Interactive analysis of single-cell data using flexible workflows with SCTK2.

Analysis of single-cell RNA sequencing (scRNA-seq) data can reveal novel insights into the heterogeneity of complex biological systems. Many tools and workflows have been developed to perform different types of analyses. However, these tools are spread across different packages or programming environments, rely on different underlying data structures, and can only be utilized by people with knowledge of programming languages. In the Single-Cell Toolkit 2 (SCTK2), we have integrated a variety of popular tools and workflows to perform various aspects of scRNA-seq analysis. All tools and workflows can be run in the R console or using an intuitive graphical user interface built with R/Shiny. HTML reports generated with Rmarkdown can be used to document and recapitulate individual steps or entire analysis workflows. We show that the toolkit offers more features when compared with existing tools and allows for a seamless analysis of scRNA-seq data for non-computational users.

Long-Term Whole-Body Vibration Stimulus Decreases Body Fat Accumulation in Rats Fed a High-Fat Diet.

Whole-body vibration (WBV) is a type of light-resistance exercise that involves exposing the body to rapid and repeated oscillations of a vibrating platform. It has been suggested that long-term WBV can improve bone mass and muscle strength. However, little is known about its effects on body composition, and the safety and efficacy of WBV have not been established. In this study, we investigated the effects of WBV on body fat loss and muscle mass maintenance or improvement in male Wistar rats fed standard or high-fat diets. We also aimed to establish a rat model for future nutritional and physiological studies. We conducted two experiments using male Wistar rats that were 3 weeks old. The rats were randomly divided into two groups: the control group and the vibration group. The rats were fed either a commercial standard diet (Experiment 1) or a high-fat diet (Experiment 2) ad libitum for 8-12 weeks. Our results showed that WBV stimulus dramatically reduced body fat accumulation in rats fed a high-fat diet but not in those fed a standard diet. This suggests that WBV may be particularly effective under dietary conditions that promote obesity. Moreover, WBV increased the mass of several skeletal muscles, which are known to have resistance exercise effects. Our findings indicate that long-term WBV is safe, with no inhibition of growth or feeding. Taken together, our results suggest that WBV may be a promising approach for preventing and treating obesity. Further research is needed to investigate the underlying mechanisms and to determine the optimal WBV for maximum benefits.

Celda: a Bayesian model to perform co-clustering of genes into modules and cells into subpopulations using single-cell RNA-seq data.

Single-cell RNA-seq (scRNA-seq) has emerged as a powerful technique to quantify gene expression in individual cells and to elucidate the molecular and cellular building blocks of complex tissues. We developed a novel Bayesian hierarchical model called Cellular Latent Dirichlet Allocation (Celda) to perform co-clustering of genes into transcriptional modules and cells into subpopulations. Celda can quantify the probabilistic contribution of each gene to each module, each module to each cell population and each cell population to each sample. In a peripheral blood mononuclear cell dataset, Celda identified a subpopulation of proliferating T cells and a plasma cell which were missed by two other common single-cell workflows. Celda also identified transcriptional modules that could be used to characterize unique and shared biological programs across cell types. Finally, Celda outperformed other approaches for clustering genes into modules on simulated data. Celda presents a novel method for characterizing transcriptional programs and cellular heterogeneity in scRNA-seq data.

Comprehensive generation, visualization, and reporting of quality control metrics for single-cell RNA sequencing data.

Single-cell RNA sequencing (scRNA-seq) can be used to gain insights into cellular heterogeneity within complex tissues. However, various technical artifacts can be present in scRNA-seq data and should be assessed before performing downstream analyses. While several tools have been developed to perform individual quality control (QC) tasks, they are scattered in different packages across several programming environments. Here, to streamline the process of generating and visualizing QC metrics for scRNA-seq data, we built the SCTK-QC pipeline within the singleCellTK R package. The SCTK-QC workflow can import data from several single-cell platforms and preprocessing tools and includes steps for empty droplet detection, generation of standard QC metrics, prediction of doublets, and estimation of ambient RNA. It can run on the command line, within the R console, on the cloud platform or with an interactive graphical user interface. Overall, the SCTK-QC pipeline streamlines and standardizes the process of performing QC for scRNA-seq data.

Elevated T cell repertoire diversity is associated with progression of lung squamous cell premalignant lesions.

