MUC4, MUC6, COL4A4, MYO7A, AKAP12, COL11A1, DSPP, ESPN, GPR98, PCDH15, BSN, CACNA1D, TPRN, and USH1C for microtia (P = 2.51 × 10−4). Among these genes, COL4A4 and COL11A1 may lead to microtia through focal adhesion pathway and ECM-receptor interaction pathway which are connected to the downstream Wnt signaling pathway. The present results indicate that certain genes may affect both external/middle and inner ear development, and demonstrate the benefits of using a capture array in microtia patients.Microtia is a congenital malformation of the external ear caused by genetic and/or environmental factors. However, no causal genetic mutations have been identified in isolated microtia patients. In this study, we utilized targeted genomic capturing combined with next-generation sequencing to screen for mutations in 307 deafness genes in 32 microtia patients. Forty-two rare heterozygous mutations in 25 genes, including 22 novel mutations in 24 isolated unilateral microtia cases were identified. Pathway analysis found five pathways especially focal adhesion pathway and ECM-receptor interaction pathway were significantly associated with microtia. The low-frequency variants association study was used and highlighted several strong candidate genes  Microtia is a congenital developmental malformation of the external ear that ranges in severity from mild differences in auricular shape and size to complete absence of the external ear with atresia or stenosis of the auditory canal ,2. PrevaMicrotia can occur as an isolated birth defect or as part of a spectrum of anomalies or a syndrome. The most common associated malformations are cleft lip and/or palate, limb reduction defects, renal anomalies, vertebral abnormalities, and cardiac defects ,4,8. AltIn addition, many syndromic microtia patients present with inner ear malformations, including branchio-oculofacial, branchio-oto-renal/branchio-otic, CHARGE , Nager , Treacher Collins, Wildervanck (cervico-oculo-acoustic), LAMM  and lacrimo-auriculodento-digital (LADD) syndromes, among others. The frequent malformations of the inner ear are common cavity, cochlear aplasia, Mondini defect, enlarged vestibular aqueduct, abnormal/absent semicircular canals, and bulbous IAC  . ConductTo date, no studies have reported screens of deafness genes in microtia patients. The technology of targeted genomic capturing combined with next-generation sequencing (NGS) has proven to be effective and feasible and has been successfully used in several studies ,11. HereA total of 4899 exons comprising 2,362,343 bases  were captured and sequenced in our study. After alignment to the reference human genome (NCBI37/hg19), 70.93% of the clean reads could be matched to the targeted regions. The average read depth for the targeted regions was 394.5-fold, and 97.68% of the targeted regions were covered by 20 or more reads, demonstrating the high quality of the sequencing. A detailed coverage analysis for each sample is provided in DIAPH1 (diaphanous-related formin 1), c.3300_3301delAA (p.K1103Afs*16) in ALMS1 (Alstrom syndrome 1), and c.6072delG (p.R2025Afs*6) in OTOG (otogelin). One mutation, c.4248C>A in MYO7A (myosin VIIA), was predicted to lead to a truncated protein owing to a premature stop codon. Mutations in ALMS1, COL4A4 , GPR98 (G protein-coupled receptor 98), MYO15A, MYO7A, and USH1C (Usher syndrome 1C) genes occurred in two or more cases (Allele frequency ≥ 1.56%) . An alignment of a selected region of the HOXA1 protein showed that amino acid 115 exhibited very high evolutionary conservation , which introduces a positively charged arginine for the negatively charged glutamine in the wild-type protein, could cause repulsion of ligands or other residues with the same charge.In addition, two intronic variants were identified; one, a c.8559-2A>G mutation in Wnt signaling pathway. Based on these considerations, we propose that both pathways are linked to microtia (http://www.genome.jp/kegg/pathway.html).To further determine the signaling pathways in which the 25 rare mutated genes found in microtia patients are involved, we performed a KEGG pathway enrichment analysis. This analysis revealed that five pathways are significantly associated with isolated unilateral microtia: the protein digestion and absorption pathway, the focal adhesion pathway, the extracellular matrix (ECM)-receptor interaction pathway, the amoebiasis pathway, and the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway , MUC4 (mucin 4), MUC6, COL4A4, MYO7A, AKAP12 (A-kinase anchoring protein 12), COL11A1, DSPP , ESPN (espin), GPR98, PCDH15 (protocadherin 15), BSN (bassoon), CACNA1D , TPRN (taperin), and USH1C were identified as significantly associated with isolated unilateral microtia .To test whether low-frequency mutations exist that might be risk factors for microtia, we conducted an association analysis of 32 isolated unilateral microtia cases and 208 healthy controls from China. After correction for multiple testing  plays a critical role in driving facial outgrowth and morphogenesis, including that of the external and inner ear . The extUSH2A, has been reported to be pathogenic; when combined with p.T3936P or p.R34fs as a compound heterozygous mutation, it results in Usher syndrome type II [In the current study, we screened all exons of 307 deafness-associated genes, identifying 42 rare or novel mutations of 25 genes in isolated unilateral microtia patients. All of these mutations add to the database of potential pathogenic mutations in isolated unilateral microtia cases. Among these muations, only one, c.8559-2A>G in  type II . This inThe wingless/INT (Wnts) signaling pathway, together with bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs), have been reported to be involved in external ear development . Here, tMUC4, MUC6, COL4A4, MYO7A, AKAP12, COL11A1, DSPP, ESPN, GPR98, PCDH15, BSN, CACNA1D, TPRN, and USH1C. Of these genes, two were related to the pathways we identified using KEGG pathway enrichment analysis. COL4A4 was related to the pathways for ECM-receptor interaction and focal adhesion, and COL11A1 was related to protein digestion and absorption. Thus we suggest that the two genes, COL4A4 and COL11A1, may be strongly involved in the mechanism of microtia and are worth exploring in the context of the pathogenesis of microtia.In addition to exploring rare mutations that may cause microtia, we also employed associated studies to identify potential pathogenic genes among low-frequency variants. The contribution of rare and low-frequency variants to human traits has been largely unexplored ,18. UsinHOXA1 identified in one isolated unilateral microtia patient also attracted our interest. It resulted in substitution of an arginine for a glutamine at position 115 (Q115R), which is highly evolutionarily conserved. A structural analysis of HOXA1 protein performed using the HOPE server indicated that this mutation, which replaces a negatively charged residue in the wild-type with a positively charge amino acid, could cause repulsion of ligands or other residues with the same charge. Hox genes encode proteins that share a 60-amino-acid domain called the homeodomain. HoxA cluster genes play a fundamental role in building the sensorimotor circuitry in vertebrates. Mutations in either HOXA1 or HOXA2 have reported to result in external ear deformities, although the phenotypes caused by HOXA1 mutations are more severe than those caused by HOXA2 mutations. Diseases associated with HOXA1 mutations include Athabaskan Brainstem Dysgenesis Syndrome and Bosley-Salih-Alorainy syndrome [Additionally, a novel heterozygous mutation (c. 344A>G) in syndrome ,22.HOXA2 gene as a genetic cause of isolated microtia. Identification of pathogenic mutations in HOXA2 was reported in three different isolated bilateral microtia families [HOXA2 may be among genes responsible for the pathogenesis of isolated microtia. However, studies that have focused on sporadic microtia patients have found no pathogenic mutations in HOXA2 [HOXA2, suggesting that there may be distinct etiologies that result in isolated microtia.A small number of studies have also focused on the families –25, promin HOXA2 ,27. ConsIn conclusion, some deafness genes may be associated with microtia, and certain genes may affect both external/middle and inner ear development. Pathway analysis showed that two pathways—the focal adhesion and ECM-receptor interaction pathways—were associated with microtia. The deafness genes panel has the potential to enable the discovery of pathogenic genes in microtia patients and can be reliably used in such patients.Thirty-two isolated unilateral microtia patients  were recruited from Peking Union Medical College Hospital (PUMCH). They were all classified as grade III external ear deformities, according to the classification by the Marx classification system , and hadEthical approval for this study was obtained from the institutional review board of PUMCH. All subjects or their parents gave written, informed consent to participate in this study.Patients’ genomic DNA (gDNA) was extracted from blood samples using a Blood DNA kit , and 1 ug of purified gDNA was fragmented into 200–300-base-pair lengths using an ultrasonoscope. Libraries were prepared by performing end-repair, adenylation, and adapter ligation using a protocol provided by the manufacturer of the NGS system .A customized capture array (Roche-NimbleGen) containing 307 deafness genes was designed to capture all exons and flanking intron (±10 bp) sequences of the target genes . The samIllumina Pipeline (version 1.9.4) was used to perform image analysis, error estimation and base calling, yielding the primary data. After removing low-quality reads from the primary data using a local algorithm, data analysis and bioinformatics processing were performed based on the reference sequences of the NCBI37/hg19 assembly of the human genome using the Burrows-Wheeler alignment tool. Single nucleotide variations (SNVs) and small insertions and deletions (Indels) were detected using GATK , and in-house scripts were used for annotation of variants.http://www.umd.be/HSF/). The HOPE server (http://www.cmbi.ru.nl/hope/) was used to analyze and predict structural variations in mutant HOXA1 (homeobox A1) protein and the ConSeq server was used for determining the conservation of HOXA1 amino acid position 115. Additionally, all rare and novel mutations were