G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

The transcription factor JUN is highly expressed in pulmonary fibrosis. Its induction in mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and secretome of cells from patients with pulmonary fibrosis. We show that JUN is activated in fibrotic fibroblasts that expressed increased CD47 and PD-L1. Using ATAC-seq and ChIP-seq, we found that activation of JUN rendered promoters and enhancers of CD47 and PD-L1 accessible. We further detect increased IL-6 that amplified JUN-mediated CD47 enhancer activity and protein expression. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47 and PD-L1 reversed fibrosis, by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote fibrosis and suggest a therapeutic approach that could be used alongside conventional anti-fibrotics for pulmonary fibrosis.

SummaryNeuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.

Neuronal differentiation plays a key role during embryogenesis. However, based on the capacity of neuronal stem cells to either generate or regenerate neurons and because differentiation stops aberrant neuroblasts proliferation, neuronal differentiation is crucial during neuropathological conditions. Although phosphatidylcholine (PtdCho) has been proposed as an important molecule for neurite growth and neuronal regeneration, the identity of the molecular target has remained elusive. This study originally describes that lysophosphatidylcholine (LPtdCho), either exogenously supplied or generated by the imbalance of PtdCho metabolism through the enzymatic action of cytosolic phospholipase A, acts as a neurotrophic-like factor. We demonstrated that LPtdCho induces neuronal differentiation by activation of the small G protein Ras followed by the Raf/MEK/ERK signaling pathway. Accordingly, LPtdCho redirects neuroblasts gene expression leading to the generation of functional mature neurons expressing βIII-tubulin and having increased acetylcholinesterase activity and membrane biosynthesis required for neuritogenesis. These findings provide mechanistic details of the role of cytidine-5-diphosphocholine (CDP-choline) and PtdCho as neuroprotectors. Furthermore, as LPtdCho recapitulates the effect of the therapeutic agent retinoic acid, these results open new avenues for drug discovery for the treatment of neuropathological conditions.

Key Points CRISPR gene editing of blood stem and progenitor cells induces high-efficiency t(9;11) chromosomal translocation and MLLr leukemia. Gene-edited leukemia analysis documents clonal selection in leukemia progression and similarity of MLLr MPALs to AMLs vs ALLs.

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In pulmonary fibrosis, the transcription factor JUN is highly expressed in the fibrotic foci. Its induction in adult mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and the secretome of individual fibroblasts and leukocytes from pulmonary fibrosis patients. We show that JUN is activated in fibroblasts derived from fibrotic lungs which also demonstrated increased CD47 and PD-L1 expression. Using ATAC-seq and ChIP-seq, we found that activation of JUN in fibroblasts rendered enhancers of CD47 and PD-L1 accessible, an observation that reporter assays corroborated.Meanwhile we detected increased IL-6 signaling which amplified both JUN-mediated CD47-enhancer activity and protein expression in fibrotic lung fibroblasts. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47, and PD-L1 reversed fibrosisincreased phagocytosis of profibrotic fibroblasts and elimination of suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote the fibrotic process and suggest a complementary therapeutic approach that could be used alongside conventional antifibrotics for pulmonary fibrosis diseases.

The application of machine learning and artificial intelligence to high‐dimensional cytometry data sets has increasingly become a staple of bioinformatic data analysis over the past decade. This is especially true in the field of cancer biology, where protocols for collecting multiparameter single‐cell data in a high‐throughput fashion are rapidly developed. As the use of machine learning methodology in cytometry becomes increasingly common, there is a need for cancer biologists to understand the basic theory and applications of a variety of algorithmic tools for analyzing and interpreting cytometry data. We introduce the reader to several keystone machine learning‐based analytic approaches with an emphasis on defining key terms and introducing a conceptual framework for making translational or clinically relevant discoveries. The target audience consists of cancer cell biologists and physician‐scientists interested in applying these tools to their own data, but who may have limited training in bioinformatics. © 2020 International Society for Advancement of Cytometry