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Background: There is a lack of randomized controlled trial (RCT) data comparing outcomes of different catheter-based interventions for intermediate-risk pulmonary embolism (PE)., Methods: PEERLESS is a prospective, multicenter, RCT that enrolled 550 intermediate-risk PE patients with right ventricular dilatation and additional clinical risk factors randomized 1:1 to treatment with large-bore mechanical thrombectomy (LBMT) or catheter-directed thrombolysis (CDT). The primary endpoint was a hierarchal win ratio (WR) composite of the following: 1) all-cause mortality, 2) intracranial hemorrhage, 3) major bleeding, 4) clinical deterioration and/or escalation to bailout, and 5) postprocedural intensive care unit (ICU) admission and length of stay, assessed at the sooner of hospital discharge or 7 days post-procedure. Assessments at the 24-hour visit included respiratory rate, mMRC dyspnea score, NYHA classification, right ventricle (RV)/left ventricle (LV) ratio reduction, and RV function. Endpoints through 30 days included total hospital stay, all-cause readmission, and all-cause mortality., Results: The primary endpoint occurred significantly less frequently with LBMT vs CDT (WR 5.01 [95% CI: 3.68-6.97]; P <0.001). There were significantly fewer episodes of clinical deterioration and/or bailout (1.8% vs 5.4%; P =0.04) with LBMT vs CDT and less postprocedural ICU utilization ( P <0.001), including admissions (41.6% vs 98.6%) and stays >24 hours (19.3% vs 64.5%). There was no significant difference in mortality, intracranial hemorrhage, or major bleeding between strategies, nor in a secondary WR endpoint including the first 4 components (WR 1.34 [95% CI: 0.78-2.35]; P =0.30). At the 24-hour visit, respiratory rate was lower for LBMT patients (18.3±3.3 vs 20.1±5.1; P <0.001) and fewer had moderate to severe mMRC dyspnea scores (13.5% vs 26.4%; P <0.001), NYHA classifications (16.3% vs 27.4%; P =0.002), and RV dysfunction (42.1% vs 57.9%; P =0.004). RV/LV ratio reduction was similar (0.32±0.24 vs 0.30±0.26; P =0.55). LBMT patients had shorter total hospital stays (4.5±2.8 vs 5.3±3.9 overnights; P =0.002) and fewer all-cause readmissions (3.2% vs 7.9%; P =0.03), while 30-day mortality was similar (0.4% vs 0.8%; P =0.62)., Conclusions: PEERLESS met its primary endpoint in favor of LBMT vs CDT in treatment of intermediate-risk PE. LBMT had lower rates of clinical deterioration and/or bailout and postprocedural ICU utilization compared with CDT, with no difference in mortality or bleeding.

CD4 + T cells play a crucial role in adaptive immune responses and have been implicated in the pathogenesis of autoimmune diseases (ADs). Despite numerous studies, the molecular mechanisms underlying T cell dysregulation in ADs remain incompletely understood. Here, we used chromatin immunoprecipitation (ChIP)-sequencing of active chromatin and transcriptomic data from CD4 + T cells of healthy donors and patients with systemic lupus erythematosus (SLE), psoriasis, juvenile idiopathic arthritis (JIA), and Graves' disease to investigate the role of enhancers in AD pathogenesis. By generating enhancer-based gene regulatory networks (eGRNs), we identified disease-specific dysregulated pathways and potential downstream target genes of enhancers harboring AD-associated single-nucleotide polymorphisms (SNPs), which we also validated using chromatin-capture (HiC) data and CRISPR interference (CRISPRi) in primary CD4 + T cells. Our results suggest that alterations in the regulatory landscapes of CD4 + T cells, including enhancers, contribute to the development of ADs and provide a basis for developing new therapeutic approaches., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Enhancers play a vital role in gene regulation and are critical in mediating the impact of noncoding genetic variants associated with complex traits. Enhancer activity is a cell-type-specific process regulated by transcription factors (TFs), epigenetic mechanisms and genetic variants. Despite the strong mechanistic link between TFs and enhancers, we currently lack a framework for jointly analysing them in cell-type-specific gene regulatory networks (GRN). Equally important, we lack an unbiased way of assessing the biological significance of inferred GRNs since no complete ground truth exists. To address these gaps, we present GRaNIE (Gene Regulatory Network Inference including Enhancers) and GRaNPA (Gene Regulatory Network Performance Analysis). GRaNIE (https://git.embl.de/grp-zaugg/GRaNIE) builds enhancer-mediated GRNs based on covariation of chromatin accessibility and RNA-seq across samples (e.g. individuals), while GRaNPA (https://git.embl.de/grp-zaugg/GRaNPA) assesses the performance of GRNs for predicting cell-type-specific differential expression. We demonstrate their power by investigating gene regulatory mechanisms underlying the response of macrophages to infection, cancer and common genetic traits including autoimmune diseases. Finally, our methods identify the TF PURA as a putative regulator of pro-inflammatory macrophage polarisation., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Competing Interests: Competing interests: None declared.