checked against the Human Gene Mutation Database (HGMD) to determine whether any identified mutations had been reported to be pathogenic.To identify the most likely pathogenic mutations, we filtered out 1) synonymous and non-coding variants (with the exception of splicing site mutations that might create an ectopic splicing site); 2) variants with allele frequencies of 0.0005 or higher in the dbSNP, 1000 Genomes Project, ESP6500 or ExAC database; and 3) missense variants that were predicted to be neutral by the Condel program, which assesses the functional effects of non-synonymous variants using a CONsensus DELeteriousness (Condel) score that combines various tools  . The strP), calculated according to the following formula: N represents the gene count in all pathways, n represents the z potential pathogenic gene count in N, M represents the gene count in each pathway, and m represents the potential pathogenic gene count in each pathway. Pathways with a q-value < 0.05, determined from the p-value through multiple comparisons, were considered to be significantly enriched.The number of potential pathogenic genes and all genes in each pathway were determined, and then the number of all potential pathogenic genes and all genes in all pathways were determined separately. Ultimately, a hypergeometric test was applied to determine the statistical significance of the enrichment pathway (P = 2.51 × 10−4).To test whether low-frequency mutations (MAF < 0.01 in the 1000 Genomes Project) that might be risk factors for microtia, we performed a gene-based test comparing the burden of low-frequency variants in cases and controls using a sequence kernel association test (SKAT) implemented in the SKAT software package. Only nonsynonymous variants were included in the analysis. Genotype data for the Han Chinese in Beijing (CHB) and Southern Han Chinese (CHS) populations were extracted from the 1000 Genomes Project. Finally, all 32 microtia cases and 208 normal controls from the 1000 Genome Project were genotyped for the selected variants. A Bonferonni correction was used to account for multiple testing (


Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal types of cancer in children. In recent years, it has become evident that these tumors are driven by epigenetic events, mainly mutations involving genes encoding Histone 3, setting them apart from their adult counterparts. These tumors are exceptionally resistant to chemotherapy and respond only temporarily to radiotherapy. Moreover, their delicate location and diffuse growth pattern make complete surgical resection impossible. In many other forms of cancer, chemo- and radioresistance, in combination with a diffuse, invasive phenotype, are associated with a transcriptional program termed the epithelial-to-mesenchymal transition (EMT). Activation of this program allows cancer cells to survive individually, invade surrounding tissues and metastasize. It also enables them to survive exposure to cytotoxic therapy, including chemotherapeutic drugs and radiation. We here suggest that EMT plays an important, yet poorly understood role in the biology and therapy resistance of pHGG and DIPG. This review summarizes the current knowledge on the major signal transduction pathways and transcription factors involved in the epithelial-to-mesenchymal transition in cancer in general and in pediatric HGG and DIPG in particular. Despite the fact that the mesenchymal transition has not yet been specifically studied in pHGG and DIPG, activation of pathways and high levels of transcription factors involved in EMT have been described. We conclude that the mesenchymal transition is likely to be an important element of the biology of pHGG and DIPG and warrants further investigation for the development of novel therapeutics. H3F3A and HIST1H3B), leading to a lysine-to-methionine substitution at position 27 in the N-terminal tail of the histone protein, causing global H3K27 hypomethylation. Supratentorial pHGG often carry a G34R/V mutation in H3F3A, resulting in decreased H3K36 methylation [Brain tumor is the number one cause of cancer-related morbidity and mortality in children and adolescents. Among pediatric brain tumors, high-grade gliomas (HGG), and especially, diffuse intrinsic pontine gliomas (DIPG) carry the worst prognosis with a 5-year survival of less than 10% . On a mohylation . In conthylation . Additiohylation . The onlhylation , 12. In hylation –15. We hEMT is induced by external factors, such as cytokines, growth factors and hypoxia. Different signaling pathways lead to activation of EMT transcription factors; most importantly SNAI1 (SNAIL), SNAI2 (SLUG), ZEB1, ZEB2 and TWIST. These transcription factors inhibit the expression of genes that promote cell–cell contact, most importantly E-cadherin, and induce genes that promote migration and stemness , 16–18. CDH1 promoter and concurrently increase the expression of other cadherins, such as N-cadherin (CDH2) [One of the most important regulators of the mesenchymal transition is the SNAIL family of master transcriptional regulators, consisting of three members; SNAIL SNAI1), SLUG (SNAI2) and SMUC (SNAI3). SNAIL family transcription factors are basic zinc finger transcription factors with a broad variety of functions during normal development and cancer progression –22. Mostn (CDH2) , 23. Furn (CDH2) , 25  transcription factors, of which TWIST1 and TWIST2 have been studied most. Basic HLH transcription factors, such as the zinc finger transcription factors, exert their action through binding to the E-boxes in the promotor regions of target genes. In this manner, as with the SNAIL and ZEB families of transcription factors, TWIST represses E-cadherin and enhances N-cadherin expression, thereby promoting tumor cell migration, invasion, metastasis and chemoresistance in a broad variety of cancer types –55.TWIST1 has been repeatedly investigated in adult glioma, in which its expression is correlated with higher tumor grade and poorer survival of patients. Mechanistically, this is attributed to the increased migratory and invasive capacities of glioma cells by TWIST1 activity . AdditioLess well studied, but nonetheless relevant, is the SIX family of homeobox transcription factors, of which only SIX1 has received considerable attention. These transcription factors are highly expressed in embryogenesis, where they have an important role in embryonal progenitor cell proliferation and survival. When SIX proteins are aberrantly expressed in non-embryonal tissue they can function as oncogenes, promoting cell proliferation and metastasis in various cancer types, partially through activation of the TGF-β and RAS/MAPK pathways –63.SIX1 has mostly been studied in breast cancer, where it correlates with worse patient survival and induces EMT in cancer stem cells, thereby causing malignant progression , 64. FurThe epithelial-to-mesenchymal transition is commonly initiated by external stimuli, such as growth factors, cytokines and/or hypoxia. These external stimuli trigger intracellular signaling cascades that ultimately result in an upregulation of transcriptional regulators, such as SNAIL and ZEB . Key pat3K/AKT, MAPK and JNK pathways, which will be discussed later on [One of the first mechanisms discovered by which external triggers could induce EMT is the TGF-β signaling pathway. The TGF-β superfamily consists of two activins, three TGF-βs and many bone morphogenetic proteins that all function through homo- or heterodimers of receptors. Binding of TGF-β or related proteins to their receptors causes phosphorylation of tyrosine, serine and/or threonine residues in the cytoplasmic domain of the receptor. Via a series of intermediate events, this results in phosphorylation of SMAD2 and SMAD3, which in turn form a complex with and phosphorylate SMAD4 , 67  in a subset of diffuse midline gliomas [In pediatric high-grade glioma, a special role seems to be reserved for the activins within the TGF-β superfamily of proteins. Recent studies have identified activating mutations gene encoding  gliomas , 70, 71. gliomas –74. Ther gliomas . This ob gliomas . This maIn the canonical WNT/β-catenin pathway, WNT first binds to Frizzled cell surface receptors and low-density lipoprotein-related receptor proteins 5 or 6 –78. The The WNT/β-catenin pathway has many interactions with other pathways involved in EMT. For example, it cooperates with TGF-β signaling to induce EMT in palate medial-edge epithelial cells , 85 and 2+ pathways [Besides the canonical WNT/β-catenin pathway, two non-canonical WNT signaling pathways have been identified; the planar cell polarity (PCP) and WNT/Capathways , 109. Alpathways –114. Botpathways –122. Dat3K/AKT and RAS/MAPK pathways. The role of the PI3K/AKT pathway in EMT is well established; after activation of the pathway, AKT inhibits GSK3β by phosphorylating serine 9. This in turn prevents GSK3β from phosphorylating SNAIL or SLUG, thus inhibiting their ubiquitination and subsequent proteasomal degradation [3K/AKT pathway causes stabilization of SNAIL family proteins, thereby inducing EMT [Activation of well-known receptor tyrosine kinases (RTK), such as the EGF, FGF and PDGF receptors, as well as G-protein coupled receptors (GPCR), is known to induce EMT via activation of the canonical PIradation , 124. Vicing EMT .3K/AKT pathway induces the transcription of ZEB1 by reducing GSK3β-mediated phosphorylation of β-catenin, thereby stabilizing the protein and inducing ZEB1 transcription via a β-catenin/TCF4 complex [3K)/AKT signaling), resulting in induction of EMT [3K/AKT pathway, which is commonly activated in malignant cells, has been shown to induce EMT in several tumor types. In certain types of cancer, inhibition of the overactive PI3K/AKT signaling resulted in a decrease of mesenchymal characteristics and (re-)sensitization of cancer cells to chemo- and radiotherapy [In addition, activation of the PI complex , 126. Fin of EMT . The PI3otherapy –131.3K/AKT/GSK3β pathway in glioma, for example the HIF-1α and the WNT/β-catenin pathways [3K pathway [3K/mTOR kinase inhibitors provide a promising glioma therapy, as those drugs sensitize tumor cells to radiation and chemotherapy [3K are found in 15–26% of patients [There are several EMT pathways that cross talk with the PIpathways –135. Mor pathway . In lineotherapy . In pedipatients –140, whiSNAIL and SLUG and regulators of cell invasion