CodonBert large language model for mRNA vaccines.

mRNA-based vaccines and therapeutics are gaining popularity and usage across a wide range of conditions. One of the critical issues when designing such mRNAs is sequence optimization. Even small proteins or peptides can be encoded by an enormously large number of mRNAs. The actual mRNA sequence can have a large impact on several properties including expression, stability, immunogenicity, and more. To enable the selection of an optimal sequence, we developed CodonBERT, a large language model (LLM) for mRNAs. Unlike prior models, CodonBERT uses codons as inputs which enables it to learn better representations. CodonBERT was trained using more than 10 million mRNA sequences from a diverse set of organisms. The resulting model captures important biological concepts. CodonBERT can also be extended to perform prediction tasks for various mRNA properties. CodonBERT outperforms previous mRNA prediction methods including on a new flu vaccine dataset.

Representations of lipid nanoparticles using large language models for transfection efficiency prediction.

MOTIVATION: Lipid nanoparticles (LNPs) are the most widely used vehicles for mRNA vaccine delivery. The structure of the lipids composing the LNPs can have a major impact on the effectiveness of the mRNA payload. Several properties should be optimized to improve delivery and expression including biodegradability, synthetic accessibility, and transfection efficiency. RESULTS: To optimize LNPs, we developed and tested models that enable the virtual screening of LNPs with high transfection efficiency. Our best method uses the lipid Simplified Molecular-Input Line-Entry System (SMILES) as inputs to a large language model. Large language model-generated embeddings are then used by a downstream gradient-boosting classifier. As we show, our method can more accurately predict lipid properties, which could lead to higher efficiency and reduced experimental time and costs. AVAILABILITY AND IMPLEMENTATION: Code and data links available at: https://github.com/Sanofi-Public/LipoBART.

Coronary bypass surgery guided by computed tomography in a low-risk population.

BACKGROUND AND AIMS: In patients with three-vessel disease and/or left main disease, selecting revascularization strategy based on coronary computed tomography angiography (CCTA) has a high level of virtual agreement with treatment decisions based on invasive coronary angiography (ICA). METHODS: In this study, coronary artery bypass grafting (CABG) procedures were planned based on CCTA without knowledge of ICA. The CABG strategy was recommended by a central core laboratory assessing the anatomy and functionality of the coronary circulation. The primary feasibility endpoint was the percentage of operations performed without access to the ICA. The primary safety endpoint was graft patency on 30-day follow-up CCTA. Secondary endpoints included topographical adequacy of grafting, major adverse cardiac and cerebrovascular (MACCE), and major bleeding events at 30 days. The study was considered positive if the lower boundary of confidence intervals (CI) for feasibility was ≥75% (NCT04142021). RESULTS: The study enrolled 114 patients with a mean (standard deviation) anatomical SYNTAX score and Society of Thoracic Surgery score of 43.6 (15.3) and 0.81 (0.63), respectively. Unblinding ICA was required in one case yielding a feasibility of 99.1% (95% CI 95.2%-100%). The concordance and agreement in revascularization planning between the ICA- and CCTA-Heart Teams was 82.9% with a moderate kappa of 0.58 (95% CI 0.50-0.66) and between the CCTA-Heart Team and actual treatment was 83.7% with a substantial kappa of 0.61 (95% CI 0.53-0.68). The 30-day follow-up CCTA in 102 patients (91.9%) showed an anastomosis patency rate of 92.6%, whilst MACCE was 7.2% and major bleeding 2.7%. CONCLUSIONS: CABG guided by CCTA is feasible and has an acceptable safety profile in a selected population of complex coronary artery disease.

Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation.

Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of "fractions" was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.

Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands.

Massively parallel reporter assays (MPRAs) are useful tools to characterize regulatory elements in human genomes. An aspect of MPRAs that is not typically the focus of analysis is their intrinsic ability to differentiate activity levels for a given sequence element when placed in both of its possible orientations relative to the reporter construct. Here, we describe pervasive strand asymmetry of MPRA signals in data sets from multiple reporter configurations in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that function related to endogenous transcription is driving the phenomenon. Similarly, we find that within Alu mobile element insertions, strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes and is more pronounced in less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and show its prediction from sequence alone. We see some evidence for RNA stabilization and transcriptional activation mechanisms and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry is a pervasive and reproducible feature in MPRA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.

