Characterizing noise structure in single-cell RNA-seq distinguishes genuine from technical stochastic allelic expression.

Single-cell RNA-sequencing (scRNA-seq) facilitates identification of new cell types and gene regulatory networks as well as dissection of the kinetics of gene expression and patterns of allele-specific expression. However, to facilitate such analyses, separating biological variability from the high level of technical noise that affects scRNA-seq protocols is vital. Here we describe and validate a generative statistical model that accurately quantifies technical noise with the help of external RNA spike-ins. Applying our approach to investigate stochastic allele-specific expression in individual cells, we demonstrate that a large fraction of stochastic allele-specific expression can be explained by technical noise, especially for lowly and moderately expressed genes: we predict that only 17.8% of stochastic allele-specific expression patterns are attributable to biological noise with the remainder due to technical noise

Corrigendum: Characterizing noise structure in single-cell RNA-seq distinguishes genuine from technical stochastic allelic expression.

Nature Communications 6: Article number: 8687 (2015); Published: 22 October 2015; Updated: 11 January 2016. The original version of this Article contained an error in the spelling of the author Tomislav Ilicic, which was incorrectly given as Tomislav Illicic. This has now been corrected in both the PDF and HTML versions of the Article.</jats:p

Flipping between Polycomb repressed and active transcriptional states introduces noise in gene expression.

Polycomb repressive complexes (PRCs) are important histone modifiers, which silence gene expression; yet, there exists a subset of PRC-bound genes actively transcribed by RNA polymerase II (RNAPII). It is likely that the role of Polycomb repressive complex is to dampen expression of these PRC-active genes. However, it is unclear how this flipping between chromatin states alters the kinetics of transcription. Here, we integrate histone modifications and RNAPII states derived from bulk ChIP-seq data with single-cell RNA-sequencing data. We find that Polycomb repressive complex-active genes have greater cell-to-cell variation in expression than active genes, and these results are validated by knockout experiments. We also show that PRC-active genes are clustered on chromosomes in both two and three dimensions, and interactions with active enhancers promote a stabilization of gene expression noise. These findings provide new insights into how chromatin regulation modulates stochastic gene expression and transcriptional bursting, with implications for regulation of pluripotency and development.Polycomb repressive complexes modify histones but it is unclear how changes in chromatin states alter kinetics of transcription. Here, the authors use single-cell RNAseq and ChIPseq to find that actively transcribed genes with Polycomb marks have greater cell-to-cell variation in expression

Single Cell RNA-Sequencing of Pluripotent States Unlocks Modular Transcriptional Variation

SummaryEmbryonic stem cell (ESC) culture conditions are important for maintaining long-term self-renewal, and they influence cellular pluripotency state. Here, we report single cell RNA-sequencing of mESCs cultured in three different conditions: serum, 2i, and the alternative ground state a2i. We find that the cellular transcriptomes of cells grown in these conditions are distinct, with 2i being the most similar to blastocyst cells and including a subpopulation resembling the two-cell embryo state. Overall levels of intercellular gene expression heterogeneity are comparable across the three conditions. However, this masks variable expression of pluripotency genes in serum cells and homogeneous expression in 2i and a2i cells. Additionally, genes related to the cell cycle are more variably expressed in the 2i and a2i conditions. Mining of our dataset for correlations in gene expression allowed us to identify additional components of the pluripotency network, including Ptma and Zfp640, illustrating its value as a resource for future discovery

Classification of low quality cells from single-cell RNA-seq data.

Single-cell RNA sequencing (scRNA-seq) has broad applications across biomedical research. One of the key challenges is to ensure that only single, live cells are included in downstream analysis, as the inclusion of compromised cells inevitably affects data interpretation. Here, we present a generic approach for processing scRNA-seq data and detecting low quality cells, using a curated set of over 20 biological and technical features. Our approach improves classification accuracy by over 30 % compared to traditional methods when tested on over 5,000 cells, including CD4+ T cells, bone marrow dendritic cells, and mouse embryonic stem cells

Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

BACKGROUND: Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. RESULTS: In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a (+/+) and Bcl11a (-/-)). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a (-/-) HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a (-/-) HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a (-/-) HSCs are myeloerythroid-restricted and defective in self-renewal. CONCLUSIONS: Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment

The role of the microbiome in NAFLD and NASH

Abstract Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of cardiometabolic syndrome, which often also includes obesity, diabetes, and dyslipidemia. It is rapidly becoming the most prevalent liver disease worldwide. A sizable minority of NAFLD patients develop nonalcoholic steatohepatitis (NASH), which is characterized by inflammatory changes that can lead to progressive liver damage, cirrhosis, and hepatocellular carcinoma. Recent studies have shown that in addition to genetic predisposition and diet, the gut microbiota affects hepatic carbohydrate and lipid metabolism as well as influences the balance between pro‐inflammatory and anti‐inflammatory effectors in the liver, thereby impacting NAFLD and its progression to NASH. In this review, we will explore the impact of gut microbiota and microbiota‐derived compounds on the development and progression of NAFLD and NASH, and the unexplored factors related to potential microbiome contributions to this common liver disease