and motility through activation of RHO-GTPases [Activation of RTKs also leads to signaling via the RAS/MAPK pathway . Within -GTPases , 142. Ad-GTPases , as well-GTPases , 145. On-GTPases , 146–151-GTPases –154. In -GTPases . In cont-GTPases . Yet, ac-GTPases . In pedi-GTPases –140, 157-GTPases , 50, 139-GTPases , whereas-GTPases .Among the receptor tyrosine kinases, the TAM family, consisting of TYRO3, AXL and MERTK, plays a particular role in the induction of EMT. TYRO3 and MERTK can both be activated by binding of their endogenous ligands protein S and Gas6, whereas AXL can only bind Gas6 . AdditioIn glioblastoma, both AXL and MERTK have been identified as potential therapeutic targets and causes of treatment resistance. Inhibition of AXL and/or MERTK leads to diminished proliferation, migration and invasion of glioma cells, as well as sensitization of glioma cells to chemotherapy –180 Alth3K/AKT and RAS/MAPK pathway, a third pathway can be activated after engagement of cell surface receptors by their ligands; the JAK/STAT pathway. Although commonly associated with GPCRs, RTKs are also capable of initiating signaling along the JAK/STAT pathway. In this pathway, receptor-associated Janus kinases (JAKs) phosphorylate and activate signal transducer and activator of transcription (STAT) proteins. Upon phosphorylation, STATs form homo- or heterodimers that can function as transcription factors [SNAIL1, ZEB1, HIF-1α, and most importantly, TWIST [Besides the PI factors . Within y, TWIST –187. Undy, TWIST . In cancy, TWIST , breast y, TWIST  and ovary, TWIST . Based oy, TWIST , 191, ovy, TWIST  and biley, TWIST . In aduly, TWIST . STAT3 ay, TWIST –196. Thiy, TWIST . Additioy, TWIST –200.SNAI1, SNAI2, ZEB1, ZEB2 and TWIST [Sonic hedgehog (SHH) is a soluble glycoprotein that can bind to the transmembrane receptor Patched1 (PTCH1), thereby inducing phosphorylation and stabilization of another transmembrane receptor, Smoothened (SMO). Activated SMO phosphorylates a variety of intracellular signaling molecules that eventually lead to increased activity of the GLI family of transcription factors , 202. Thnd TWIST , 204. Innd TWIST , 206. Abnd TWIST , 208. Innd TWIST , 209. Mond TWIST .Inhibition of SMO represents a promising strategy to overcome therapy resistance in HGG; the Smoothened inhibitor cyclopamine has been shown to induce radiosensitization in glioblastoma cells and Erismodegib, another SMO inhibitor, was capable of inhibiting self-renewal and EMT in glioblastoma initiating cells , 212. AdThe hedgehog pathway has been found to be active in DIPG cells, where it plays a crucial role in self-renewal of malignant cells . Its conSNAIL1, SNAIL2, ZEB1, FN1 and PDGFRs [Mammalian cells express four transmembrane NOTCH receptors, NOTCH 1–4, and five canonical transmembrane ligands: Delta-like 1, Delta- like 3, Delta- like 4, Jagged-1 and Jagged-2.  When a Nd PDGFRs –218. Furd PDGFRs , 220. Asd PDGFRs . Howeverd PDGFRs . NOTCH sd PDGFRs –224. In d PDGFRs , 226. Cod PDGFRs –231. Mord PDGFRs . AlthougNOTCH, CTNNB1 (β-catenin), SNAIL1 and TWIST [Another external factor, other than growth factors and cytokines that is capable of inducing mesenchymal transition is hypoxia. Exposure of tumor cells to hypoxia induces stabilization of hypoxia-inducible factor 1α (HIF-1α), which dimerizes with HIF-1β to form a transcription factor complex Fig. . Activitnd TWIST –239. Addnd TWIST .Besides EMT, these hypoxia-inducible factors induce expression of genes responsible for stem cell properties, such as self-renewal and multipotency via mechanisms common to EMT, such as the NOTCH signaling pathway . Further3K/AKT, MAPK and NF-kB pathways, both directly and through modulation of receptor tyrosine kinases such as FGFR, EGFR, PDGFR and integrin-linked kinases [The ultimate result of activation of the EMT-related signaling pathways described above is the so-called cadherin switch –257 Fig. Cadheri kinases –263.In adult glioblastoma (GBM), loss of E-cadherin expression and concurrent increase in either N- or R-cadherin expression is related to a more mesenchymal phenotype, tumor invasiveness and worse overall survival , 264–266Over the past years, the mesenchymal transition has received considerable attention in the field of oncology, due to its relationship with invasion, metastasis and therapy resistance of cancer cells. Components of the mesenchymal transition have often been put forward as therapeutic targets and/or therapy-sensitizing agents, not in the least in the treatment of adult glioblastoma. However, little research is available on the role of the mesenchymal transition in pediatric high-grade glioma and diffuse intrinsic pontine glioma. The therapy resistance and diffusely infiltrative nature that these tumors often possess imply that mesenchymal gene expression may play a larger role in their biology than is known so far. We have reviewed current knowledge about the major signal transduction pathways involved in initiation and maintenance of the mesenchymal transition in cancer in general, and high-grade glioma in particular.HIF2A in both pediatric and adult HGG [HIF2A overexpression in the datasets we analyzed, whereas HIF1A was overexpressed in all three tumor types. Tables When comparing published RNA expression datasets available from normal brain tissue , adult Gdult HGG , 253, onACVR1 mutations and a mesenchymal phenotype can potentially be chemo- and radio-sensitized using these compounds [3K and/or MAPK pathway can potentially be sensitized to therapy by the use of PI3K or MEK inhibitors, many of which are already clinically approved for other indications. Also, considering the perceived importance of the hedgehog pathway in the development and progression of DIPG, currently available Smoothened inhibitors may be used to interfere with the mesenchymal transition in this type of cancer. Within the paradigm of the mesenchymal transition, these drugs would only serve as therapy sensitizers, and should therefore always be combined with other cytotoxic therapeutics or radiotherapy. Finally, over the past years, it has become clear that a large part of pediatric HGG and DIPG are driven by mutations in genes encoding the Histone 3 protein. These mutations either induce a K27M or G34R/V substitution in the N-terminal end of the Histone 3 protein, thereby preventing histone methyltransferases from methylating H3K27 or H3K36 and causing a global epigenetic shift [The differences in mechanisms of mesenchymal transition employed by pHGG and DIPG as compared to adult GBM will provide researchers with important clues in the identification of therapeutic targets and the design of novel therapeutic strategies to overcome therapy resistance and metastasis in these hard-to-treat brain tumors. With the currently available knowledge, several promising therapeutic targets for interfering with the mesenchymal transition in pHGG and DIPG can already be identified. With the development of selective ACVR1 inhibitors, tumors harbouring activating ompounds . Furtheric shift . This hyic shift –273. Desic shift . These p


Gossypium klotzschianum. To elucidate the salt-tolerant mechanisms in G. klotzschianum, we firstly detected the changes in hormones, H2O2 and glutathione (GSSH and GSH), then investigated the gene expression pattern of roots and leaves treated with 300 mM NaCl for 0, 3, 12, 48 h, and each time control by RNA-seq on the Illumina-Solexa platform. Physiological determination proved that the significant increase in hormone ABA at 48 h, while that in H2O2 was at 12 h, likewise, the GSH content decrease at 48 h and the GSSH content increase at 48 h, under salt stress. In total, 37,278 unigenes were identified from the transcriptome data, 8,312 and 6,732 differentially expressed genes (DEGs) were discovered to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation and expression analysis elucidated hormone biosynthesis and signal transduction, reactive oxygen species (ROS), and salt overly sensitive (SOS) signal transduction related genes revealed the important roles of them in signal transmission, oxidation balance and ion homeostasis in response to salinity stress. This is a report which focuses on primary response to highly salty stress (upto 300 mM NaCl) in cotton using a wild diploid Gossypium species, broadening our understanding of the salt tolerance mechanism in cotton and laying a solid foundation of salt resistant for the genetic improvement of upland cotton with the resistance to salt stress.Cotton is a pioneer of saline land crop, while salt stress still causes its growth inhibition and fiber production decrease. Phenotype identification showed better salt tolerance of a wild diploid cotton species  Arabidopsis and rice [Abiotic stresses such as salt excess (NaCl), drought, and temperature are among factors most limiting to plant productivity . Plants and rice . The strand rice , 5.