Effective gene expression prediction from sequence by integrating long-range interactions.

How noncoding DNA determines gene expression in different cell types is a major unsolved problem, and critical downstream applications in human genetics depend on improved solutions. Here, we report substantially improved gene expression prediction accuracy from DNA sequences through the use of a deep learning architecture, called Enformer, that is able to integrate information from long-range interactions (up to 100 kb away) in the genome. This improvement yielded more accurate variant effect predictions on gene expression for both natural genetic variants and saturation mutagenesis measured by massively parallel reporter assays. Furthermore, Enformer learned to predict enhancer-promoter interactions directly from the DNA sequence competitively with methods that take direct experimental data as input. We expect that these advances will enable more effective fine-mapping of human disease associations and provide a framework to interpret cis-regulatory evolution.

How to assess nonresponsiveness to vasodilator stress.

Myocardial perfusion imaging (MPI) is a powerful tool for the functional assessment of ischemia in patients with suspected or known coronary artery disease (CAD). Given that the diagnostic accuracy and prognostic value of MPI and post-test management are highly dependent on achieving an adequate stress vasodilatory response, it is critical to identify those who may not have adequately responded to vasodilator pharmacological stress agents such as adenosine, dipyridamole, and regadenoson. Caffeine, a potent inhibitor of the adenosine receptor, is a compound that can affect vasodilatory hemodynamics, result in false negative studies, and potentially alter management in cases of inaccurate test results. Vasodilator non-responsiveness can be suspected by examining hemodynamics, quantitative positron emission tomography (PET) metrics such as myocardial flow reserve (MFR), and splenic response to stress. Quantitative MFR values of 1-1.2 should raise suspicion for nonresponsiveness in the setting of normal perfusion, along with the absence of a splenic switch off. Newer metrics, such as splenic response ratio, can be used to aid in the identification of potential nonresponders to pharmacologic vasodilators.

Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression.

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.

A systematic evaluation of the design and context dependencies of massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there are limited studies that systematically compare differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate that enhancer activity is largely independent of orientation, at least for our library and designs. Finally, we assemble and test the same enhancers as 192-mers, 354-mers and 678-mers and observe sizable differences. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements and to a lesser degree the precise assay, influence MPRA results.

Uncommon spectral Doppler findings in patients with heart failure.

Spectral Doppler examination is necessary for full hemodynamic assessment of patients with systolic heart failure. It is fully incorporated into comprehensive echocardiographic examination. In this manuscript, we describe two uncommon findings in patients with established severe left ventricular systolic dysfunction: notched aortic regurgitation and merged mitral regurgitation.

Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (≥1.5 × 10(5) added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect.

Global analyses of the effect of different cellular contexts on microRNA targeting.

MicroRNA (miRNA) regulation clearly impacts animal development, but the extent to which development-with its resulting diversity of cellular contexts-impacts miRNA regulation is unclear. Here, we compared cohorts of genes repressed by the same miRNAs in different cell lines and tissues and found that target repertoires were largely unaffected, with secondary effects explaining most of the differential responses detected. Outliers resulting from differential direct targeting were often attributable to alternative 3' UTR isoform usage that modulated the presence of miRNA sites. More inclusive examination of alternative 3' UTR isoforms revealed that they influence ∼10% of predicted targets when comparing any two cell types. Indeed, considering alternative 3' UTR isoform usage improved prediction of targeting efficacy significantly beyond the improvements observed when considering constitutive isoform usage. Thus, although miRNA targeting is remarkably consistent in different cell types, considering the 3' UTR landscape helps predict targeting efficacy and explain differential regulation that is observed.

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair.

Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.

Comparison of myocardial fibrosis quantification methods by cardiovascular magnetic resonance imaging for risk stratification of patients with suspected myocarditis.

