Sequencing of TGF-beta pathway genes in familial cases of intracranial aneurysm.

BACKGROUND AND PURPOSE: Familial aggregation of intracranial aneurysms (IA) strongly suggests a genetic contribution to pathogenesis. However, genetic risk factors have yet to be defined. For families affected by aortic aneurysms, specific gene variants have been identified, many affecting the receptors to transforming growth factor-beta (TGF-beta). In recent work, we found that aortic and intracranial aneurysms may share a common genetic basis in some families. We hypothesized, therefore, that mutations in TGF-beta receptors might also play a role in IA pathogenesis. METHODS: To identify genetic variants in TGF-beta and its receptors, TGFB1, TGFBR1, TGFBR2, ACVR1, TGFBR3, and ENG were directly sequenced in 44 unrelated patients with familial IA. Novel variants were confirmed by restriction digestion analyses, and allele frequencies were analyzed in cases versus individuals without known intracranial disease. Similarly, allele frequencies of a subset of known SNPs in each gene were also analyzed for association with IA. RESULTS: No mutations were found in TGFB1, TGFBR1, TGFBR2, or ACVR1. Novel variants identified in ENG (p.A60E) and TGFBR3 (p.W112R) were not detected in at least 892 reference chromosomes. ENG p.A60E showed significant association with familial IA in case-control studies (P=0.0080). No association with IA could be found for any of the known polymorphisms tested. CONCLUSIONS: Mutations in TGF-beta receptor genes are not a major cause of IA. However, we identified rare variants in ENG and TGFBR3 that may be important for IA pathogenesis in a subset of families.

Relationship between onset-to-door time and door-to-thrombolysis time: a pooled analysis of 10 dedicated stroke centers

BACKGROUND AND PURPOSE Inverse relationship between onset-to-door time (ODT) and door-to-needle time (DNT) in stroke thrombolysis was reported from various registries. We analyzed this relationship and other determinants of DNT in dedicated stroke centers. METHODS Prospectively collected data of consecutive ischemic stroke patients from 10 centers who received IV thrombolysis within 4.5 hours from symptom onset were merged (n=7106). DNT was analyzed as a function of demographic and prehospital variables using regression analyses, and change over time was considered. RESULTS In 6348 eligible patients with known treatment delays, median DNT was 42 minutes and kept decreasing steeply every year (P<0.001). Median DNT of 55 minutes was observed in patients with ODT ≤30 minutes, whereas it declined for patients presenting within the last 30 minutes of the 3-hour time window (median, 33 minutes) and of the 4.5-hour time window (20 minutes). For ODT within the first 30 minutes of the extended time window (181-210 minutes), DNT increased to 42 minutes. DNT was stable for ODT for 30 to 150 minutes (40-45 minutes). We found a weak inverse overall correlation between ODT and DNT (R(2)=-0.12; P<0.001), but it was strong in patients treated between 3 and 4.5 hours (R(2)=-0.75; P<0.001). ODT was independently inversely associated with DNT (P<0.001) in regression analysis. Octogenarians and women tended to have longer DNT. CONCLUSIONS DNT was decreasing steeply over the last years in dedicated stroke centers; however, significant oscillations of in-hospital treatment delays occurred at both ends of the time window. This suggests that further improvements can be achieved, particularly in the elderly.

Predictors of adverse events among patients undergoing primary percutaneous coronary intervention: insights from a pooled analysis of the COMFORTABLE AMI and EXAMINATION trials

Aims: The aim of this study was to identify predictors of adverse events among patients with ST-elevation myocardial infarction (STEMI) undergoing contemporary primary percutaneous coronary intervention (PCI). Methods and results: Individual data of 2,655 patients from two primary PCI trials (EXAMINATION, N=1,504; COMFORTABLE AMI, N=1,161) with identical endpoint definitions and event adjudication were pooled. Predictors of all-cause death or any reinfarction and definite stent thrombosis (ST) and target lesion revascularisation (TLR) outcomes at one year were identified by multivariable Cox regression analysis. Killip class III or IV was the strongest predictor of all-cause death or any reinfarction (OR 5.11, 95% CI: 2.48-10.52), definite ST (OR 7.74, 95% CI: 2.87-20.93), and TLR (OR 2.88, 95% CI: 1.17-7.06). Impaired left ventricular ejection fraction (OR 4.77, 95% CI: 2.10-10.82), final TIMI flow 0-2 (OR 1.93, 95% CI: 1.05-3.54), arterial hypertension (OR 1.69, 95% CI: 1.11-2.59), age (OR 1.68, 95% CI: 1.41-2.01), and peak CK (OR 1.25, 95% CI: 1.02-1.54) were independent predictors of all-cause death or any reinfarction. Allocation to treatment with DES was an independent predictor of a lower risk of definite ST (OR 0.35, 95% CI: 0.16-0.74) and any TLR (OR 0.34, 95% CI: 0.21-0.54). Conclusions: Killip class remains the strongest predictor of all-cause death or any reinfarction among STEMI patients undergoing primary PCI. DES use independently predicts a lower risk of TLR and definite ST compared with BMS. The COMFORTABLE AMI trial is registered at: http://www.clinicaltrials.gov/ct2/show/NCT00962416. The EXAMINATION trial is registered at: http://www.clinicaltrials.gov/ct2/show/NCT00828087.