+ causes osmotic imbalance, membrane disorganization, reduction in growth, inhibition of cell division and expansion, which also leads to reduction in photosynthesis and production of reactive oxygen species[2+-related and reactive oxygen species (ROS) signaling pathways play the key role in signaling transmission [2+ signaling in response to salt stress [+ ions out of the cell via the plasma membrane Na+/H+ antiporter and helps in reinstating cellular ion homeostasis. A rapid increase in the rate of ROS production, known as ‘the oxidative burst’, occurs as a response to stress conditions, which always depends on the respiratory burst oxidase homolog protein (RBOH) [2+ signaling pathway [2O2, one kind of mild ROS, also plays as the signal molecule in the antioxidation [High concentrations of Nan species, 7. It ismission . It is wsmission –11. Espesmission , 13. Brit stress , 15, resn (RBOH) . High ROn (RBOH) –19. To cn (RBOH) , 21. Mann (RBOH) , 23, Ca2 pathway . H2O2, oxidation . These sxidation .GhCIPK6) and SnRK2 could enhance abiotic stress tolerance [AtNHX1 gene in cotton can also improve salt tolerance [GhWRKY39-1 and GhDREB1 enhance abiotic stress tolerance in transgenic plant [SNAC1, could also improve salt tolerance in transgenic cotton [GhSOD1, GhCAT1, and GhMT3a, showed the high salt stress tolerance in cotton [Cotton is a relatively salt tolerant species, but growth inhibition still occurs when the plant is exposed to saline stress. Germination and seedling growth are the two growing procedures particularly with salt sensitive , 28. As olerance , 30. Furolerance . Evidencic plant , 33 and c cotton . Overexpn cotton , 36.G. raimondii [G. arboretum [G. hirsutum [G. barbadense [G. aridum [G. davidsonii [G. klotzschianum to decipher the mechanism of cotton’s rapid response to 300 mM NaCl salinity stress.In addition, the completion of the aimondii , 38, G. rboretum , G. hirshirsutum , 41, andrbadense  whole gerbadense , such asrbadense . Howeverrbadense –49, ther. aridum  and G. dvidsonii  showed tG. klotzschianum, was selected for transcriptome analysis to identify the regulation networks involved in responses to salinity stress. On account of its sister species G. davidsonii has already been proved salt tolerance by physiological and RNA-Seq analysis under mild stress [G. klotzschianum and G. davidsonii in various concentration salt stresses demonstrated the salt tolerance of G. klotzschianum. Likewise, the contents of four kinds of phytohormones , H2O2, AsA, and glutathione in leaves were measured to exam the response mechanism of high salinity tolerance in cotton. The transcriptome analysis of leaves and roots over 48 h  in response to 300 mM NaCl stress were designed to investigate regulatory network at early stage under severe salt stress. The study not only provides supplement data for cotton salt tolerance research but also lays a solid foundation on engineering breeding for salt resistance in cotton.Here, a cotton D-genome diploid species, d stress , the samG. klotzschianum (accession D3k-01) and G. davidsonii (accession D3d-01), which were grown in the greenhouses at the Institute of Cotton Research, Chinese Academy of Agricultural Sciences (ICR-CAAS)  and also perennially maintained in the National Wild Cotton Nursery which locates at Sanya City, Hainan Island, China. The accession seeds were delinted and sterilized by using 1% of sodium hypochlorite for 15 min, followed by washing with sterile water for three times. Sterilized seeds were germinated at 28°C under long day conditions in a 16 h light/8 h dark cycle with a light intensity of 150μmol m-2s-1 on 17% water content sands. Three days after germination, the properly plants were potted in soil and placed in a growth room at 28°C, 60–70% relative humidity, a photoperiod of 16 h/8 h (day/night) and light intensity of 150μmol m-2s-1. Seedlings containing two simple leaves and one heart-shaped leaf were randomly selected to four groups, one group with three pots, then the plants were watered with 0, 200mM, 250mM, and 300 mM NaCl for 3 days, three biological replicates were prepared for each time point. The leaf and root samples of 300 mM NaCl treatment were harvested for varying durations  after exposing to salt stress, the control plants were also harvested at the same time point, three plants were collected and mixed, to minimize the effect of transcriptome unevenness among plants. For the treated leaves and roots, parts of them were used to RNA-seq, and parts of them were used to quantitative real-time PCR (qRT-PCR), parts of leaves were used to detect hormones  and other biochemistry indexes . All the samples were frozen and stored in liquid nitrogen and stored at −70°C for further use .The plant materials in the study were two diploid D genome wild cotton species The concentration of abscisic acid (ABA), ethylene (ETH), gibberellic acid (GA), and indole acetic acid (IAA) in leaves were determined using the ELISA method. ABA , ETH , GA , IAA , these four kind of kits were utilized for the determination of each hormone contents. 0.5 gram for each sample applied for the measurement ELISA . Three replicated experiments  were performed.2O2. A series of enzymatic reactions were activated by GSH in the homogenate which subsequently led to the conversion of GSH (reduced glutathione) to oxidize glutathione (GSSG). The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 405 nm. Three replicated experiments  were performed.The contents of GSH (reduced glutathione) and GSSG (oxidized glutathione) were measured using a GSH and GSSG assay kit (Nanjing Jiancheng Bioengineering Institute). The reaction was initiated by the addition of H2O2 in leaves was assessed using a commercially available kit (Nanjing Jiancheng Bioengineering Institute). H2O2 bound with molybdenic acid to form a complex, which was measured at 405 nm and the content of H2O2 was then calculated. The content of AsA in leaves was assessed using a commercially available kit (Nanjing Jiancheng Bioengineering Institute), which was measured at 265 nm and the content of AsA was then calculated. Three replicated experiments  were performed.The content of HTotal RNA of each sample was extracted from the cotton samples according to the instruction manual of the TRlzol Reagent . RNA integrity and concentration were checked using an Agilent 2100 Bioanalyzer . The mRNA was isolated by NEB Next Poly (A) mRNA Magnetic Isolation Module . The cDNA library was constructed following the manufacturer’s instructions of NEB Next Ultra RNA Library Prep Kit for Illumina  and NEB Next Multiplex Oligos for Illumina . In briefly, the enriched mRNA was fragmented into approximately 200nt RNA inserts, which were used to synthesize the first-strand cDNA and the second cDNA. The double-stranded cDNA were performed end-repair/dA-tail and adaptor ligation. The suitable fragments were isolated by Agencourt AMPure XP beads , and enriched by PCR amplification. Finally, the constructed cDNA libraries of the cotton were sequenced on a flow cell using an Illumina HiSeq™ 2500 sequencing platform.G. raimondii; JGI v2.1) using Tophat2 software [−5. Furthermore, genes were searched against the NCBI non-redundant nucleotide sequence (Nt) database using BLASTn by a cut-off E-value of 10−5. Genes were retrieved based on the best BLAST hit (highest score) along with their protein functional annotation. To annotate the gene with gene ontology (GO) terms, the Nr BLAST results were imported into the Blast2GO program [Low quality reads, such as only adaptor, unknown nucleotides>5%, or Q20 <20% (percentage of sequences with sequencing error rates <1%), were removed by per script. The clean reads that were filtered from the raw reads were mapped to cotton genome  gene was used as a control. Each reaction was carried out in a final volume of 20 μL, containing 10 μL of SYBR Green PCR master mix, 0.5 μL of each gene-specific primer and 2 μL of diluted cDNA. The PCR thermal cycling conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 30 s. Data were collected during the extension step: 95°C for 15 s, 60°C for 1 min, 95°C for 30 s and 60°C for 15 s. Three biological replicates were performed, and three technical replicates were designed per cDNA sample.Real-time RT-PCR (qRT-PCR) was performed on a new set of 3 replicates for each sample. A set of 38 genes was chosen randomly  from rooG. klotzschianum was considered as sister species of G. davidsonii in Gossypum taxonomy and G. davidsonii has been proved with better salt-tolerances by the previous study [G. klotzschianum owned the same phenotypic feature to G. davidsonii after post in various NaCl concentrations of salt stress for 3 days  using Tophat2 software [G. raimondii 2.1) [Here, we used high-throughput RNA sequencing (RNA-seq) to investigate the changes in gene expression of the diploid D-genome species obtained . After tsoftware , and 64.dii 2.1) . The RNAG. klotzschianum transcriptional responses to salt stress, we carried out the identification of the unigenes with expression level significantly changed upon NaCl-treatments. The transcript abundance of each gene was estimated by FPKM value. DESeq and Q-value were employed and used to evaluate differential gene expression pattern. A cutoff P-value < 0.01 adjusted by false discovery rate (FDR) and fold change ≥ 2 was used for identifying differentially expressed genes. A total of 11417 genes showed differential expression under salt stress conditions . In total, respectively, 8,312 and 6,732 DEGs were found in roots and leaves in response to salt stress. To identify candidate genes that respond to salt stress in G. klotzschianum, we adapted the DEGs identified by comparing the gene expression levels under salt stress vs. control conditions at the same time points. Meanwhile, 3,603 DEGs were commonly identified in the two tissue types, which highlights that 1,406 and 782 common genes with different expressions at three time points in roots (RSC RS vs. RC) and leaves (LSC LS vs. LC), respectively. The distribution of these genes is shown in a Venn diagram . The results revealed that ABA plays the important role in cotton under salt stress.A cluster analysis of the common different expression genes at three time points in roots (1406) and leaves (782), respectively, resulted a dynamic change of gene expression by a heat map , which i2 = 0.801 and 0.905) indicates the reliability of RNA-Seq quantification of gene expression  as the cutoff. 