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Small nuclear RNAs (snRNAs) combine with specific proteins to generate small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. U4 snRNA forms a duplex with U6 and, together with U5, contributes to the tri-snRNP spliceosomal complex. Variants in RNU4-2, which encodes U4, have recently been implicated in neurodevelopmental disorders. Here we show that heterozygous inherited and de novo variants in RNU4-2 and in four RNU6 paralogs (RNU6-1, RNU6-2, RNU6-8 and RNU6-9), which encode U6, recur in individuals with nonsyndromic retinitis pigmentosa (RP), a genetic disorder causing progressive blindness. These variants cluster within the three-way junction of the U4/U6 duplex, a site that interacts with tri-snRNP splicing factors also known to cause RP (PRPF3, PRPF8, PRPF31), and seem to affect snRNP biogenesis. Based on our cohort, deleterious variants in RNU4-2 and RNU6 paralogs may explain up to ~1.4% of otherwise undiagnosed RP cases. This study highlights the contribution of noncoding RNA genes to Mendelian disease and reveals pleiotropy in RNU4-2, where distinct variants underlie neurodevelopmental disorder and retinal degeneration.

Vascular smooth muscle cell (VSMC) diversification drives atherosclerotic coronary artery disease (CAD). Mechanisms governing these cell state transitions remain unclear. We applied multiomic single-cell profiling, epitope mapping, and spatial transcriptomics across 27 human coronary arteries, identifying fibroblast activation protein (FAP) as a marker of modulated VSMCs. Lineage tracing in mice indicated that FAP+ cells originate from Myh11+ VSMCs, and FAP PET imaging in CAD patients showed plaque uptake. FAP+ cells states resided in the macrophage-rich neo-intima. Therapeutically, we developed an anti-FAP bispecific T-cell engager, which reduced plaque burden and remodeled the stromal-immune microenvironment through T-cell clonal expansion. Our study delivers a single-cell and spatial atlas of human CAD, establishes FAP as a marker of modulated VSMCs, and highlights immunotherapy for lipid-independent targets.

Importance:Intensive lowering of low-density lipoprotein cholesterol (LDL-C) levels with PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors for cardiovascular event reduction has largely been reserved for patients with significant atherosclerosis. Objective:To investigate whether evolocumab could prevent a first major cardiovascular event (MACE) in patients without known significant atherosclerosis. Design, Setting, and Participants:VESALIUS-CV was a randomized, double-blind, placebo-controlled trial of evolocumab conducted across 774 sites in 33 countries and enrolling 12 257 patients with no prior myocardial infarction or stroke, LDL-C level 90 mg/dL or greater, and qualifying atherosclerosis or high-risk diabetes. This prespecified subgroup analysis examined outcomes in patients without known significant atherosclerosis (none of the following: prior arterial revascularization, arterial stenosis ≥50%, or coronary artery calcium score ≥100 Agatston units), all of whom had diabetes. Enrollment started in June 2019 and the last patient visit was July 2025, with a median follow-up of 4.8 years. Intervention:Patients were randomized in a 1:1 ratio to subcutaneous administration of either evolocumab (140 mg every 2 weeks) or matching placebo added to optimally tolerated statin therapy. Main Outcomes and Measures:The dual primary end points were composites of coronary heart disease death, myocardial infarction, or ischemic stroke (3-P MACE) and 3-P MACE plus ischemia-driven arterial revascularization (4-P MACE). Secondary end points included all-cause mortality. Results:This predefined subgroup included 3655 patients (1849 in the evolocumab group and 1806 in the placebo group) with a median age of 65 years (57% female). Among those in the lipid substudy, the median LDL-C level at 48 weeks was 52 mg/dL in the evolocumab group vs 111 mg/dL in the placebo group (P < .001). A 3-P MACE event occurred in 83 patients (5-year Kaplan-Meier estimate, 5.0%) in the evolocumab group compared with 117 patients (5-year Kaplan-Meier estimate, 7.1%) in the placebo group (hazard ratio [HR], 0.69 [95% CI, 0.52-0.91]; P = .009; between-group difference, 2.1% [95% CI, 0.4%-3.8%]). A 4-P MACE event occurred in 127 patients (5-year Kaplan-Meier estimate, 7.6%) in the evolocumab group compared with 178 patients (5-year Kaplan-Meier estimate, 10.5%) in the placebo group (HR, 0.69 [95% CI, 0.55-0.86]; P = .001; between-group difference, 2.9% [95% CI, 0.9%-4.9%]). There were 136 deaths (5-year Kaplan-Meier estimate, 7.8%) in the evolocumab group compared with 172 deaths (5-year Kaplan-Meier estimate, 10.1%) in the placebo group (HR, 0.76 [95% CI, 0.61-0.95]). Conclusions and Relevance:In high-risk patients without known significant atherosclerosis and with diabetes, evolocumab reduced the risk of a first major cardiovascular event. Trial Registration:ClinicalTrials.gov Identifier: NCT03872401.