Bioactivity Potential of Prunus spinosa L. Flower Extracts: Phytochemical Profiling, Cellular Safety, Pro-inflammatory Enzymes Inhibition and Protective Effects Against Oxidative Stress In Vitro

Flower extracts of Prunus spinosa L. (blackthorn)—a traditional medicinal plant of Central and Eastern Europe indicated for the treatment of urinary tract disorders, inflammation, and adjunctive therapy of cardiovascular diseases—were evaluated in terms of chemical composition, antioxidant activity, potential anti-inflammatory effects, and cellular safety in function of fractionated extraction. The UHPLC-PDA-ESI-MS3 fingerprinting led to full or partial identification of 57 marker constituents (36 new for the flowers), mostly flavonoids, A-type proanthocyanidins, and phenolic acids, and provided the basis for authentication and standardization of the flower extracts. With the contents up to 584.07 mg/g dry weight (dw), 490.63, 109.43, and 66.77 mg/g dw of total phenolics (TPC), flavonoids, proanthocyanidins, and phenolic acids, respectively, the extracts were proven to be rich sources of polyphenols. In chemical in vitro tests of antioxidant (DPPH, FRAP, TBARS) and enzyme (lipoxygenase and hyaluronidase) inhibitory activity, the extracts effects were profound, dose-, phenolic-, and extraction solvent-dependent. Moreover, at in vivo-relevant levels (1–5 μg/mL) the extracts effectively protected the human plasma components against peroxynitrite-induced damage (reduced the levels of oxidative stress biomarkers: 3-nitrotyrosine, lipid hydroperoxides, and thiobarbituric acid-reactive substances) and enhanced the total antioxidant status of plasma. The effects observed in biological models were in general dose- and TPC-dependent; only for protein nitration the relationships were not significant. Furthermore, in cytotoxicity tests, the extracts did not affect the viability of human peripheral blood mononuclear cells (PBMC), and might be regarded as safe. Among extracts, the defatted methanol-water (7:3, v/v) extract and its diethyl ether and ethyl acetate fractions appear to be the most advantageous for biological applications. As compared to the positive controls, activity of the extracts was favorable, which might be attributed to some synergic effects of their constituents. In conclusion, this research proves that the antioxidant and enzyme inhibitory capacity of phenolic fractions should be counted as one of the mechanisms behind the activity of the flowers reported by traditional medicine and demonstrates the potential of the extracts as alternative ingredients for functional products supporting the treatment of oxidative stress-related pathologies cross-linked with inflammatory changes, especially in cardiovascular protection

Polyphenol-Rich Extracts from Cotoneaster Leaves Inhibit Pro-Inflammatory Enzymes and Protect Human Plasma Components against Oxidative Stress In Vitro

The present study investigated the phenolic profile and biological activity of dry extracts from leaves of C. bullatus, C. zabelii and C. integerrimus&mdash;traditional medicinal and dietary plants&mdash;and evaluated their potential in adjunctive therapy of cardiovascular diseases. Complementary UHPLC-PDA-ESI-MS3, HPLC-PDA-fingerprint, Folin-Ciocalteu, and n-butanol/HCl assays of the extracts derived by fractionated extraction confirmed that they are rich in structurally diverse polyphenols (47 analytes, content up to 650.8 mg GAE/g dw) with proanthocyanidins (83.3&ndash;358.2 mg CYE/g) dominating in C. bullatus and C. zabelii, and flavonoids (53.4&ndash;147.8 mg/g) in C. integerrimus. In chemical in vitro tests of pro-inflammatory enzymes (lipoxygenase, hyaluronidase) inhibition and antioxidant activity (DPPH, FRAP), the extracts effects were dose-, phenolic- and extraction solvent-dependent. The most promising polyphenolic extracts were demonstrated to be effective antioxidants in a biological model of human blood plasma&mdash;at in vivo-relevant levels (1&ndash;5 &micro;g/mL) they normalized/enhanced the non-enzymatic antioxidant capacity of plasma and effectively prevented peroxynitrite-induced oxidative/nitrative damage of plasma proteins and lipids. As demonstrated in cytotoxicity tests, the extracts were safe&mdash;they did not affect viability of human peripheral blood mononuclear cells. In conclusion, Cotoneaster leaves may be useful in development of natural-based products, supporting the treatment of oxidative stress/inflammation-related chronic diseases, including cardiovascular disorders