6,698 (80.58%) and 5,535 (82.22%) of the DEGs in roots and leaves, respectively, were annotated to GO terms. Further GO category enrichment analysis was performed using these DEGs. Respectively, 6,698 and 5,535 DEGs in roots and leaves were classified into 51 and 51 GO terms based on biological process, cellular component and molecular function . In the Further GO category enrichment analysis was performed using common DEGs. In our data, 1,191 (84.71%) and 680 (86.96) of the common DEGs in roots and leaves were classified into 44 and 46 GO terms based on biological process, cellular component and molecular function. For the molecular function, most DEGs were sorted into binding  and catalytic activity . The GO term of response to stimulus in the biological process section, was significant enriched with the number of 941 genes, 66.93% of the 1406 DEGs, and 510 genes, 65.22% of the 782 DEGs, respectively. Furthermore, in order to understand the dynamic expression patterns of DEGs under salt stress, several key terms were investigated . The resArabidopsis thaliana and Oryza sativa, highlighting the involvement of several hormone and ROS-related pathways in cotton  and 1270 (18.87%) of the DEGs in roots and leaves, respectively, were annotated to KEGG pathway. A set of these DEGs were mapped onto KEGG pathways in n cotton . The plaIn order to comprehend the dynamic change of gene expression among the two organs, 117, 120, 118, 119, 102, and 115 pathways were individual categorized in R3, R12, R48, L3, L12, and L48, respectively . Based oAnalyses on both the GO and KEGG enrichments demonstrated that the genes related to ion transport, ROS, hormones and signal transmission were significantly affected during the early stage of salt stress in cotton. Hence, we further explored that DEGs were involved in the metabolism of ROS and hormones, along with the signal transduction pathways systematically.Plant could perceive and respond to stresses rapidly via signal transduction pathways mediated by stress hormones , 11. HorPSY), two beta-carotene hydroxylase genes (BCH), nine 9-cis-epoxycarotenoid dioxygenase genes (NCED), and seven abscisic acid 8 hydroxylase genes (CYP707A) were significant different expression in the ABA biosynthetic pathway. Furthermore, most of NCED genes, an important biosynthetic gene which has been proved the important function on ABA biosynthesis [Gorai.002G038100; Gorai.013G177100) were completely activated in two tissues. By contrast, the CYP707A genes, which were the key ABA catabolic genes, maintained higher expression at early stage under salt stress, while keep unchanged or down-regulated after exposure in salt stress 48h. Twenty-four genes involved in the ethylene biosynthetic pathway were detected as DEGs. Among them, two S-adenosylmethionine synthase (SAM) genes , precursor synthesis, were continuously upregulated. Meanwhile all seven 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (ACS) were significantly induced, while half of ten 1-aminocyclopropane-1-carboxylate oxidase genes (ACO) were induced, and other five were inhibited gradually. These results showed that the key regulatory components of the biosynthetic pathways of two stress hormones, ABA and ethylene, were changed significantly during cotton’s response to salt stress  genes participating in BRs biosynthesis in cotton, two CYP749A22  continuous expression in roots, while CYP90A1 was only up-regulated before 12 h treatment in roots, and one of two CYP85A was activated. Three CYP734A1 genes and two CYP724B1 genes significant decreased. Only one genes encoding steroid 5-alpha-reductase (DET2), a major rate-limiting enzyme in BRs biosynthesis, showed decrease to more than 2-fold expression levels at 12h treatment in roots. Twenty-seven DEGs were identified involved JA biosynthesis pathway. And the expression of five allene oxide synthase (AOS) genes, which are the first enzyme in the branch pathway leading to JA biosynthesis, were significant increase after 3h treatment. These findings indicated that BRs and JA also actively participate in the stress response of cotton.Many studies reported that BRs and JA play essential roles in plant responses to abiotic stress. The expression of thirteen genes involved in BRs biosynthetic pathways changed under salt stress . Ten cytIn addition, 14, 16, and 8 EDGs were also found in the biosynthetic pathway of GA, cytokinin, and auxin, which were widely considered to participate in regulating plant development. Interestingly, many of their genes showed increased transcript abundances in response to salt .Arabidopsis, PP2C family genes were demonstrated to regulate ABA signaling pathways negatively by opposing the action of particular protein kinases and eight PP2Cs were characterized as key factors in ABA signaling transduction, otherwise PP2Cs could inactivate SnRK2 via dephosphorylation, and this inactivation is inhibited by PYR/PYL/RCRA, ABA receptors, in an ABA-dependent manner [PYLs including PYL1, 4 and 8 were significantly down-regulated, especially with 3 members of the PYL4 continuously down-regulated at both two organs under salt stress  and three MYC2s increased significantly. In particular, most of the JAZs’ expressions increased at 3h in roots , which take the responsibility for the accumulation of H2O2, were significantly up-regulated in both root and leaf tissues when suffering salt stress (G. davidsonii transcriptome results [POD) family were up-regulated in the two organs and most of them were up-regulated at 12h treatment in roots. In addition, two catalase (CAT) genes were up-regulated one in roots and one in leaves, respectively, and the superoxide dismutase (SOD) maintained no change. Most of the twenty-one glutathione S-transferase (GST) genes were significantly upregulated. Seven glutaredoxins (GRXs) genes and one Glutathione reductase (GR) were also upregulated, whereas a completely opposite expression pattern of two PRX genes with one upregulated in roots and another down-regulated in leaves under salt treatments was examined. These results were consistent with the high level of GSH , which leads to secondary oxidation stress, disturbs cellular redox homeostasis and damages cell components and structures. The alleviation of oxidative damage and increased resistance to salt stresses was often correlated with the balance between ROS producing and ROS scavenging . In the t stress . Simulta results . In total of GSH  and sugg+ will immediately activated Ca2+ signal pathway, Ca2+ acts as a secondary messenger in salt stress responses [2+ concentration changes within seconds in plants subjected to salt stress. The salt overly sensitive (SOS) signaling pathway, a kind of Ca2+-related pathway, has been well documented at the cellular level, play the vital role in maintain ion homeostasis. In our study, six SOS2 were upregulated in roots and leaves after post in salt stress , is more remarkable in roots in Arabidopsis than SOS3 [G. davidsonii transcriptome consequence that the significant activation of CBL10 under salt stress in cotton [+/H+ antiporter is an important protein in excluding Na+ at cellular level. In our data, NHX7/SOS1 remain unchanged, while the vacuolar location gene NHX2 (Gorai.007G264500) was found up-regulated in both tissues, especially in roots. Moreover, other genes involving the ion homeostasis were also found in our data. Such as the Na+ transporter gene HKT6  were down-regulated in roots. Such as the potassium transporters KUP , the plasma membrane ATPase  were also found to be up-regulated under salty stress.The damage of ion homeostasis by excess Naesponses . The cytt stress . Whereashan SOS3 . Interesn cotton . FurtherN. benthamiana [CIPKs, eighteen CMLs and eight Ca2+-ATPase genes showed significant activating by salt stress, and most of them up-regulated in roots, which revealed the important role of roots in perception and transmission salt signal.In addition, four CIPK6 genes were upregulated in roots . As the thamiana , FurtherPSY, one BCH, two NCEDs, and one CYP707A, five ABA signal transduction genes included one PYL, two PP2Cs, one SnRK2, and one ABF, six ROS-related genes included one RBOH, one CAT, two PODs, one GR, and one GST, and three SOS-related genes consisted of one CBL10, one SOS2, and one NHX2. These genes have been proved the important roles in response to salt stress by the previous studies [To further verify the correctness of the analysis results, nineteen typical genes were selected and performed by RT-PCR, which contained ten ABA-related genes included five biosynthesis genes and five signal transduction genes, six ROS-related genes including one ROS-producing gene and five ROS-scavenging genes, three SOS-related genes , which i studies , 67, 69,AP2/ERF, MYB, WRKY, and NAC families were shown to regulate salt tolerance and many studies have shown that TFs could enhance salt tolerance through ABA signaling and the modulation of ROS production [G. klotzschianum differentially expressed TFs families were MYB, bHLH, ERF, NAC, bZIP, and WRKY, which are known to mediate stress responses in plants, and the results were consistent with previous studies [NAC family TFs, which is closely related with ABA signal pathway, showed significant up-regulated at the organs under salt stress. Moreover, six tissue-specific regulated TFs YABBY family proteins were also found to be altered in our data, as like the previous studies.Transcriptional modulation is vital aspects of the complex genetic and biochemical networks that plants use to respond to stress. TFs are master regulators that control gene clusters involved ABA-independent pathway and ABA-dependent pathway . The incoduction , 74. In  studies , 76. The2+, ROS and hormones [When salt stress occurs, two main factors are responsible for salt stress-induced inhibition of plant growth: osmotic stress and ionic stress . As it ihormones , 77. Thehormones . In presRBOHs  were activated under salt stress  by Na+/H+ exchangers located in the plasma membrane, or sequestered into the vacuole by Na+/H+ exchangers (e.g. NHX proteins) located in the tonoplast [2+ concentration changes within seconds, then SOS3 appeared to function as a primary calcium sensor by binding Ca2+ and activating SOS2, and the latest the SOS3–SOS2 complex stimulated the Na+/H+ exchanger activity of SOS [+. In present study, seven antiporting ATPases were up-regulated. Whereas, the expression of SOS3 and SOS1 was unchanged, and several SOS2, two CBL10, and one tonoplast location Na+/H+ antiporter gene NHX2 were up-regulated. The results were consistent with those in G. davidsonii at the early stage under salt stress [+/H+ NHX antiporters are activated by AtCIPK24/SOS2 through the mechanism related to the AtCBL10 to sequester intracellular extra Na+ in the vacuole and AtCBL10–AtCIPK24/SOS2 was reported to regulate Na+ homeostasis in the vacuolar membrane in shoots and leaves [+ exclusion and cellular ion homeostasis at early stage in cotton. In addition, HKT6, which encodes a K+ transporter, was down-regulated at late stages under salt stress treatment in roots, likely leading to a decrease in Na+ and K+ uptake. Interestingly, all KUPs in roots and KUP11 in leaves were up-regulated, likely leading to further K+ influx and maintaining the K+ balance of the cell. Together, these signaling pathways are involved in maintaining ion homeostasis to keep the plant away from ion toxicity.Once Naonoplast –88. The y of SOS , resultit stress . As prevt stress , 89, 90.t stress , 92. It d leaves . Therefo2O2, ABA signal molecules transductions and SOS-related genes to mediate salinity stress in cotton Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 TableData Section. RNA-Seq data in this study have been deposited at the NCBI under the accessions: SRR5500539.(XLSX)Click here for additional data file.