OBJECTIVE: The immune response to invasive carcinoma has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological, and molecular features. METHODS: Endobronchial biopsies (n=295) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via autofluorescence bronchoscopy and CT, were profiled by RNA-seq. We applied the TCR Repertoire Utilities for Solid Tissue/Tumor tool to the RNA-seq data to identify TCR CDR3 sequences across all samples. In the biopsies, we measured the correlation of TCR diversity with previously derived immune-associated PML transcriptional signatures and PML outcome. We also quantified the spatial and temporal distribution of shared and clonally expanded TCRs. Using the biopsies and brushes, the ratio of private (ie, found in one patient only) and public (ie, found in two or more patients) TCRs was quantified, and the CDR3 sequences were compared with those found in curated databases with known antigen specificities. RESULTS: We detected 39,303 unique TCR sequences across all samples. In PML biopsies, TCR diversity was negatively associated with a transcriptional signature of T cell mediated immune activation (p=4e-4) associated with PML outcome. Additionally, in lesions of the proliferative molecular subtype, TCR diversity was decreased in regressive versus progressive/persistent PMLs (p=0.045). Within each patient, TCRs were more likely to be shared between biopsies sampled at the same timepoint than biopsies sampled at the same anatomic location at different times. Clonally expanded TCRs, within a biopsied lesion, were more likely to be expanded at future time points than non-expanded clones. The majority of TCR sequences were found in a single sample, with only 3396 (8.6%) found in more than one sample and 1057 (2.7%) found in two or more patients (ie, public); however, when compared with a public database of CDR3 sequences, 4543 (11.6%) of TCRs were identified as public. TCRs with known antigen specificities were enriched among public TCRs (p<0.001). CONCLUSIONS: Decreased TCR diversity may reflect nascent immune responses that contribute to PML elimination. Further studies are needed to explore the potential for immunoprevention of PMLs.

Whole-genome doubling confers unique genetic vulnerabilities on tumour cells.

Whole-genome doubling (WGD) is common in human cancers, occurring early in tumorigenesis and generating genetically unstable tetraploid cells that fuel tumour development1,2. Cells that undergo WGD (WGD+ cells) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can be exploited therapeutically, is unclear. Here, using sequencing data from roughly 10,000 primary human cancer samples and essentiality data from approximately 600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent than WGD- cells on signalling from the spindle-assembly checkpoint, DNA-replication factors and proteasome function. We also identify KIF18A, which encodes a mitotic kinesin protein, as being specifically required for the viability of WGD+ cells. Although KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD- cells, its loss induces notable mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results suggest new strategies for specifically targeting WGD+ cancer cells while sparing the normal, non-transformed WGD- cells that comprise human tissue.

Prediction Error of Intraocular Lens Power Calculation in Very Elderly Patients over 90 Years Old.

BACKGROUND: To investigate the influence of age on prediction error (PE) after cataract surgery in very elderly (VE) patients aged more than 90 years. METHODS: We retrospectively analyzed 66 eyes of patients aged ≥90 years (VE group) who underwent phacoemulsification and intraocular lens (IOL) implantation. As the control group (CG), we investigated 121 eyes of patients aged 70-89 years who underwent the same surgery. PE was calculated 1 month post-surgery as the actual postoperative spherical equivalent minus the target diopter, which was calculated using the Sanders-Retzlaff-Kraff/T formula. The absolute and arithmetic PE were compared between the two groups. The factors affecting absolute PE outside ±0.5 diopter (D) and ±1.0 D were determined through logistic regression analysis with the variables age, sex, axial length (AL), average corneal power, preoperative best-corrected visual acuity, target diopter, and coexisting pseudoexfoliation syndrome. RESULTS: The absolute PE was significantly larger in the VE group than that in the CG (0.60 ± 0.52 D and 0.34 ± 0.25 D, respectively; P < .001). There was no significant difference in terms of arithmetic PE between the two groups (-0.06 ± 0.79 D and -0.07 ± 0.42 D, respectively; P = .653). In the logistic regression analysis, age was significantly associated with absolute PE outside ±0.50 D (Odds ratio [OR]: 1.05). Age and AL were significantly associated with absolute PE outside ±1.0 D (OR: 1.24 and 0.20, respectively). CONCLUSIONS: Absolute PE tended to increase in the cataract surgery of VE patients.

Genomic Profiling of Prostate Cancers from Men with African and European Ancestry.