A mechanistic basis for luciferase bioluminescence provides a glimpse into its evolutionary role for organism survival, as it provides a blueprint to engineer luciferase enzymes for advanced technological applications. Gaussia Luciferase is among the brightest natural luciferases, but (1) the evolutionary development of its luminescence behavior remains unclear, (2) recent fundamental studies utilized Escherichia coli expression systems instead of eukaryotic expression systems, and (3) notable mutants have been discovered but not integrated into a comprehensive mechanistic analysis. We describe new mechanistic observations from GLuc by addressing these gaps. We monitored the fluorescent coelenterazine-to-coelenteramide conversion to study turnover kinetics of mammalian-derived GLuc; this assay characterized the positive cooperativity kinetics of GLuc. The nonluminescent mutants, R76A and R147A, still turn over the substrate with high efficiency, each demonstrating sustained positive cooperativity. Through mass spectrometry, mutational analysis, and analytical liquid chromatography, we demonstrate that GLuc undergoes methionine oxidation during substrate turnover and that this impacts the luciferase's flash-type luminescence; we did not observe indications of covalent attachment with the substrate, product, or their intermediates. Chromatography of luciferases derived from ancestral sequence reconstruction highlighted that the extent of methionine-induced surface changes was greater for earlier ancestral luciferases. Ancestral sequence reconstruction also revealed that earlier ancestral copepod luciferases produced less light when compared to GLuc.

Kp,uu,brain is a critical parameter for evaluating the brain penetration of CNS-targeted compounds, reflecting the ratio of unbound drug concentration in the brain to that in the plasma. While Kp,uu,brain is widely used in the pharmaceutical industry to assess brain exposure, the fidelity of translating Kp,uu,brain to target coverage and pharmacodynamic (PD) effect remains uncertain. This study explores the effectiveness of Kp,uu,brain-based strategies in identifying drug candidates with sufficient target coverage and substantial PD effect. By analyzing reported Kp,uu,brain, unbound drug concentrations in the brain and IC50 values against pharmacological targets for 17 drugs including anticonvulsants, antidepressants, antipsychotics, and antimicrobials, our study demonstrated that while in vitro and in vivo models work well for rank ordering compounds with high Kp,uu,brain, this parameter does not necessarily translate into adequate target coverage (Cu/IC50). In addition, by leveraging PK and PD profiles of 18 drugs measured from human glioblastoma tumors, our study showed that target coverage (glioblastoma Cu/5xIC50) generally correlates well with PD effect. Additionally, Kp,uu,brain tumor is a better indicator for glioblastoma PD effect than Kp,uu,brain, suggesting that intact BBB model may not adequately reflect the barrier heterogeneity in brain tumors such as glioblastoma. In conclusion, while Kp,uu,brain provides an insight on the extent of brain penetration, our study highlighted the need for integrative approaches combining Kp,uu,brain data with comprehensive PK/PD analysis to prioritize CNS-targeted drug candidates with sufficient target coverage and substantial PD effect. a-c: in vitro trans-well, in vivo rodent and non-human primate models, and CSF from non-human primate or human, to predict human Kp,uu,brain. The arrows with solid line represent reasonable prediction. d: For oncology programs that develop drugs targeting brain tumors, Kp,uu,brain is commonly used for prioritizing compounds, assuming that Kp,uu,brain tumor would be higher or similar to Kp,uu,brain, which provides conservative estimate of brain tumor penetration. The arrow with dashed line represents that there is limited information about the effectiveness of Kp,uu,brain as a surrogate of Kp,uu,brain tumor. e–f: Translation of Kp,uu,brain and Kp,uu,brain tumor to target coverage. Arrows with dashed line represent that there is limited information about the direct correlation. g: Translation of target coverage to PD effect. The arrow with solid line represents reasonable translation.