Amplification ratio control system for copy number variation genotyping

We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP′) in each strand form a hairpin. The bases immediately beyond the 3′-end of the UP and 5′ of UP′ are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3′-ends of amplicon strands. The resultant ‘amplification ratio control system’ (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and ‘chemokine (C-C motif) ligand 3-like 1’ gene

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

Temporal Network Based Analysis of Cell Specific Vein Graft Transcriptome Defines Key Pathways and Hub Genes in Implantation Injury

Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12–24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention

Antiplatelet therapy with aspirin, clopidogrel, and dipyridamole versus clopidogrel alone or aspirin and dipyridamole in patients with acute cerebral ischaemia (TARDIS): a randomised, open-label, phase 3 superiority trial

Background: Intensive antiplatelet therapy with three agents might be more effective than guideline treatment for preventing recurrent events in patients with acute cerebral ischaemia. We aimed to compare the safety and efficacy of intensive antiplatelet therapy (combined aspirin, clopidogrel, and dipyridamole) with that of guideline-based antiplatelet therapy. Methods: We did an international, prospective, randomised, open-label, blinded-endpoint trial in adult participants with ischaemic stroke or transient ischaemic attack (TIA) within 48 h of onset. Participants were assigned in a 1:1 ratio using computer randomisation to receive loading doses and then 30 days of intensive antiplatelet therapy (combined aspirin 75 mg, clopidogrel 75 mg, and dipyridamole 200 mg twice daily) or guideline-based therapy (comprising either clopidogrel alone or combined aspirin and dipyridamole). Randomisation was stratified by country and index event, and minimised with prognostic baseline factors, medication use, time to randomisation, stroke-related factors, and thrombolysis. The ordinal primary outcome was the combined incidence and severity of any recurrent stroke (ischaemic or haemorrhagic; assessed using the modified Rankin Scale) or TIA within 90 days, as assessed by central telephone follow-up with masking to treatment assignment, and analysed by intention to treat. This trial is registered with the ISRCTN registry, number ISRCTN47823388. Findings: 3096 participants (1556 in the intensive antiplatelet therapy group, 1540 in the guideline antiplatelet therapy group) were recruited from 106 hospitals in four countries between April 7, 2009, and March 18, 2016. The trial was stopped early on the recommendation of the data monitoring committee. The incidence and severity of recurrent stroke or TIA did not differ between intensive and guideline therapy (93 [6%] participants vs 105 [7%]; adjusted common odds ratio [cOR] 0·90, 95% CI 0·67–1·20, p=0·47). By contrast, intensive antiplatelet therapy was associated with more, and more severe, bleeding (adjusted cOR 2·54, 95% CI 2·05–3·16, p<0·0001). Interpretation: Among patients with recent cerebral ischaemia, intensive antiplatelet therapy did not reduce the incidence and severity of recurrent stroke or TIA, but did significantly increase the risk of major bleeding. Triple antiplatelet therapy should not be used in routine clinical practice

The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead