A unified model-based framework for doublet or multiplet detection in single-cell multiomics data.

Droplet-based single-cell sequencing techniques rely on the fundamental assumption that each droplet encapsulates a single cell, enabling individual cell omics profiling. However, the inevitable issue of multiplets, where two or more cells are encapsulated within a single droplet, can lead to spurious cell type annotations and obscure true biological findings. The issue of multiplets is exacerbated in single-cell multiomics settings, where integrating cross-modality information for clustering can inadvertently promote the aggregation of multiplet clusters and increase the risk of erroneous cell type annotations. Here, we propose a compound Poisson model-based framework for multiplet detection in single-cell multiomics data. Leveraging experimental cell hashing results as the ground truth for multiplet status, we conducted trimodal DOGMA-seq experiments and generated 17 benchmarking datasets from two tissues, involving a total of 280,123 droplets. We demonstrated that the proposed method is an essential tool for integrating cross-modality multiplet signals, effectively eliminating multiplet clusters in single-cell multiomics data-a task at which the benchmarked single-omics methods proved inadequate.

Nasal epithelial gene expression and total IgE in children and adolescents with asthma.

BACKGROUND: Little is known about nasal epithelial gene expression and total IgE in youth. OBJECTIVE: We aimed to identify genes whose nasal epithelial expression differs by total IgE in youth, and group them into modules that could be mapped to airway epithelial cell types. METHODS: We conducted a transcriptome-wide association study of total IgE in 469 Puerto Ricans aged 9 to 20 years who participated in the Epigenetic Variation and Childhood Asthma in Puerto Ricans study, separately in all subjects and in those with asthma. We then attempted to replicate top findings for each analysis using data from 3 cohorts. Genes with a Benjamini-Hochberg-adjusted P value of less than .05 in the Epigenetic Variation and Childhood Asthma in Puerto Ricans study and a P value of less than .05 in the same direction of association in 1 or more replication cohort were considered differentially expressed genes (DEGs). DEGs for total IgE in subjects with asthma were further dissected into gene modules using coexpression analysis, and such modules were mapped to specific cell types in airway epithelia using public single-cell RNA-sequencing data. RESULTS: A higher number of DEGs for total IgE were identified in subjects with asthma (n = 1179 DEGs) than in all subjects (n = 631 DEGs). In subjects with asthma, DEGs were mapped to 11 gene modules. The top module for positive correlation with total IgE was mapped to myoepithelial and mucus secretory cells in lower airway epithelia and was regulated by IL-4, IL5, IL-13, and IL-33. Within this module, hub genes included CDH26, FETUB, NTRK2, CCBL1, CST1, and CST2. Furthermore, an enrichment analysis showed overrepresentation of genes in signaling pathways for synaptogenesis, IL-13, and ferroptosis, supporting interactions between interleukin- and acetylcholine-induced responses. CONCLUSIONS: Our findings for nasal epithelial gene expression support neuroimmune coregulation of total IgE in youth with asthma.

Methyl-TWAS: A powerful method for in silico transcriptome-wide association studies (TWAS) using long-range DNA methylation.

In silico transcriptome-wide association studies (TWAS) are commonly used to test whether expression of specific genes is linked to a complex trait. However, genotype-based in silico TWAS such as PrediXcan, exhibit low prediction accuracy for a majority of genes because genotypic data lack tissue- and disease-specificity and are not affected by the environment. Because methylation is tissue-specific and, like gene expression, can be modified by environment or disease status, methylation should predict gene expression with more accuracy than SNPs. Therefore, we propose Methyl-TWAS, the first approach that utilizes long-range methylation markers to impute gene expression for in silico TWAS through penalized regression. Methyl-TWAS 1) predicts epigenetically regulated/associated expression (eGReX), which incorporates tissue-specific expression and both genetically- (GReX) and environmentally-regulated expression to identify differentially expressed genes (DEGs) that could not be identified by genotype-based methods; and 2) incorporates both cis- and trans- CpGs, including various regulatory regions to identify DEGs that would be missed using cis- methylation only. Methyl-TWAS outperforms PrediXcan and two other methods in imputing gene expression in the nasal epithelium, particularly for immunity-related genes and DEGs in atopic asthma. Methyl-TWAS identified 3,681 (85.2%) of the 4,316 DEGs identified in a previous TWAS of atopic asthma using measured expression, while PrediXcan could not identify any gene. Methyl-TWAS also outperforms PrediXcan for expression imputation as well as in silico TWAS in white blood cells. Methyl-TWAS is a valuable tool for in silico TWAS, leveraging a growing body of publicly available genome-wide DNA methylation data for a variety of human tissues.

Cis- and trans-eQTM analysis reveals novel epigenetic and transcriptomic immune markers of atopic asthma in airway epithelium.