Campylobacter jejuni FJ3124 and ZP3204 isolated from retail chicken gizzards and Campylobacter jejuni TS1218 isolated from retail chicken showed the presence of 1,694,324-, 1,763,161-, and 1,762,596-bp circular chromosomes, respectively. Campylobacter jejuni ZP3204 and TS1218 harbored large tetracycline resistance plasmids with type IV secretion systems.Genome sequences of  Campylobacter spp. have been reported to be prevalent in retail chicken livers and gizzards; however, the prevalence of these foodborne pathogens in chicken livers was higher than that in chicken gizzards  was used to prepare the library according to the manufacturer’s protocol. It was then run on a MiSeq desktop sequencer using an Illumina V2 reagent kit (Illumina). Coverage of 200× was obtained from the paired-end reads. CLC Genomics Workbench version 7.5.1 (Qiagen) was applied for raw data assembly, and genome alignment was performed using the Microbial Genome Finishing Module (Qiagen).To our knowledge, only one reported . Hence,  poultry , 3. TotaCampylobacter jejuni FJ3124 and ZP3204 revealed the presence of circular chromosomes of 1,694,324 and 1,763,161 bp, respectively. Campylobacter jejuni FJ3124 contained a total of 1,784 genes, 1,733 coding sequences (CDS), and 1,662 coding genes along with 56 RNAs and 71 pseudogenes. Campylobacter jejuni ZP3204 contained 1,906 genes, 1,852 CDS, and 1,764 coding genes with 54 RNAs and 88 pseudogenes. On the other hand, the chicken isolate Campylobacter jejuni TS1218 contained a circular chromosome of 1,762,596 bp and has 1,896 genes, 1,840 CDS, and 1,757 coding genes with 56 RNAs and 63 pseudogenes.The complete genome sequences of the chicken gizzard isolates Campylobacter jejuni ZP3204 also contained two circular plasmids, pCJDM204L and PCJDM204S, of 44,436 and 5,257 bp, respectively. The larger plasmid contained a type IV secretion system and a tetracycline resistance gene with 47 CDS and 2 pseudogenes. The smaller plasmid contained 6 CDS with some hypothetical and replication proteins. Campylobacter jejuni TS1218 also contained one tetracycline resistance plasmid  with a type IV secretion system and had 48 CDS and 2 pseudogenes.Campylobacter jejuni ZP3204 and the chicken isolate Campylobacter jejuni TS1218 contain the genes coding for the type VI secretion system, which are homologous to previously reported plasmid-borne type VI secretion genes (It is noteworthy that the chromosomes of the chicken gizzard isolate on genes .CP017856 (Campylobacter jejuni ZP3204), CP017862 (Campylobacter jejuni FJ3124), and CP017860 (Campylobacter jejuni TS1218); plasmids, CP017854 (Campylobacter jejuni ZP3204 pCJDM204L), CP017855 (Campylobacter jejuni ZP3204 pCJDM204S), and CP017861 (Campylobacter jejuni TS1218 pCJDM218).The genome sequences of the chromosomes and plasmids for the 3 strains are available in GenBank with accession numbers as follows: chromosomes,


Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’. Of all 2.The detection and signalling of DSBs involves the MRE11-RAD50-NBS1 (MRN) and Ku70-Ku80 (Ku) complexes, which bind DSBs and recruit the ATM and DNA-PKcs kinases, respectively ,7. ATM, 3.Cells use two main pathways for the repair of DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR) ,11,12. DHR on the other hand is initiated by the MRN complex, which recognizes and binds broken DNA ends. MRN acts in concert with CtIP to initiate DNA end-resection, a process that leads to the formation of 3′ single-stranded (ss) DNA overhangs. Further resection of the ends is mediated by the EXO1 and DNA2 nucleases. The stretches of ssDNA are covered and stabilized by RPA, which allows for recruitment of the BRCA1-PALB2-BRCA2 complex. Finally, BRCA2 facilitates removal of RPA and loading of RAD51 recombinase, which searches for an undamaged homologous template, usually a sister chromatid. Following strand invasion and duplication of the sister chromatid, the DSB is repaired in an error-free manner .NHEJ and HR are tightly regulated throughout the cell cycle. While NHEJ is dominant in G1 and late G2 phase , HR occu4.DNA is packaged through histone and non-histone proteins into a higher order structure called chromatin . ConsequIt has become clear that both histone PTMs such as phosphorylation (e.g. γH2AX), acetylation and methylation, as well as ATP-dependent chromatin remodelling play important roles in modulating chromatin structure during the DSB response ,30,32,335.Several SWI/SNF2 family members are known by a SMARCA  acronym, classifying them in the SMARCA class of chromatin remodellers. This concerns SMARCA1 (SNF2L), SMARCA2 (BRM), SMARCA3 (LTF), SMARCA4 (BRG1), SMARCA5 (SNF2H), SMARCA6 (HELLS), SMARCAD1 and SMARCAL1, which belong to three different SWI/SNF2 subfamilies: SWI/SNF, ISWI and INO80. SMARCA chromatin remodellers act in different multi-protein complexes that play important roles in several distinct biological processes such as transcription, cell division and development, as well as DNA repair ,32. Here6.Saccharomyces cerevisiae [SMARCA2 (BRM) and SMARCA4 (BRG1) were originally identified as Snf2 and Sth1, respectively, in revisiae . Biochemrevisiae –65, suggrevisiae ,65,66. Irevisiae . RSC recrevisiae ,65. Consrevisiae ,65,69. Rrevisiae . Moreoverevisiae . AlthougThe human counterparts of the yeast SWI/SNF and RSC complexes are called BAF (BRG1-Associated Factors) and PBAF (Polybromo-Associated BAF) . SMARCA2SMARCA2 and SMARCA4 have been implicated in DSB repair. However, the exact mechanism by which these chromatin remodellers affect this process is not completely understood due to conflicting findings. One study demonstrated that SMARCA4 facilitates HR rather than NHEJ as it was shown to specifically promote RAD51-mediated DNA strand invasion by regulating the replacement of RPA with RAD51 on ssDNA . Two oth7.Drosophila melanogaster. It is the catalytic subunit of the CHRAC, ACF, RSF, WHICH and NoRC chromatin remodelling complexes in mammalian cells. SMARCA5 has the ability to remodel chromatin by sliding and displacing nucleosomes [SMARCA5 (SNF2H) is a member of the ISWI family of chromatin remodellers, which was first purified from leosomes ,32. It aleosomes ,73,74. Dleosomes ,73. Secoleosomes  , with which it forms the RSF complex. Indeed, one report showed that SMARCA5 and RSF1 cooperate to promote NHEJ by depositing the centromeric histone-fold proteins CENP-S and CENP-X at DSBs. These CENP proteins in turn recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI . Mono-ub8.Saccharomyces cerevisiae [Human SMARCAD1 and its orthologue in yeast, Fun30, belong to the INO80 family of chromatin remodellers, whose founding member Ino80 was first discovered in revisiae . INO80 wrevisiae –54,78,79revisiae –49,80,81revisiae ,48,80,82revisiae ,79,83,84revisiae ,48,80  family of chromatin remodellers was originally identified in s laevis . The fams laevis ,86. A ges laevis . The cla10.A recent study showed that CHD1 is recruited to chromatin in response to DSB induction and that it is required for HR-mediated repair of DSBs . CHD1 re11.ALC1 (amplified in liver cancer 1) is an ATP-dependent chromatin remodeller that is often classified as a CHD family member despite the absence of an identifiable chromodomain. Instead biochemical and structural analyses revealed a distinct C-terminal macrodomain ,88. This12.A recent study demonstrated an important role for the CHD2 chromatin remodeller in the DSB response . CHD2 ra13.CHD3 and CHD4 are the mutually exclusive catalytic ATPase subunits of the Nucleosome Remodelling and Deacetylase (NuRD) complex. Additionally, this complex contains the histone deacetylases HDAC1 and HDAC2 as subunits, which help to repress transcription of NuRD target genes by histone deacetylation ,91. Whil14.in vitro through an N-terminal domain located between residues 145–225, which is structurally similar to the high mobility group box (HMG) domain [CHD4 is rapidly recruited to sites of laser- and IR-induced DSBs ,93–96 an) domain ,97. Loss) domain . Whether) domain ,68,99. H) domain ,99, rais) domain  (figure ) domain ,96,100. CHD4 has been shown to affect both the signalling and repair of DSBs ,93,94. T15.It has become clear that chromatin reorganization during the DSB response is not simply a matter of switching chromatin from a ‘closed’ to ‘open’ state and vice versa. The DSB response is a complex, multi-step procedure, that requires distinct chromatin changes at each step. Only this way cells can properly detect, signal and repair DSBs in the context of chromatin. Evidently, ATP-dependent chromatin remodellers induce chromatin structural changes that are crucial for these DSB responses .For instance, SMARCA2 and SMARCA4 act at DSBs to facilitate the ATM-dependent spreading of γH2AX. In addition, CHD4 and SMARCA5 team-up with RNF8 and RNF168, which are recruited in an ATM/γH2AX-dependent manner, to locally unfold chromatin, thereby facilitating ubiquitylation of the damaged chromatin and the recruitment of DNA repair proteins such as BRCA1 to the DSB . Thus, iHR requires extensive chromatin remodelling to enable DNA end-resection. To overcome the nucleosomal barrier, INO80 ejects core histones as well as variant histone H2A.Z, while CHD1 ‘opens’ chromatin around DSBs. This facilitates the loading of factors such as MRE11 and CtIP, thereby triggering the initiation of end-resection . FurtherIt has been long thought that NHEJ simply relies on direct sealing of the broken ends without the need of extensive chromatin structural changes. However, recent studies have implicated several chromatin remodellers in NHEJ. For instance, CHD2 and ALC1 work in a PARP1-dependent pathway that facilitates chromatin relaxation ,58. AddiCHD2 gene have been associated with lymphoma [Emerging evidence shows that ATP-dependent chromatin remodellers are not only critical for a proper DSB response, but also key in preventing cancer and disorders associated with intellectual disability or ageing. For instance, the inactivation of SMARCA2 and SMARCA4 due to somatic mutations or epigenetic silencing led to the development of various tumours . In contlymphoma , whereaslymphoma . It is s


Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family.The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm ( The Na+ electrochemical potential difference is the driving force used to transport substrates. In insects, some SLC6 members also depend on the existence of K+ [The solute carrier 6 (SLC6) gene family of membrane transporters shuttle extracellular neurotransmitters, amino acids and osmolytes across the plasma membrane into the cytoplasm through co-transport of Nace of K+ ,2,3,4. Gce of K+ ,6. SLC6 ce of K+ ,8. Thesece of K+ .The SLC6 family is currently divided into four subfamilies that are monoamine transporters, γ-aminobutyric acid (GABA) transporters, amino acid transporters (I), and amino acid transporters (II)/orphan transporters . MonoamiDrosophila melanogaster and the serotonin transporter of humans were reported, which provide blueprints for future drug design [D. melanogaster SLC6 transporters through RT-PCR and in situ hybridization [Anopheles gambiae, transport phenol-branched and indole-branched substrates [Manduca sexta have been shown to have narrow substrates spectrum and are responsible for proline and threonine transport [Drosophila mediates the absorption of d-amino acids [Neurotransmitters are important in the regulation of homeostasis and biological processes. Thus, many earlier studies have focused on NTTs, and their functions have been demonstrated with abundant evidence. For example, a dopamine transporter mediates dopamine homeostasis and is related with schizophrenia, Parkinson’s disease, and ADHD ,16. In ag design ,22. To adization , furtherbstrates ,24; MsCAransport ; and DmNno acids .Bombyx mori. To investigate the role of SLC6 family genes in silkworm, bioinformatic analyses were performed to identify SLC6 transporters homologs in the silkworm genome. Moreover, phylogenetic relationships among insect SLC6 members and spatio-temporal expression profiles of silkworm SLC6 family members were determined. In addition, starvation experiments and juvenile hormone analog (JHA) treatment were performed to functionally assess midgut-enriched and midgut-specific SLC6 genes. Together, our results will further enhance studies of the evolution and function of SLC6 genes in insects.Compared with the studies of other insect SLC6 transporters, there is little knowledge about SLC6 family genes in the silkworm, BmDAT and BmSERT  using known SLC6 protein sequences from other species. Subsequently, a total of 16 transporters were identified in the silkworm genome, including two previously identified SLC6 genes, namely d BmSERT . These s studies ,28.Aquifex aeolicus LeuTAa reveals important residues for substrate binding and ion binding [Aa as a control. Amino acid sequence alignments of B. mori SLC6 transporters with A. aeolicus LeuTAa are presented in B. mori SLC6 transporters contain an inverted structural repeat of transmembrane helices TMs1-5 and TMs6-10  showed 45% sequence identity.The reported crystal structure of  binding . As a re TMs6-10 . MultiplB. mori and 85 SLC6 proteins from other insects: 21 from D. melanogaster, 20 from A. aegypti, 16 from A. gambiae, 11 from Tribolium castaneum (T. castaneum), eight from Danaus plexippus (D. plexippus), six from M. sexta, and three from Trichoplusia ni (T. ni)  . All ami(T. ni) , similarBmNAT1, BmNAT2, BmGT1-L, and BmSLC6-15 were specifically expressed in the silkworm midgut. The level of BmINE mRNA expression was high in midgut and Malpighian tubules, but lower in other tissues. Moreover, BmGT1 was expressed in all tissues except for the hemocytes. Compared to other tissues, BmGT1 showed high levels of mRNA expression in midgut. Six genes AT3-1, and BmB(0)AT3-2) were highly expressed in the heads of silkworms, particular BmGAT1 and BmGT2. Subsequently, we further found that the aforementioned six genes are highly expressed in silkworm brain. BmDAT, the dopamine transporter gene, is also expressed at high levels in gonads. In addition, BmSERT, the serotonin transporter gene, is also expressed in the cuticle. Similarities were found in the expression patterns of BmGT2 and BmB(0)AT3-2, which were found to be expressed in the ovaries and testis. BmCT1-L and BmOctT were specifically expressed in testis. The level of BmSLC6-14 mRNA expression was high in testis and the silk gland, but low in the ovaries, head, and Malpighian tubules. Furthermore, BmSLC6-16 was found to be highly expressed in Malpighian tubules and less so in other tissues.Tissue expression profiles of silkworm SLC6 genes in nine tissues collected from third day of the fifth instar larvae were performed using quantitative real-time PCR . BmNAT1,BmINE, BmNAT2, BmGT1-L, and BmSLC6-15 in newly molted larvae were found to be higher than in molting larvae, and there was a significantly difference between newly molted larvae and molting larvae AT3-2 were found to show differential expression between the feeding and wandering stages. The third day of the fifth instar (3d5I), the boundary of larval development [BmGAT1, BmDAT, and BmB(0)AT3-1) showed an identical trend during the feeding and wandering stages. Compared to other developmental stages, these genes were highly expressed at the middle pupal and adult stages. Brain-enriched genes, except for BmB(0)AT3-2 and BmDAT, were expressed at lower levels in the early pupal stage. In summary, these findings serve as further validation that head-enriched SLC6 genes are highly expressed in brain tissue. Together, these results imply that brain-enriched genes may be involved in wandering and mating behaviors.As a result of increased feeding for the last instar larvae and the behavior changes during the wandering and adult stages, we further investigated mRNA expression patterns of head-enriched SLC6 genes in silkworm brain during larval-pupal-adult development. The cDNA templates were derived from the silkworm brains in this experiment and levels of gene expression were examined in dissected brains. Levels of mRNA transcripts were used to screen for differential expression of genes related to wandering and mating behaviors . As a reelopment , was selBmGT1-L, BmNAT2 and BmGT1 were significantly down-regulated by starvation and up-regulated again by re-feeding. In contrast, with a starvation time of 6 h, BmNAT1 and BmSLC6-15 were up-regulated; afterwards, these genes were down-regulated by starvation. However, BmINE was up-regulated by starvation in most of treatments and down-regulated by re-feeding. These results indicated that midgut-enriched and midgut-specific SLC6 genes may be closely related to silkworm nutrient absorption.Genes in the SLC6 family were found to be highly and specifically expressed in the alimentary canal, mediating nutrient absorption, such as essential and neutral amino acids or amino acid-like substrates ,33. TherBmNAT1, BmNAT2, BmGT1-L, and BmINE were significantly induced after incubation with JHA for 6 h  were found to be highly expressed in brain and midgut, indicating that the glycine transporters may differ functionally between B. mori and D. melanogaster; Finally, the NAT includes numerous neutral amino acid transporters and a few B0+ transporters, which are expressed in the alimentary canal and mediate nutrient absorption [BmNAT1, BmNAT2, BmB(0)AT3-1, and BmB(0)AT3-2) were classified into the NAT subfamily. Among midgut-specific and midgut-enriched genes, expression levels of BmGT1-L and BmNAT1 were higher than the others tested. This result implies that these genes may play important roles in nutrient absorption.Phylogenetic analyses inferred the classification of insect SLC6 genes into five major classes: NTT, INE, AAT, NAT, and Orphan . Firstlynogaster ,42, and  hindgut . Interesctively. ,44,45. Ssorption . In thisBmGT2 and BmSERT mRNA expression levels are very low at the wandering stage AT3-1, BmDAT, and BmGAT were highly expressed during mating, which implies that these genes may be involved in mating behavior and reproduction. Moreover, it is noteworthy that the expression level of BmGAT