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      Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model

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          Abstract

          Sensorineural hearing loss is a common disability often caused by the loss of sensory hair cells in the cochlea. Hair cell (HCs) regeneration has long been the main target for the development of novel therapeutics for sensorineural hearing loss. In the mammalian cochlea, hair cell regeneration is limited, but the auditory epithelia of non-mammalian organisms retain the capacity for hair cell regeneration. In the avian basilar papilla (BP), supporting cells (SCs), which give rise to regenerated hair cells, are usually quiescent. Hair cell loss induces both direct transdifferentiation and mitotic division of supporting cells. Here, we established an explant culture model for hair cell regeneration in chick basilar papillae and validated it for investigating the initial phase of hair cell regeneration. The histological assessment demonstrated hair cell regeneration via direct transdifferentiation of supporting cells. Labeling with 5-ethynyl-2′-deoxyuridine (EdU) revealed the occurrence of mitotic division in the supporting cells at specific locations in the basilar papillae, while no EdU labeling was observed in newly generated hair cells. RNA sequencing indicated alterations in known signaling pathways associated with hair cell regeneration, consistent with previous findings. Also, unbiased analyses of RNA sequencing data revealed novel genes and signaling pathways that may be related to the induction of supporting cell activation in the chick basilar papillae. These results indicate the advantages of our explant culture model of the chick basilar papillae for exploring the molecular mechanisms of hair cell regeneration.

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          Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

          In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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            STAR: ultrafast universal RNA-seq aligner.

            Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.
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              Fast gapped-read alignment with Bowtie 2.

              As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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                Author and article information

                Contributors
                Journal
                Front Cell Neurosci
                Front Cell Neurosci
                Front. Cell. Neurosci.
                Frontiers in Cellular Neuroscience
                Frontiers Media S.A.
                1662-5102
                12 November 2020
                2020
                : 14
                : 583994
                Affiliations
                [1] 1Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kyoto University , Kyoto, Japan
                [2] 2Medical Innovation Center, Kyoto University , Kyoto, Japan
                Author notes

                Edited by: Taha A. Jan, University of California, San Francisco, United States

                Reviewed by: Zheng-Yi Chen, Massachusetts Eye and Ear Infirmary and Harvard Medical School, United States; Mark E. Warchol, Washington University School of Medicine in St. Louis, United States; Andy Groves, Baylor College of Medicine, United States

                *Correspondence: Takayuki Nakagawa tnakagawa@ 123456ent.kuhp.kyoto-u.ac.jp

                Specialty section: This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience

                Article
                10.3389/fncel.2020.583994
                7688741
                33281558
                2987a332-44c0-48a2-abaf-f0962d4ea4b4
                Copyright © 2020 Matsunaga, Kita, Yamamoto, Yamamoto, Okano, Omori, Sakamoto and Nakagawa.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 16 July 2020
                : 14 October 2020
                Page count
                Figures: 6, Tables: 0, Equations: 0, References: 86, Pages: 19, Words: 13419
                Funding
                Funded by: Japan Society for the Promotion of Science 10.13039/501100001691
                Funded by: Japan Agency for Medical Research and Development 10.13039/100009619
                Categories
                Cellular Neuroscience
                Original Research

                Neurosciences
                atoh1,basilar papilla,regeneration,hair cell,supporting cell,transdifferentiation,type 1 interferon

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