A comprehensive assessment of cell type-specific differential expression methods in bulk data.

Accounting for cell type compositions has been very successful at analyzing high-throughput data from heterogeneous tissues. Differential gene expression analysis at cell type level is becoming increasingly popular, yielding biomarker discovery in a finer granularity within a particular cell type. Although several computational methods have been developed to identify cell type-specific differentially expressed genes (csDEG) from RNA-seq data, a systematic evaluation is yet to be performed. Here, we thoroughly benchmark six recently published methods: CellDMC, CARseq, TOAST, LRCDE, CeDAR and TCA, together with two classical methods, csSAM and DESeq2, for a comprehensive comparison. We aim to systematically evaluate the performance of popular csDEG detection methods and provide guidance to researchers. In simulation studies, we benchmark available methods under various scenarios of baseline expression levels, sample sizes, cell type compositions, expression level alterations, technical noises and biological dispersions. Real data analyses of three large datasets on inflammatory bowel disease, lung cancer and autism provide evaluation in both the gene level and the pathway level. We find that csDEG calling is strongly affected by effect size, baseline expression level and cell type compositions. Results imply that csDEG discovery is a challenging task itself, with room to improvements on handling low signal-to-noise ratio and low expression genes.

cypress: an R/Bioconductor package for cell-type-specific differential expression analysis power assessment.

SUMMARY: Recent methodology advances in computational signal deconvolution have enabled bulk transcriptome data analysis at a finer cell-type level. Through deconvolution, identifying cell-type-specific differentially expressed (csDE) genes is drawing increasing attention in clinical applications. However, researchers still face a number of difficulties in adopting csDE genes detection methods in practice, especially in their experimental design. Here we present cypress, the first experimental design and statistical power analysis tool in csDE genes identification. This tool can reliably model purified cell-type-specific (CTS) profiles, cell-type compositions, biological and technical variations, offering a high-fidelity simulator for bulk RNA-seq convolution and deconvolution. cypress conducts simulation and evaluates the impact of multiple influencing factors, by various statistical metrics, to help researchers optimize experimental design and conduct power analysis. AVAILABILITY AND IMPLEMENTATION: cypress is an open-source R/Bioconductor package at https://bioconductor.org/packages/cypress/.

imply: improving cell-type deconvolution accuracy using personalized reference profiles.

Using computational tools, bulk transcriptomics can be deconvoluted to estimate the abundance of constituent cell types. However, existing deconvolution methods are conditioned on the assumption that the whole study population is served by a single reference panel, ignoring person-to-person heterogeneity. Here, we present imply, a novel algorithm to deconvolute cell type proportions using personalized reference panels. Simulation studies demonstrate reduced bias compared with existing methods. Real data analyses on longitudinal consortia show disparities in cell type proportions are associated with several disease phenotypes in Type 1 diabetes and Parkinson's disease. imply is available through the R/Bioconductor package ISLET at https://bioconductor.org/packages/ISLET/ .

ISLET: individual-specific reference panel recovery improves cell-type-specific inference.

We propose a statistical framework ISLET to infer individual-specific and cell-type-specific transcriptome reference panels. ISLET models the repeatedly measured bulk gene expression data, to optimize the usage of shared information within each subject. ISLET is the first available method to achieve individual-specific reference estimation in repeated samples. Using simulation studies, we show outstanding performance of ISLET in the reference estimation and downstream cell-type-specific differentially expressed genes testing. We apply ISLET to longitudinal transcriptomes profiled from blood samples in a large observational study of young children and confirm the cell-type-specific gene signatures for pancreatic islet autoantibody. ISLET is available at https://bioconductor.org/packages/ISLET .

imply: improving cell-type deconvolution accuracy using personalized reference profiles.

Real-world clinical samples are often admixtures of signal mosaics from multiple pure cell types. Using computational tools, bulk transcriptomics can be deconvoluted to solve for the abundance of constituent cell types. However, existing deconvolution methods are conditioned on the assumption that the whole study population is served by a single reference panel, which ignores person-to-person heterogeneity. Here we present imply, a novel algorithm to deconvolute cell type proportions using personalized reference panels. imply can borrow information across repeatedly measured samples for each subject, and obtain precise cell type proportion estimations. Simulation studies demonstrate reduced bias in cell type abundance estimation compared with existing methods. Real data analyses on large longitudinal consortia show more realistic deconvolution results that align with biological facts. Our results suggest that disparities in cell type proportions are associated with several disease phenotypes in type 1 diabetes and Parkinson's disease. Our proposed tool imply is available through the R/Bioconductor package ISLET at https://bioconductor.org/packages/ISLET/.