Tracking a tuberculosis outbreak over 21 years: strain-specific single nucleotide polymorphism-typing combined with targeted whole genome sequencing.

BACKGROUND  Whole genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single nucleotide polymorphism (SNP)-typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland. METHODS  Based on genome sequences of three historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1,642 patient isolates, and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected. RESULTS  We identified 68 patients associated with the outbreak strain. Most were diagnosed in 1991-1995, but cases were observed until 2011. Two thirds belonged to the homeless and substance abuser milieu. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into three sub-clusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs. CONCLUSIONS  Strain-specific SNP-genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real-time and at high resolution.

Highlights in Analytical Chemistry in Switzerland: OMA & OPA - A Software Tool for Mass Spectrometric Sequencing of Nucleic Acids

Oligonucleotides comprising unnatural building blocks, which interfere with the translation machinery, have gained increased attention for the treatment of gene-related diseases (e.g. antisense, RNAi). Due to structural modifications, synthetic oligonucleotides exhibit increased biostability and bioavailability upon administration. Consequently, classical enzyme-based sequencing methods are not applicable to their sequence elucidation and verification. Tandem mass spectrometry is the method of choice for performing such tasks, since gas-phase dissociation is not restricted to natural nucleic acids. However, tandem mass spectrometric analysis can generate product ion spectra of tremendous complexity, as the number of possible fragments grows rapidly with increasing sequence length. The fact that structural modifications affect the dissociation pathways greatly increases the variety of analytically valuable fragment ions. The gas-phase dissociation of oligonucleotides is characterized by the cleavage of one of the four bonds along the phosphodiester chain, by the accompanying loss of nucleases, and by the generation of internal fragments due to secondary backbone cleavage. For example, an 18-mer oligonucleotide yields a total number of 272’920 theoretical fragment ions. In contrast to the processing of peptide product ion spectra, which nowadays is highly automated, there is a lack of tools assisting the interpretation of oligonucleotide data. The existing web-based and stand-alone software applications are primarily designed for the sequence analysis of natural nucleic acids, but do not account for chemical modifications and adducts. Consequently, we developed a software to support the interpretation of mass spectrometric data of natural and modified nucleic acids and their adducts with chemotherapeutic agents.

Prognostic implications of coronary calcification in patients with obstructive coronary artery disease treated by percutaneous coronary intervention: a patient-level pooled analysis of 7 contemporary stent trials

OBJECTIVE To investigate the long-term prognostic implications of coronary calcification in patients undergoing percutaneous coronary intervention for obstructive coronary artery disease. METHODS Patient-level data from 6296 patients enrolled in seven clinical drug-eluting stents trials were analysed to identify in angiographic images the presence of severe coronary calcification by an independent academic research organisation (Cardialysis, Rotterdam, The Netherlands). Clinical outcomes at 3-years follow-up including all-cause mortality, death-myocardial infarction (MI), and the composite end-point of all-cause death-MI-any revascularisation were compared between patients with and without severe calcification. RESULTS Severe calcification was detected in 20% of the studied population. Patients with severe lesion calcification were less likely to have undergone complete revascularisation (48% vs 55.6%, p<0.001) and had an increased mortality compared with those without severely calcified arteries (10.8% vs 4.4%, p<0.001). The event rate was also high in patients with severely calcified lesions for the combined end-point death-MI (22.9% vs 10.9%; p<0.001) and death-MI- any revascularisation (31.8% vs 22.4%; p<0.001). On multivariate Cox regression analysis, including the Syntax score, the presence of severe coronary calcification was an independent predictor of poor prognosis (HR: 1.33 95% CI 1.00 to 1.77, p=0.047 for death; 1.23, 95% CI 1.02 to 1.49, p=0.031 for death-MI, and 1.18, 95% CI 1.01 to 1.39, p=0.042 for death-MI- any revascularisation), but it was not associated with an increased risk of stent thrombosis. CONCLUSIONS Patients with severely calcified lesions have worse clinical outcomes compared to those without severe coronary calcification. Severe coronary calcification appears as an independent predictor of worse prognosis, and should be considered as a marker of advanced atherosclerosis.