PURPOSE: African American (AFR) men have the highest mortality rate from prostate cancer (PCa) compared with men of other racial/ancestral groups. Differences in the spectrum of somatic genome alterations in tumors between AFR men and other populations have not been well-characterized due to a lack of inclusion of significant numbers in genomic studies. EXPERIMENTAL DESIGN: To identify genomic alterations associated with race, we compared the frequencies of somatic alterations in PCa obtained from four publicly available datasets comprising 250 AFR and 611 European American (EUR) men and a targeted sequencing dataset from a commercial platform of 436 AFR and 3018 EUR men. RESULTS: Mutations in ZFHX3 as well as focal deletions in ETV3 were more frequent in tumors from AFR men. TP53 mutations were associated with increasing Gleason score. MYC amplifications were more frequent in tumors from AFR men with metastatic PCa, whereas deletions in PTEN and rearrangements in TMPRSS2-ERG were less frequent in tumors from AFR men. KMT2D truncations and CCND1 amplifications were more frequent in primary PCa from AFR men. Genomic features that could impact clinical decision making were not significantly different between the two groups including tumor mutation burden, MSI status, and genomic alterations in select DNA repair genes, CDK12, and in AR. CONCLUSIONS: Although we identified some novel differences in AFR men compared with other populations, the frequencies of genomic alterations in current therapeutic targets for PCa were similar between AFR and EUR men, suggesting that existing precision medicine approaches could be equally beneficial if applied equitably.

Decontamination of ambient RNA in single-cell RNA-seq with DecontX.

Droplet-based microfluidic devices have become widely used to perform single-cell RNA sequencing (scRNA-seq). However, ambient RNA present in the cell suspension can be aberrantly counted along with a cell's native mRNA and result in cross-contamination of transcripts between different cell populations. DecontX is a novel Bayesian method to estimate and remove contamination in individual cells. DecontX accurately predicts contamination levels in a mouse-human mixture dataset and removes aberrant expression of marker genes in PBMC datasets. We also compare the contamination levels between four different scRNA-seq protocols. Overall, DecontX can be incorporated into scRNA-seq workflows to improve downstream analyses.

Pipeliner: A Nextflow-Based Framework for the Definition of Sequencing Data Processing Pipelines.

The advent of high-throughput sequencing technologies has led to the need for flexible and user-friendly data preprocessing platforms. The Pipeliner framework provides an out-of-the-box solution for processing various types of sequencing data. It combines the Nextflow scripting language and Anaconda package manager to generate modular computational workflows. We have used Pipeliner to create several pipelines for sequencing data processing including bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, as well as digital gene expression data. This report highlights the design methodology behind Pipeliner that enables the development of highly flexible and reproducible pipelines that are easy to extend and maintain on multiple computing environments. We also provide a quick start user guide demonstrating how to setup and execute available pipelines with toy datasets.

Role of 14-3-3η protein on cardiac fatty acid metabolism and macrophage polarization after high fat diet induced type 2 diabetes mellitus.

Diabetic cardiomyopathy (DCM), a metabolic disorder, is one of the leading causes of mortality around the world and its pathogenesis involves cardiac inflammation and altered metabolic profile. Altered fatty acid metabolism during DCM can cause macrophage polarization in which inflammatory M1 phenotype dominates over the anti-inflammatory M2 phenotype. Hence, it is essential to identify a specific target, which could revert the metabolic profile and thereby reducing the M1 macrophage polarization. 14-3-3η protein has several cellular protective functions especially in the heart as plenty of reports available in various animal models of heart failure including diabetes mellitus. However, its role in the cardiac fatty acid metabolism and macrophage polarization remains unidentified. The present study has been designed to delineate the effect of cardiospecific dominant negative mutation of 14-3-3η protein (DN14-3-3) on various lipid metabolism related marker proteins expressions and cardiac macrophage phenotype in high fat diet (HFD) fed mice. Feeding HFD for 12 weeks has produced significant increase in body weight in the DN14-3-3 (TG) mice than C57BL6/J (WT) mice. Western blotting and immunohistochemical staining analysis of the heart tissue has revealed an increase in the expression of markers of cardiac fatty acid synthesis related proteins in addition to the reduced expression of fatty acid oxidation related proteins in TG mice fed HFD than WT mice fed HFD. Furthermore, the M1 macrophage marker proteins were increasingly expressed while M2 markers expressions were reduced in the hearts of TG mice fed HFD. In conclusion, our current study has identified that there is a definite role for the 14-3-3η protein against the pathogenesis of heart failure via regulation of cardiac fatty acid metabolism and macrophage polarization.