BACKGROUND: Although the presence of late gadolinium enhancement (LGE) using cardiovascular magnetic resonance imaging (CMR) is a significant discriminator of events in patients with suspected myocarditis, no data are available on the optimal LGE quantification method. METHODS: Six hundred seventy consecutive patients (48 ± 16 years, 59% male) with suspected myocarditis were enrolled between 2002 and 2015. We performed LGE quantitation using seven different signal intensity thresholding methods based either on 2, 3, 4, 5, 6, 7 standard deviations (SD) above remote myocardium or full width at half maximum (FWHM). In addition, a LGE visual presence score (LGE-VPS) (LGE present/absent in each segment) was assessed. For each of these methods, the strength of association of LGE results with major adverse cardiac events (MACE) was determined. Inter-and intra-rater variability using intraclass-correlation coefficient (ICC) was performed for all methods. RESULTS: Ninety-eight (15%) patients experienced a MACE at a medium follow-up of 4.7 years. LGE quantification by FWHM, 2- and 3-SD demonstrated univariable association with MACE (hazard ratio [HR] 1.05, 95% confidence interval [CI]:1.02-1.08, p = 0.001; HR 1.02, 95%CI:1.00-1.04; p = 0.001; HR 1.02, 95%CI: 1.00-1.05, p = 0.035, respectively), whereas 4-SD through 7-SD methods did not reach significant association. LGE-VPS also demonstrated association with MACE (HR 1.09, 95%CI: 1.04-1.15, p < 0.001). In the multivariable model, FWHM, 2-SD methods, and LGE-VPS each demonstrated significant association with MACE adjusted to age, sex, BMI and LVEF (adjusted HR of 1.04, 1.02, and 1.07; p = 0.009, p = 0.035; and p = 0.005, respectively). In these, FWHM and LGE-VPS had the highest degrees of inter and intra-rater reproducibility based on their high ICC values. CONCLUSIONS: FWHM is the optimal semi-automated quantification method in risk-stratifying patients with suspected myocarditis, demonstrating the strongest association with MACE and the highest technical consistency. Visual LGE scoring is a reliable alternative method and is associated with a comparable association with MACE and reproducibility in these patients. TRIAL REGISTRATION NUMBER: NCT03470571 . Registered 13th March 2018. Retrospectively registered.

Isolated cardiac sarcoidosis: A focused review of an under-recognized entity.

There is accumulating evidence for the existence of a phenotype of isolated cardiac sarcoidosis (ICS), or sarcoidosis that only involves the heart. In the absence of biopsy-confirmed cardiac sarcoidosis (CS), existing diagnostic criteria require the presence of extra-cardiac sarcoidosis as an inclusion criterion for the diagnosis of CS. Consequently, in the absence of a positive endomyocardial biopsy, ICS is not diagnosable by current guidelines. Therefore, there is uncertainty regarding the epidemiology, pathobiology, clinical characteristics, prognosis, and optimal treatment of ICS. This review will summarize the available data related to the prevalence and prognosis of ICS and will discuss challenges surrounding the diagnosis and management of this under-recognized entity.

Independent regulation of vertebral number and vertebral identity by microRNA-196 paralogs.

The Hox genes play a central role in patterning the embryonic anterior-to-posterior axis. An important function of Hox activity in vertebrates is the specification of different vertebral morphologies, with an additional role in axis elongation emerging. The miR-196 family of microRNAs (miRNAs) are predicted to extensively target Hox 3' UTRs, although the full extent to which miR-196 regulates Hox expression dynamics and influences mammalian development remains to be elucidated. Here we used an extensive allelic series of mouse knockouts to show that the miR-196 family of miRNAs is essential both for properly patterning vertebral identity at different axial levels and for modulating the total number of vertebrae. All three miR-196 paralogs, 196a1, 196a2, and 196b, act redundantly to pattern the midthoracic region, whereas 196a2 and 196b have an additive role in controlling the number of rib-bearing vertebra and positioning of the sacrum. Independent of this, 196a1, 196a2, and 196b act redundantly to constrain total vertebral number. Loss of miR-196 leads to a collective up-regulation of numerous trunk Hox target genes with a concomitant delay in activation of caudal Hox genes, which are proposed to signal the end of axis extension. Additionally, we identified altered molecular signatures associated with the Wnt, Fgf, and Notch/segmentation pathways and demonstrate that miR-196 has the potential to regulate Wnt activity by multiple mechanisms. By feeding into, and thereby integrating, multiple genetic networks controlling vertebral number and identity, miR-196 is a critical player defining axial formulae.

