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Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases 1,2 . Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired 3,4 . However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10 -8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors., (© 2023. The Author(s).)

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases 1-4 . Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired 5 . However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging., Competing Interests: Competing Interests A provisional patent application on HiDEF-seq has been filed (NYU Grossman School of Medicine). G.D.E. owns stock in DNA sequencing companies (Illumina, Oxford Nanopore Technologies, and Pacific Biosciences).

Neural precursor cells (NPCs) transplanted into the adult neocortex generate neurons that synaptically integrate with host neurons, supporting the possibility of achieving functional tissue repair. However, poor survival and functional neuronal recovery of transplanted NPCs greatly limits engraftment. Here, we test the hypothesis that combining blood vessel-forming vascular cells with neuronal precursors improves engraftment. By transplanting mixed embryonic neocortical cells into adult mice with neocortical strokes, we show that transplant-derived neurons synapse with appropriate targets while donor vascular cells form vessels that fuse with the host vasculature to perfuse blood within the graft. Although all grafts became vascularized, larger grafts had greater contributions of donor-derived vessels that increased as a function of their distance from the host-graft border. Moreover, excluding vascular cells from the donor cell population strictly limited graft size. Thus, inclusion of vessel-forming vascular cells with NPCs is required for more efficient engraftment and ultimately for tissue repair., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis. SIGNIFICANCE STATEMENT How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells., (Copyright © 2021 the authors.)

Fibroblast Growth Factor Receptors (FGFRs) play crucial roles in promoting dendrite growth and branching during development. In mice, three FGFR genes, Fgfr1, Fgfr2, and Fgfr3, remain expressed in the adult neurogenic niche of the hippocampal dentate gyrus. However, the function of FGFRs in the dendritic maturation of adult-born neurons remains largely unexplored. Here, using conditional alleles of Fgfr1, Fgfr2, and Fgfr3 as well as Fgfr1 alleles lacking binding sites for Phospholipase-Cγ (PLCγ) and FGF Receptor Substrate (FRS) proteins, we test the requirement for FGFRs in dendritogenesis of adult-born granule cells. We find that deleting all three receptors results in a small decrease in proximal dendrite elaboration. In contrast, specifically mutating Tyr766 in FGFR1 (a PLCγ binding site) in an Fgfr2;Fgfr3 deficient background results in a dramatic increase of overall dendrite elaboration, while blocking FGFR1-FRS signaling causes proximal dendrite deficits and, to a lesser extent than Tyr766 mutants, increases distal dendrite elaboration. These findings reveal unexpectedly complex roles for FGFRs and their intracellular mediators in regulating proximal and distal dendrite elaboration, with the most notable role in suppressing distal elaboration through the PLCγbinding site., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

L1 is an immunoglobulin domain (Ig)-containing protein essential for a wide range of neurodevelopmental processes highly conserved across species from worms to humans. L1 can act as a cell adhesion molecule by binding to other Ig-containing proteins or as a ligand for certain tyrosine kinase receptors such as FGFRs and TRKs, which are required not only during neurodevelopment but also in hippocampal neurogenesis. Yet, the role of L1 itself in adult hippocampal neurogenesis remains unaddressed. Here, we used several Cre-driver lines in mice to conditionally delete a floxed allele of L1cam at different points along the differentiation lineage of new neurons and in surrounding neurons in the adult dentate gyrus of the hippocampus. We found that L1cam deletion in stem/progenitor cells increased: 1) the differentiation of progenitors into new neurons, 2) the complexity of dendritic arbors in immature neurons, and 3) anxiety-related behavior. In addition, deletion of L1cam in neurons leads to an earlier age-related decline in hippocampal neurogenesis. These data suggest that L1 is not only important for normal nervous system development, but also for maintaining certain neural processes in adulthood., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)