Profiling of transcription factor binding events by chromatin immunoprecipitation sequencing (ChIP-seq).

In multi-cellular organisms, gene expression is orchestrated by thousands of transcription factors (TF). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a robust tool to investigate gene expression because this technique profiles in vivo protein-DNA interaction at a genome-wide scale. Eight years after the first ChIP-seq paper, there are limited reports of ChIP-seq experiments in plants, especially for sequence-specific DNA binding TFs This lag greatly prevents our understanding of transcriptional regulation in an entire kingdom. In order to bridge the technical gap, we describe a ChIP-seq procedure that we have successfully applied to dozens of sequence-specific DNA binding TFs. The basic protocol includes procedures to isolate nuclei, sonicate chromatin, immunoprecipitate TF-DNA complex, and recover ChIP-enriched DNA fragments. The support protocol also describes practices to optimize library preparation by a gel-free DNA size selection. Lastly, examples are given to optimize library amplification using real-time PCR.

Association between reduction of plasma adiponectin levels and risk of bacterial infection after gastric cancer surgery.

BACKGROUND AND PURPOSE: Infections are important causes of postoperative morbidity after gastric surgery; currently, no factors have been identified that can predict postoperative infection. Adiponectin (ADN) mediates energy metabolism and functions as an immunomodulator. Perioperative ADN levels and perioperative immune functioning could be mutually related. Here we evaluated a potential biological marker to reliably predict the incidence of postoperative infections to prevent such comorbidities. METHODS: We analyzed 150 consecutive patients who underwent elective gastric cancer surgery at the Shiga University of Medical Science Hospital (Shiga, Japan) from 1997 to 2009; of these, most surgeries (n = 100) were performed 2008 onwards. The patient characteristics and surgery-related factors between two groups (with and without infection) were compared by the paired t-test and χ(2) test, including preoperative ADN levels, postoperative day 1 ADN levels, and ADN ratio (postoperative ADN levels/preoperative ADN levels) as baseline factors. Logistic regression analysis was performed to access the independent association between ADN ratio and postoperative infection. Finally, receiver operating curves (ROCs) were constructed to examine its clinical utility. RESULTS: Sixty patients (40%) experienced postoperative infections. The baseline values of age, American Society of Anesthesiologists physical status, total operating time, blood loss, surgical procedure, C-reactive protein (CRP) levels, preoperative ADN levels, and ADN ratio were significantly different between groups. Logistic regression analysis using these factors indicated that type 2 diabetes mellitus (T2DM) and ADN ratio were significantly independent variables (*p<0.05, ** p<0.01, respectively). ROC analysis revealed that the useful cutoff values (sensitivity/specificity) for preoperative ADN levels, ADN ratio, blood loss, operating time, and CRP levels were 8.81(0.567/0.568), 0.76 (0.767/0.761), 405 g (0.717/0.693), 342 min (0.617/0.614), and 8.94 mg/dl (0.583/0.591), respectively. CONCLUSION: T2DM and ADN ratio were independent predictors of postoperative infection and ADN ratio was the most useful predictor for postoperative infection.

Changes in catecholamines and dopaminergic metabolites in pigeon brain during development from the late embryonic stage toward hatch.

While brain development during embryogenesis has been extensively studied in precocial birds, there is no information available on altricial birds. Thus, the concentrations of the catecholamines norepinephrine (NE), epinephrine (E), and dopamine (DA), and the dopaminergic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA) were determined at several stages during the late embryonic period (E13, E14, E15, E16, E17 and E18) and the day-of-hatch (P0) in the pigeon telencephalon, cerebellum, optic lobe, and brainstem. The concentrations of all catecholamines were higher than those reported in chicken embryos. During embryogenesis, NE, E, DOPAC and HVA concentrations in the various brain parts increased throughout embryonic development until shortly before hatching at which time they decreased. DA, however, continued to increase through hatching in the brainstem, and the changes in DA concentrations varied in several brain parts. In conclusion, catecholamine concentrations in the various brain parts tended to increase with embryonic age, and the concentrations were higher than those in chickens. Furthermore, brain catcholamine metabolism changed at hatch in pigeons.

Beta 3-adrenergic receptor is involved in feeding regulation in chicks.

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.