Comparison of the current reasons for undergoing pharmacologic stress during echocardiographic and radionuclide stress testing.

BACKGROUND: Symptom-limited exercise is the preferred method of cardiac stress testing, but pharmacologic testing has been increasing over time. The exact reasons for pharmacologic stress testing have not been rigorously categorized. Thus, we systematically explored the reasons for pharmacologic stress testing in patients referred for cardiac stress imaging. METHODS: We studied consecutive patients referred for stress imaging [stress echocardiography or radionuclide myocardial perfusion imaging (MPI)] at Mount Sinai St Luke's hospital between August 2013 and April 2014. Baseline information was obtained using a standardized questionnaire and a trained physician triaged the patient for symptom-limited exercise stress testing or pharmacologic stress testing. RESULTS: In total, 551(48%) of our entire stress cohort underwent cardiac imaging following initial exercise testing and 589 (52%) underwent imaging with initial pharmacologic stress testing. Deconditioning and inability to walk (primarily due to musculoskeletal conditions) constituted the top two reasons for performing pharmacologic stress, followed by frailty, left bundle branch block (for MPI), resting wall motion abnormality (for echocardiography), and failed exercise attempts. The reasons for performing pharmacologic stress testing were similar in the MPI and echocardiography patients, despite a much higher level of disease acuity in the MPI group. CONCLUSIONS: We have applied a systematic approach for categorizing the reasons for pharmacologic stress. These reasons are heterogeneous, but similar across MPI and echo stress laboratories.

Challenges in Diagnosis, Management, and Treatment of Allopurinol-Induced DRESS Syndrome: Case Report and Literature Review.

We describe the presentation, diagnosis, management, and treatment of a 62-year-old woman with a medical history of gout who presented with a maculopapular rash, facial and tongue edema. Her initial presentation, coupled with a history of recent allopurinol use for systematic relief, led to the diagnosis of allopurinol-induced drug rash with eosinophilia and systemic symptoms (DRESS) syndrome, further confirmed by the RegiSCAR scoring criteria including a skin biopsy. The patient was initially treated conservatively but required systemic corticosteroid therapy as she developed severe multi-organ dysfunction. This article will highlight the challenges involved in diagnosing DRESS syndrome from other adverse cutaneous drug reactions, delayed systemic complications, and the need for evidence-based treatment modalities and regimens using the most recent published literature and analysis of case reports. Among treatment modalities, pulsed parenteral steroids show promise in a few case reports. We also discuss the newer alternative gout therapies since the mainstay of gout treatment, allopurinol, is potentially associated with morbidity and mortality risks as manifested in our patient with DRESS.

Prognostic Value of Stress Echocardiography in Patients Presenting with Syncope.

BACKGROUND: Evaluation for ischemia is appropriate in patients at risk for or with a history of coronary artery disease presenting with syncope. The aim of this study is to determine the prognostic value of stress echocardiography in patients presenting with syncope. METHODS: We examined our database of all patients undergoing stress echocardiography at our institution. Patients referred due to syncope were grouped as high risk based on any of the following: (1) known history of coronary artery disease, (2) left ventricular ejection fraction <50%, (3) moderate or severe mitral or aortic valve regurgitation, and (4) moderate mitral or aortic valve stenosis. The main outcomes were the presence of ischemia on stress imaging and all-cause mortality using the social security death index. RESULTS: A total of 225 patients were identified; mean age was 64.3 ± 14.5 years, the mean follow-up duration was 29.2 ± 13.8 months. There were 163 patients in the low-risk group and 62 patients in the high-risk group. On imaging, 7% of the overall cohort had ischemia. The low-risk group had 5 (3%) patients with ischemia and the high-risk group had 10 patients (16%) with ischemia (P < 0.01). The mortality rate was significantly higher in the high-risk group (3.99%/year vs. 1.02%/year; P = 0.02); this difference was not affected by the presence of ischemia. CONCLUSIONS: High-risk patients with syncope as defined by appropriateness criteria and existing evidence carry a higher risk of ischemia and all-cause mortality. The presence of ischemia may not be predictive of long-term outcome in this group.