A comparison of analytical strategies for cluster randomized trials with survival outcomes in the presence of competing risks.

While statistical methods for analyzing cluster randomized trials with continuous and binary outcomes have been extensively studied and compared, little comparative evidence has been provided for analyzing cluster randomized trials with survival outcomes in the presence of competing risks. Motivated by the Strategies to Reduce Injuries and Develop Confidence in Elders trial, we carried out a simulation study to compare the operating characteristics of several existing population-averaged survival models, including the marginal Cox, marginal Fine and Gray, and marginal multi-state models. For each model, we found that adjusting for the intraclass correlations through the sandwich variance estimator effectively maintained the type I error rate when the number of clusters is large. With no more than 30 clusters, however, the sandwich variance estimator can exhibit notable negative bias, and a permutation test provides better control of type I error inflation. Under the alternative, the power for each model is differentially affected by two types of intraclass correlations-the within-individual and between-individual correlations. Furthermore, the marginal Fine and Gray model occasionally leads to higher power than the marginal Cox model or the marginal multi-state model, especially when the competing event rate is high. Finally, we provide an illustrative analysis of Strategies to Reduce Injuries and Develop Confidence in Elders trial using each analytical strategy considered.

Crosstalk between RNA-Binding Proteins and Immune Microenvironment Revealed Two RBP Regulatory Patterns with Distinct Immunophenotypes in Periodontitis.

Periodontitis is an inflammatory disease whose pathogenesis is closely related with immunology. RNA-binding proteins (RBPs) were found to play crucial roles in immunity. Therefore, we aimed to investigate the potential impact of RBPs in the immune microenvironment in periodontitis. The differential expressions of RBPs in periodontitis and healthy samples were determined and were used to construct an RBP-based classifier for periodontitis using logistic regression. The correlations between RBPs and immune characteristics were investigated by the Spearman correlation. Unsupervised clustering was conducted to identify the RBP regulatory patterns. RBP-related genes were identified by WGCNA, while biological distinctions were revealed by GSVA and GO. 24 dysregulated RBPs were identified, from which a 12-RBP classifier was established to distinguish periodontitis with AUC of 0.942. Close protein-protein interactions and expression correlations were observed especially between SPATS2 and ISG20. ISG20 and ESRP1 were found to be highly correlated with immunocyte infiltration, immune signaling activation, and HLA expressions in periodontitis. Two distinct RBP regulatory patterns were identified with different immune and other biological characteristics in periodontitis. Our findings indicate a significant impact of RBPs in shaping the immune microenvironment in periodontitis, which might bring new insights into the understanding of immune mechanisms in the pathogenesis of periodontitis.

Nanoscale electrostatic gating of molecular transport through nuclear pore complexes as probed by scanning electrochemical microscopy.

The nuclear pore complex (NPC) is a large protein nanopore that solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell. There is a long-standing consensus that selective transport barriers of the NPC are exclusively based on hydrophobic repeats of phenylalanine and glycine (FG) of nucleoporins. Herein, we reveal experimentally that charged residues of amino acids intermingled between FG repeats can modulate molecular transport through the NPC electrostatically and in a pathway-dependent manner. Specifically, we investigate the NPC of the Xenopus oocyte nucleus to find that excess positive charges of FG-rich nucleoporins slow down passive transport of a polycationic peptide, protamine, without affecting that of a polyanionic pentasaccharide, Arixtra, and small monovalent ions. Protamine transport is slower with a lower concentration of electrolytes in the transport media, where the Debye length becomes comparable to the size of water-filled spaces among the gel-like network of FG repeats. Slow protamine transport is not affected by the binding of a lectin, wheat germ agglutinin, to the peripheral route of the NPC, which is already blocked electrostatically by adjacent nucleoporins that have more cationic residues than anionic residues and even FG dipeptides. The permeability of NPCs to the probe ions is measured by scanning electrochemical microscopy using ion-selective tips based on liquid/liquid microinterfaces and is analysed by effective medium theory to determine the sizes of peripheral and central routes with distinct protamine permeability. Significantly, nanoscale electrostatic gating at the NPC can be relevant not only chemically and biologically, but also biomedically for efficient nuclear import of genetically therapeutic substances.