nature at 1.5-4.3°C during seasons was analyzed through short-term (1 h at ten different temperatures from 26°C to 35°C) and long-term (1-14 h at 26°C and 1-4 h at 32°C) heat shocks. A high thermotolerance was detected (LT50=30.9-32.8°C and LT100=34.0-37.8°C). However, survival decreased consistently with increasing exposure time, especially at higher temperature (LTime50=7.64 h at 26°C and LTime50=1.73 h at 32°C). The relationship between such heat resistance and HSP70 expression appeared evident because a relationship between HSP70 level and larval survival rate was generally found. A heat shock response (HSR) was consistent only in the summer larvae. The absence of HSR in the other populations coupled with even higher amounts of HSP70 than in summer, led us to hypothesize that other macromolecules and other adaptive mechanisms, apart from biochemical ones, are involved in the response of D. cinerella larvae to high temperature. Altogether these results stressed how in this midge the HSP70 protein family confers resistance against cold, being detected under natural conditions in control larvae collected in all seasons, but also against warm under experimental heat shocks. These results give new insights into possible responses to climate changes in freshwater insects within the context of global warming.

Clinical populations of Pseudomonas aeruginosa isolated from acute infections show a wide virulence range partially correlated with population structure and virulence gene expression.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium responsible for a variety of infections in humans, as well as in animal hosts. While the evolution of virulence in P. aeruginosa strains isolated from chronic lung infection in cystic fibrosis (CF) patients has been extensively studied, the virulence phenotype of P. aeruginosa isolated from other infection types or from the environment is currently not well characterized. Here we report an extensive analysis of the virulence of P. aeruginosa strains isolated from acute infections compared with population structure. Virulence profiles of individual strains were also compared with the expression levels of the rhlR gene, the transcriptional regulator of the rhl quorum-sensing system, and the gene encoding Crc, a global regulator controlling catabolite repression and carbon metabolism. Additionally, the presence/absence of the two mutually exclusive genes, exoU and exoS, encoding effectors of the type III secretion system, was assessed. In order to capture the widest range of genetic variability, a collection of 120 clinical strains was initially characterized by repetitive element-based PCR genotyping, and a selection of 27 strains belonging to different clonal lineages was subsequently tested using three different virulence assays, including two Dictyostelium discoideum assays on different growth media, and a Caenorhabditis elegans fast-killing assay. We show that the parallel application of virulence assays can be used to quantitatively assess this complex, multifactorial phenotypic trait. We observed a wide spectrum of virulence phenotypes ranging from weakly to highly aggressive, indicating that clinical strains isolated from acute infections can present a reduced or altered virulence phenotype. Genotypic associations only partially correlated with virulence profiles and virulence gene expression, whereas the presence of either exoU or exoS was not significantly correlated with virulence. Interestingly, the expression of rhlR showed a significant and positive correlation with the virulence profiles obtained with the three assays, while the expression of crc was either negatively or not correlated with virulence, depending on the assay.

Thermotolerance and hsp70 heat shock response in the cold-stenothermal chironomid Pseudodiamesa branickii (NE Italy).

To better understand the physiological capability of cold-stenothermal organisms to survive high-temperature stress, we analyzed the thermotolerance limits and the expression level of hsp70 genes under temperature stress in the alpine midge Pseudodiamesa branickii (Diptera Chironomidae). A lethal temperature (LT(100)) of 36°C and a lethal temperature 50% (LT(50)) of 32.2°C were found for the cold-stenothermal larvae after short-term shocks (1 h). Additional experiments revealed that the duration of the exposure negatively influenced survival, whereas a prior exposure to a less severe high temperature generated an increase in survival. To investigate the molecular basis of this high thermotolerance, the expression of the hsp70 gene family was surveyed via semi-quantitative reverse transcription-polymerase chain reaction analysis in treated larvae. The constitutive (hsc70) and inducible (hsp70) forms were both analyzed. Larvae of P. branickii showed a significant up-regulation of inducible hsp70 gene with increasing temperatures and an over-expression of both hsp70 and hsc70 by increasing the time of exposure. Different from that was shown in many cold-stenothermal Antarctic organisms, P. branickii was able to activate hsp70 genes transcription (equal to heat shock response) in response to thermal stress. Finally, the unclear relationship between hsp70 expression and survival led us to surmise that genes other than hsp70 and other processes apart from the biochemical processes might generate the high thermaltolerance of P. branickii larvae. These results and future high-throughput studies at both the transcriptome and proteome level will improve our ability to predict the future geographic distribution of this species within the context of global warming.

Syncope and risk of sudden death in hypertrophic cardiomyopathy.

BACKGROUND: The prognostic significance of syncope has not been investigated systematically in hypertrophic cardiomyopathy, and treatment strategies have been based largely on intuition and experience. METHODS AND RESULTS: We assessed the relationship between syncope and sudden death in 1511 consecutive patients with hypertrophic cardiomyopathy. Unexplained (n=153) or neurally mediated (n=52) syncope occurred in 205 patients (14%). Over a 5.6+/-5.2-year follow-up, 74 patients died suddenly. Relative risk of sudden death was 1.78 (95% confidence interval 0.88 to 3.51, P=0.08) in patients with unexplained syncope and 0.91 (95% confidence interval 0.00 to 3.83, P=1.0) in those with neurally mediated syncope compared with patients without syncope. In multivariable analysis, the temporal proximity of unexplained syncope to initial patient evaluation was independently associated with risk of sudden death (P=0.006). Patients with unexplained syncope within 6 months before the initial evaluation showed a 5-fold increase in risk compared with patients without syncope (adjusted hazard ratio 4.89, 95% confidence interval 2.19 to 10.94), a relationship that was maintained throughout all age groups (<18, 18 to 39, and > or =40 years). Older patients (> or =40 years of age) with remote episodes of syncope (>5 years before initial evaluation) did not show an increased risk of sudden death (adjusted hazard ratio 0.38, 95% confidence interval 0.05 to 2.74). CONCLUSIONS: In the present large cohort of patients with hypertrophic cardiomyopathy, unexplained syncope was a risk factor for sudden death. Patients with syncopal events that occurred in close temporal proximity to the initial evaluation showed a substantially higher risk of sudden death than patients without syncope. Older patients with remote syncopal events did not show an increased risk.