Postmenopausal women are at increased risk of osteoporosis. Osteoporotic fractures carry an increased risk of morbidity and mortality. Denosumab is a monoclonal antibody widely used for the treatment of osteoporosis by inhibiting osteoclast-induced bone resorption. Hypocalcaemia is a known side-effect of denosumab treatment. The majority of such cases have been described in patients with underlying metastatic cancer or chronic kidney disease. We present a patient who developed severe hypocalcaemia after administration of denosumab in the context of severe vitamin D deficiency and a normal kidney function. The management was further complicated by hypophosphatemia. Following replacement of vitamin D, the patient's calcium and phosphate levels stabilised. The patient required intensive care monitoring for replacement of electrolytes. This case report emphasises the importance of screening and ongoing monitoring of risk factors for iatrogenic hypocalcaemia with denosumab treatment., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Perturbation of gene expression by means of synthetic small interfering RNAs (siRNAs) is a powerful way to uncover gene function. However, siRNA technology suffers from sequence-specific off-target effects and from limitations in knock-down efficiency. In this study, we assess a further problem: unintended effects of siRNA transfections on cellular fitness/proliferation. We show that the nucleotide compositions of siRNAs at specific positions have reproducible growth-restricting effects on mammalian cells in culture. This is likely distinct from hybridization-dependent off-target effects, since each nucleotide residue is seen to be acting independently and additively. The effect is robust and reproducible across different siRNA libraries and also across various cell lines, including human and mouse cells. Analyzing the growth inhibition patterns in correlation to the nucleotide sequence of the siRNAs allowed us to build a predictor that can estimate growth-restricting effects for any arbitrary siRNA sequence. Competition experiments with co-transfected siRNAs further suggest that the growth-restricting effects might be linked to an oversaturation of the cellular miRNA machinery, thus disrupting endogenous miRNA functions at large. We caution that competition between siRNA molecules could complicate the interpretation of double-knockdown or epistasis experiments, and potential interactions with endogenous miRNAs can be a factor when assaying cell growth or viability phenotypes.

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.

Background: Currently, American Diabetes Association guidelines suggest statin use among persons with diabetes mellitus aged >40 years. The presence of calcified plaque in coronary arteries is a sensitive surrogate of coronary artery disease and has been shown to be an independent predictor of mortality and cardiac events., Objective: We aimed to assess the prevalence and severity of calcified plaque in coronary arteries in patients aged <40 years with and without diabetes., Methods: We included 3723 asymptomatic patients aged <40 years who had undergone coronary calcium scanning. Clinical and demographic data were collected. Agatston score was categorized into Agatston score 0 as normal, 1 to 99 as low, 100 to 399 as intermediate, and ≥400 as severe; and statistical analysis was performed., Results: The study population consisted of 4% persons with diabetes (n = 142) and 56% men with a mean age of 35 ± 5 years. Young persons with diabetes had greater prevalence of Agatston score > 0 than persons without diabetes (43% vs 24%; P < .0001). In addition, 12% of persons with diabetes vs 2.5% of persons without diabetes had an Agatston score ≥ 100 (P < .0001). The prevalence of calcified plaque in coronary arteries was >50% in persons with diabetes aged >35 years. After taking into account risk factors, the presence of diabetes was associated with a 4-fold higher odds of an Agatston score ≥ 100 (odds ratio, 4.19; 95% CI, 2.29-7.65; P < .0001)., Conclusion: Our study found that 43% of young patients with diabetes have detectable coronary atherosclerosis. Given the known clinical implications of calcified plaque in coronary arteries, future studies are needed to evaluate interventions in persons aged <40 years who exhibit subclinical atherosclerosis to reduce future cardiovascular disease events in this vulnerable population., (Copyright © 2013 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.)

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome., (Copyright © 2011 Elsevier B.V. All rights reserved.)