BACKGROUND: Expression quantitative trait methylation (eQTM) analyses uncover associations between DNA methylation markers and gene expression. Most eQTM analyses of complex diseases have focused on cis-eQTM pairs (within 1 megabase). OBJECTIVES: This study sought to identify cis- and trans-methylation markers associated with gene expression in airway epithelium from youth with and without atopic asthma. METHODS: In this study, the investigators conducted both cis- and trans-eQTM analyses in nasal (airway) epithelial samples from 158 Puerto Rican youth with atopic asthma and 100 control subjects without atopy or asthma. The investigators then attempted to replicate their findings in nasal epithelial samples from 2 studies of children, while also examining whether their results in nasal epithelium overlap with those from an eQTM analysis in white blood cells from the Puerto Rican subjects. RESULTS: This study identified 9,108 cis-eQTM pairs and 2,131,500 trans-eQTM pairs. Trans-associations were significantly enriched for transcription factor and microRNA target genes. Furthermore, significant cytosine-phosphate-guanine sites (CpGs) were differentially methylated in atopic asthma and significant genes were enriched for genes differentially expressed in atopic asthma. In this study, 50.7% to 62.6% of cis- and trans-eQTM pairs identified in Puerto Rican youth were replicated in 2 smaller cohorts at false discovery rate-adjusted P < .1. Replicated genes in the trans-eQTM analysis included biologically plausible asthma-susceptibility genes (eg, HDC, NLRP3, ITGAE, CDH26, and CST1) and are enriched in immune pathways. CONCLUSIONS: Studying both cis- and trans-epigenetic regulation of airway epithelial gene expression can identify potential causal and regulatory pathways or networks for childhood asthma. Trans-eQTM CpGs may regulate gene expression in airway epithelium through effects on transcription factor and microRNA target genes.

Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection.

Influenza-associated bacterial superinfections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly, promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory before adulthood. Due to this, we sought to understand the role of immune memory during bacterial superinfections. An influenza heterotypic immunity model was established using influenza A/Puerto Rico/8/34 and influenza A/X31. We report in this article that influenza-experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naive animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer-specific CD8+ T cells. The protection that was seen in the memory-experienced mouse model is associated with the reduction in inflammatory mechanisms, making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity reshapes the lung environment and the immune response to a rechallenge event, which is highly relevant to the context of human infection.

Robust and accurate estimation of cellular fraction from tissue omics data via ensemble deconvolution.

MOTIVATION: Tissue-level omics data such as transcriptomics and epigenomics are an average across diverse cell types. To extract cell-type-specific (CTS) signals, dozens of cellular deconvolution methods have been proposed to infer cell-type fractions from tissue-level data. However, these methods produce vastly different results under various real data settings. Simulation-based benchmarking studies showed no universally best deconvolution approaches. There have been attempts of ensemble methods, but they only aggregate multiple single-cell references or reference-free deconvolution methods. RESULTS: To achieve a robust estimation of cellular fractions, we proposed EnsDeconv (Ensemble Deconvolution), which adopts CTS robust regression to synthesize the results from 11 single deconvolution methods, 10 reference datasets, 5 marker gene selection procedures, 5 data normalizations and 2 transformations. Unlike most benchmarking studies based on simulations, we compiled four large real datasets of 4937 tissue samples in total with measured cellular fractions and bulk gene expression from different tissues. Comprehensive evaluations demonstrated that EnsDeconv yields more stable, robust and accurate fractions than existing methods. We illustrated that EnsDeconv estimated cellular fractions enable various CTS downstream analyses such as differential fractions associated with clinical variables. We further extended EnsDeconv to analyze bulk DNA methylation data. AVAILABILITY AND IMPLEMENTATION: EnsDeconv is freely available as an R-package from https://github.com/randel/EnsDeconv. The RNA microarray data from the TRAUMA study are available and can be accessed in GEO (GSE36809). The demographic and clinical phenotypes can be shared on reasonable request to the corresponding authors. The RNA-seq data from the EVAPR study cannot be shared publicly due to the privacy of individuals that participated in the clinical research in compliance with the IRB approval at the University of Pittsburgh. The RNA microarray data from the FHS study are available from dbGaP (phs000007.v32.p13). The RNA-seq data from ROS study is downloaded from AD Knowledge Portal. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Outcomes after Bone-Ligament-Bone Intercarpal Ligament Reconstruction.

BACKGROUND: Numerous surgical reconstructive techniques have been described for chronic scapholunate and lunotriquetral interosseous ligament instability. METHODS: The authors retrospectively reviewed 16 consecutive patients who underwent bone-ligament-bone reconstruction for scapholunate or lunotriquetral intraosseous ligament predynamic and dynamic instability at a single tertiary care institution from 2013 to 2019. Clinical and radiographic outcomes, and complications, were recorded. RESULTS: Eleven patients had bone-ligament-bone reconstruction for scapholunate ligament injuries and five for lunotriquetral instability. Fourteen patients (87.5 percent) underwent diagnostic arthroscopy before bone-ligament-bone reconstruction, with nine of 14 having grade 3 and four of 14 having grade 4 injury. Capitohamate bone-ligament-bone grafts were used in nine patients (56 percent) and the graft was taken from Lister tubercle in seven (44 percent). The average age at surgery was 37 years. The average follow-up was 60.6 weeks. There were no significant differences between preoperative and postoperative radiographic parameters. Median postoperative wrist flexion was 45 degrees, and mean postoperative wrist extension was 53 degrees, which were significantly less than contralateral flexion (85 degrees; p < 0.0001) and extension (78 degrees; p < 0.0001). Thirty-eight percent of patients complained of persistent pain at final follow-up, and two patients (13 percent) underwent salvage procedures, both at approximately 6.5 months after the index reconstruction. CONCLUSION: Short-term outcomes of bone-ligament-bone reconstruction for early-stage scapholunate and lunotriquetral ligament injuries reveal many patients with residual postoperative wrist pain and disability, and almost uniform limitations in flexion-extension motion. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Preventable Hospitalization Trends Before and After the Affordable Care Act.