Cloning, sequencing and partial functional characterization of the 5' region of the human p75 tumor necrosis factor receptor-encoding gene (TNF-R)

A 1887-bp region at the 5' flank of the human p75 tumor necrosis factor receptor (p75 TNF-R)-encoding gene was found to be active in driving expression of the luc (luciferase-encoding) reporter gene, suggesting that it contains the promoter for the receptor. Rather unexpectedly, a 1827-bp region at the 3' end of the first intron of the p75 TNF-R gene also displayed promoter activity. This activity may be artefactual, reflecting only the presence of an enhancer in this region; yet it also raises the possibility that p75 TNF-R is controlled by more than one promoter and that it encodes various forms of the receptor, or even other proteins. We present here the nucleotide sequences of the 5' flanking and intron regions. Possible implications for the transcriptional regulation of the p75 TNF-R gene are discussed.

cDNA Library Generation for the Analysis of Small RNAs by High-Throughput Sequencing

The RNome of a cell is highly diverse and consists besides messenger RNAs (mRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) also of other small and long transcript entities without apparent coding potential. This class of molecules, commonly referred to as non-protein-coding RNAs (ncRNAs), is involved in regulating numerous biological processes and thought to contribute to cellular complexity. Therefore, much effort is put into their identification and further functional characterization. Here we provide a cost-effective and reliable method for cDNA library construction of small RNAs in the size range of 20-500 residues. The effectiveness of the described method is demonstrated by the analysis of ribosome-associated small RNAs in the eukaryotic model organism Trypanosoma brucei.

Tramadol and o-desmethyl tramadol clearance maturation and disposition in humans: a pooled pharmacokinetic study.

BACKGROUND AND OBJECTIVES We aimed to study the impact of size, maturation and cytochrome P450 2D6 (CYP2D6) genotype activity score as predictors of intravenous tramadol disposition. METHODS Tramadol and O-desmethyl tramadol (M1) observations in 295 human subjects (postmenstrual age 25 weeks to 84.8 years, weight 0.5-186 kg) were pooled. A population pharmacokinetic analysis was performed using a two-compartment model for tramadol and two additional M1 compartments. Covariate analysis included weight, age, sex, disease characteristics (healthy subject or patient) and CYP2D6 genotype activity. A sigmoid maturation model was used to describe age-related changes in tramadol clearance (CLPO), M1 formation clearance (CLPM) and M1 elimination clearance (CLMO). A phenotype-based mixture model was used to identify CLPM polymorphism. RESULTS Differences in clearances were largely accounted for by maturation and size. The time to reach 50 % of adult clearance (TM50) values was used to describe maturation. CLPM (TM50 39.8 weeks) and CLPO (TM50 39.1 weeks) displayed fast maturation, while CLMO matured slower, similar to glomerular filtration rate (TM50 47 weeks). The phenotype-based mixture model identified a slow and a faster metabolizer group. Slow metabolizers comprised 9.8 % of subjects with 19.4 % of faster metabolizer CLPM. Low CYP2D6 genotype activity was associated with lower (25 %) than faster metabolizer CLPM, but only 32 % of those with low genotype activity were in the slow metabolizer group. CONCLUSIONS Maturation and size are key predictors of variability. A two-group polymorphism was identified based on phenotypic M1 formation clearance. Maturation of tramadol elimination occurs early (50 % of adult value at term gestation).

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef)

Background: Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. Results: The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. Conclusions: It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Chromosomally and extrachromosomally mediated high-level gentamicin resistance in Streptococcus agalactiae

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in non-pregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to loss of a synergistic effect. We therefore performed a multicentre study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centres in four countries, 1128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. Though, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon, designated Tn3706. In the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing a plasmid mediated HLGR in GBS. Thus, in our clinical GBS isolates HLGR is mediated both chromosomally and extrachromosomally.