COTAN : ScRNA-seq data analysis based on gene co-expression

Estimating the co-expression of cell identity factors in single-cell is crucial. Due to the low efficiency of scRNA-seq methodologies, sensitive computational approaches are critical to accurately infer transcription profiles in a cell population. We introduce COTAN, a statistical and computational method, to analyze the co-expression of gene pairs at single cell level, providing the foundation for single-cell gene interactome analysis. The basic idea is studying the zero UMI counts' distribution instead of focusing on positive counts; this is done with a generalized contingency tables framework. COTAN can assess the correlated or anti-correlated expression of gene pairs, providing a new correlation index with an approximate p-value for the associated test of independence. COTAN can evaluate whether single genes are differentially expressed, scoring them with a newly defined global differentiation index. Similarly to correlation network analysis, it provides ways to plot and cluster genes according to their co-expression pattern with other genes, effectively helping the study of gene interactions, becoming a new tool to identify cell-identity markers. We assayed COTAN on two neural development datasets with very promising results. COTAN is an R package that complements the traditional single cell RNA-seq analysis and it is available at https://github.com/seriph78/COTAN

Assessment of antibody library diversity through next generation sequencing and technical error compensation

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.Funded by European Union Seventh Framework Program [grant No. 604102 A.C.] (Human Brain Project). https://www.humanbrainproject.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

BACKGROUND: Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. RESULTS: We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. CONCLUSIONS: We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.This work was funded by: FIRB RBAP10L8TY (MIUR), Fondazione Roma and PAINCAGE FP7 Collaborative Project number 603191 (RB,MD); Flagship Project InterOmics PB.05 and MIUR-PRIN-2012 (FC); Wellcome Trust Core Grant reference 092096 and Cancer Research UK Grant Reference C6946/A14492 (LP); CRUK-Cambridge Institute Core Grant reference C14303/A17197 (DB)

MicroRNAs and cell fate in cortical and retinal development.

MicroRNAs (miRNAs) are involved in crucial steps of neurogenesis, neural differentiation, and neuronal plasticity. Here we review experimental evidence suggesting that miRNAs may regulate the histogenesis of the cerebral cortex and neural retina. Both cortical and retinal early progenitor cells are multipotent, that is, they can generate different types of cortical or retinal cells, respectively, in one lineage. In both cortical and retinal development, the precise timing of activation of cell fate transcription factors results in a stereotyped schedule of generation of the different types of neurons. Emerging evidence indicates that miRNAs may play an important role in regulating such temporal programing of neuronal differentiation. Neuronal subtypes of the cortex and retina exhibit distinct miRNA signatures, implying that miRNA codes may be used to specify different types of neurons. Interfering with global miRNA activity changes the ratio of the different types of neurons produced. In fact, there are examples of cell fate genes that are regulated at the translational level, both in retinogenesis and in corticogenesis. A model depicting how miRNAs might orchestrate both the type and the birth of different neurons is presented and discussed. Glossary. • Lineage: the temporally ordered cell progeny of an individual progenitor cell. • Specification: the (reversible) process by which a cell becomes capable of, and biased toward, a particular fate. • Commitment: the process by which cell fate is fully determined and can no longer be affected by external cues. • Potency: the entire complement of cells that a progenitor can ultimately produce. • Multipotency: the ability to give rise to more than one cell type. • Progenitor: a dividing cell that, in contrast to a stem cell, cannot proliferate indefinitely. • Antago-miR: modified antisense oligonucleotide that blocks the activity of a miRNA. • Heterochronic neuron: type of neurons that is generated at inappropriate times of development. • Neuron birth date: the time of the last mitosis of a neuronal cell

Timing neurogenesis by cell cycle

Animals; Anura; Cell Cycle; Hedgehog Proteins; Hedgehog Proteins: metabolism; Mice; MicroRNAs; MicroRNAs: metabolism; Models; Biological; Neurogenesis; Retinal Neurons; Retinal Neurons: cytology; Retinal Neurons: metabolism; Time Factor

Dystroglycan is required for proper retinal layering

Dystroglycan (DG) is a transmembrane receptor linking the extracellular matrix to the internal cytoskeleton. Its structural function has been mainly characterized in muscle fibers, but DG plays signaling and developmental roles also in different tissues and cell types. We have investigated the effects of dystroglycan depletion during eye development of Xenopus laevis. We have injected a specific morpholino (Mo) antisense oligonucleotide in the animal pole of one dorsal blastomere of embryos at four cells stage. Mo-mediated loss of DG function caused disruption of the basal lamina layers, increased apoptosis and reduction of the expression domains of specific retinal markers, at early stages. Later in development, morphants displayed unilateral ocular malformations, such as microphtalmia and retinal delayering with photoreceptors and ganglion cells scattered throughout the retina or aggregated in rosette-like structures. These results recall the phenotypes observed in specific human diseases and suggest that DG presence is crucial at early stages for the organization of retinal architecture