Introduction: The Patient Protection and Affordable Care Act aimed to increase the number of individuals with health insurance, which may lead to adequate primary care management and reduced rates of preventable hospitalizations. To investigate the rates of preventable hospitalization after the passing of the Affordable Care Act in 2010 and Medicaid expansion in 2014 across 26 states, a population-based study was conducted using the Healthcare Cost and Utilization Project National Inpatient Sample database from 2005-2017. Methods: A logistic regression and trend analysis was performed to assess the changes in preventable hospitalization rates over time and the impact of policy changes on the rate of preventable hospitalization. Individuals were included if they were aged between 18 and 64 years and had a preventable quality indicator International Classification of Diseases, Ninth or Tenth Revision code as determined by the Agency for Healthcare Research and Quality. Results: More than 45 million preventable-hospitalization admissions were reported between 2005 and 2017. There was a significant decrease in preventable hospitalization rates after the passing of the Affordable Care Act from 12.0% to 10.8% (p<0.01) and from 11.5% to 10.6% (p<0.01) after Medicaid expansion. Bacterial pneumonia declined from 1.5% to 0.6% (p<0.01), along with chronic obstructive pulmonary disease and asthma in older adults from 1.9% to 1.7% (p=0.01) after the expansion. Conclusions: States that have not implemented Medicaid expansion should make it a priority because it may lead to a reduction in preventable hospitalization rates. Furthermore, preventable hospitalization rates may be considered a quality measure to examine the accessibility and effectiveness of primary care intervention.

Patterns of Upper Extremity Reconstruction for Patients With Tetraplegia Across the United States: A Retrospective Study.

PURPOSE: The rates of upper extremity reconstruction for patients with tetraplegia remain low. We performed a retrospective study to assess recent reconstruction rates and delineate factors associated with the occurrence of reconstruction. METHODS: We examined the National Inpatient Sample database (2012-2017) for the rate of reconstruction for patients with tetraplegia. The details of provider distribution characteristics and neighborhood attributes were obtained from the American Medical Association Physician Masterfile and based on the area deprivation index, respectively. We calculated the mean reconstruction rate per year and generated multivariable logistic regression models to examine the influence of patient factors, hospital characteristics, and provider distribution on the odds of undergoing functional reconstruction for tetraplegia patients. RESULTS: Among 404,660 encounters with patients with tetraplegia, only 1,430 (0.4%) patients underwent upper extremity reconstruction from 2012 to 2017, with a mean rate of 238 procedures per year. We identified 5,450 hand surgeons, 12,751 physiatrists, and 444 spinal cord injury specialists, with variation in their national distribution. A greater number of surgeons near SCIS was associated with increased probability of reconstruction (odds ratio [OR] 1.07, 95% confidence interval [CI] 1.03-1.12). The odds of surgery were greater for patients receiving care at urban teaching (OR 5.00, 95% CI 3.35-7.47) or urban nonteaching (OR 1.71, 95% CI 1.11-2.63) hospitals, whereas those at private nonprofit (OR 0.67, 95% CI 0.58-0.78) or investor-owned (OR 0.65, 95% CI 0.52-0.82) hospitals had lower odds. Although most patients had insurance coverage, patients with a higher income or those who received subsidized care had greater odds of undergoing reconstruction. CONCLUSIONS: Reconstruction rates remain low and are correlated with the environment of care, financial factors, and provider availability. Policies that focus on reducing these factors in addition to increasing interspecialty collaboration could improve access to surgery for patients with tetraplegia. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic I.

Discrepancies in conservative treatment for thumb carpometacarpal arthritis: a comparison between different specialities and patient characteristics.

Non-surgical treatment is successful in controlling pain and preventing disease progress in treating thumb carpometacarpal arthritis. We used Optum's de-identified Clinformatics® Data Mart Databases between 2015 and 2018 to conduct a study of the patient and provider characteristics associated with three types of non-surgical treatment (hand therapy, splinting and corticosteroid injection) prior to surgery. In this population-based cohort study, we found that non-surgical providers were more likely to provide three different types of non-surgical treatments, as compared with hand surgeons. In addition, women and patients with comorbid conditions, including carpal tunnel syndrome, obesity, chronic pain and depression, were less likely to exhaust the available non-surgical management options for thumb carpometacarpal arthritis. Therefore, we suggest that these specific patient populations can potentially benefit from additional non-surgical treatments that may delay or obviate surgery for this disease. These groups are target populations for future efforts to ensure that all patients receive equitable care.Level of evidence: II.