is greatly higher than BmB(0)AT3-1 and BmDAT, implying that BmGAT may play an important roles in the genetic basis of mating behavior.Wandering behavior is important for pupation  and mating stage . InteresInR, IRS, PI3K110, and PDK in the fat body. These genes play key roles in the insulin/insulin growth factor signaling (IIS) pathway that regulates nutrition-dependent growth rates of insects [BmGT1-L, BmGT1 and BmNAT2) were induced by starvation or re-feeding  are JH-inducible [Leptinotarsa decemlineata, JH and a JH analog can activate LdNAT1 expression [BmNAT1, BmNAT2, BmGT1-L, and BmINE) were up-regulated by JHA  was provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China. The larvae were routinely reared on fresh mulberry leaves at 25 ± 1 °C and humidity of 75% ± 5% under 14 h light/10 h dark cycles. For tissue expression analysis, different tissue samples from the third day of the fifth instar larvae were dissected on ice. For temporal expression analysis, samples were collected at various instars. Each sample contained at least 3 larvae. The larval brains were collected at different time point that is related to wandering behavior and mating behavior. All samples were stored at −80 °C for RNA isolation.http://www.silkdb.org/silkdb/) to identify the candidate genes of the silkworm SLC6 family. Monoamine transporters, GABA transporters, orphan transporters, and Nutrient Amino acid transporters from other species were used for sequence searches. Subsequently, the SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.sanger.ac.uk/) databases were used to validate each candidate transporter. Candidate transporters, which have the SNF domain and high sequence identity, were considered as SLC6 gene family members. The sequences of SLC6 transporters from D. melanogaster, A. gambiae, A. aegypti, M. sexta, T. ni, D. plexippus and T. castaneum were downloaded from the NCBI GenBank, VectorBase and FlyBase. A multiple sequence alignment, including 16 putative SLC6 genes from B. mori and the leucine transporter from A. aeolicus [www.psc.edu/biomed/genedoc).BLASTP searches of predicted proteins were performed against NCBI and the Silkworm Genome Database . The phylogeny was constructed according to the Poisson correlation model generated by the neighbor-joining (NJ) method, and the bootstrap value was set to 1000. To describe evolutionary relationships among insect SLC6 members, the alignment of 101 SLC6 family transporters was selected. Homologs protein sequences were derived from Coleopteran, Lepidoptera, and Diptera, including D. melanogaster and B. mori.Phylogenetic analyses were performed using MEGA version 5.02  of B. mori was used as an internal control. The oligonucleotide primers corresponding to silkworm SLC6 family genes used in this study are listed in Total RNA from various samples was isolated using the EZNA Total RNA Kit  according to the manufacturer’s instructions. Then, DNaseI  was used to remove residual DNA. Four micrograms of total RNA were used to synthesize first-strand of cDNA with M-MLV Reverse Transcriptase  at 42 °C. Silkworm SLC6 genes mRNA sequences were obtained from the NCBI GenBank sequence database. Forward and reverse primers for silkworm SLC6 genes were designed using the Primer Premier version 5.0 . Eukaryotic translation initiation factor 4A  were used according to the manufacturer’s instructions for qPCR. The qPCR was performed under the following conditions: initial denaturation for 3 min at 95 °C, and 40 cycles for 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Reaction specificity was confirmed through generation of a melting curve. Relative expression levels of silkworm SLC6 genes were calculated according to the n = 3. A student’s t-test was used to evaluate statistical significance (* p < 0.05 and ** p < 0.01).The 7500 Fast Real Time PCR System  and a SYBRTo explore the potential function of SLC6 family genes that were highly and specifically expressed in the silkworm midgut, the starvation experiment was introduced. Newly molted larvae on the fifth instar were divided into 3 groups including feeding, starvation, and re-feeding. The feeding group (control group) was reared on fresh mulberry under the aforementioned conditions. The starvation group was subjected to the same conditions but without any diet. The larvae from the last group were starved for 24 or 36 h, and then fed with fresh mulberry for an extra 24 and 36 h, respectively. Midgut samples were collected from the three groups at 6, 24, 48, and 72 h and placed on ice. The samples were frozen in liquid nitrogen and stored at the −80 °C for total RNA isolation.To determine the impact of hormones on mRNA transcript expression of silkworm SLC6 genes, the JHA treatment experiment was performed. Midguts were dissected derive from fourth molting larvae. The isolated midguts were cultured in Grace’s medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and JHA (1 μM) in the optimal atmosphere. Midguts incubated in culture with the same volume of acetone were used as controls. Samples from each group were collected in triplicate at 6, 12, and 18 h, for total RNA extraction.


Spinal cord injury (SCI) results in fatal damage and currently has no effective treatment. The pathological mechanisms of SCI remain unclear. In this study, genome-wide transcriptional profiling of spinal cord samples from injured rats at different time points after SCI was performed by RNA-Sequencing (RNA-Seq). The transcriptomes were systematically characterized to identify the critical genes and pathways that are involved in SCI pathology.n = 3 in every group). Compared with the sham-control group, the number of differentially expressed genes was 1797 in the acute phase (1223 upregulated and 574 downregulated), 6590 in the subacute phase (3460 upregulated and 3130 downregulated), and 3499 in the chronic phase (1866 upregulated and 1633 downregulated), with an adjusted P-value <0.05 by DESeq. Gene ontology (GO) enrichment analysis showed that differentially expressed genes were most enriched in immune response, MHC protein complex, antigen processing and presentation, translation-related genes, structural constituent of ribosome, ion gated channel activity, small GTPase mediated signal transduction and cytokine and/or chemokine activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most enriched pathways included ribosome, antigen processing and presentation, retrograde endocannabinoid signaling, axon guidance, dopaminergic synapses, glutamatergic synapses, GABAergic synapses, TNF, HIF-1, Toll-like receptor, NF-kappa B, NOD-like receptor, cAMP, calcium, oxytocin, Rap1, B cell receptor and chemokine signaling pathway.RNA-Seq results were obtained from total RNA harvested from the spinal cords of sham control rats and rats in the acute, subacute, and chronic phases of SCI .The sequence data from this study have been deposited into Sequence Read Archive (The online version of this article (doi:10.1186/s12864-017-3532-x) contains supplementary material, which is available to authorized users. Spinal cord injury (SCI) results in a devastating loss of motor and sensory functions. In the United States, it has been reported that some 300,000 people are living with SCI and nearly 12,000 new patients are diagnosed annually [The pathological changes following traumatic SCI can be divided into two processes: primary injury and secondary injury. Primary injury is the immediate effect of mechanical damage on the spinal cord, which directly damages various tissue components. Secondary injury is a cascade of effects triggered by the primary injury affecting multiple biological processes and producing extensive temporal changes in gene expression . ConsideIn this study, temporal genome-wide gene expression profiles in injured spinal cords of adult rats were examined using RNA-Seq, and the results were confirmed by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Based on these data, the functions and pathways involved in the pathologic process of SCI in the acute, subacute, and chronic phases were characterized. The bioinformatics analysis confirmed that some of the expressed genes in our study have been shown to play important roles in SCI, thus supporting the reliability of our data. We also identified many additional expressed genes whose functions in SCI have not been well studied. These additional genes may be involved in the pathological process of SCI and deserve further study and discussion.We used 12 female Sprague-Dawley rats  in this study. Animal care in surgical procedures and after operation were in accordance with Regulations for the Administration of Affairs Concerning Experimental Animals  and Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care and Use Committees of Bengbu Medical College.To perform the contusive SCI, a New York University Impactor was used as described . Rats weBBB locomotor rating scale was used to assess the hind-limb movements based on observation of the rat’s freely moving in an open field , 11. Then = 3 in every group). Rats were perfused with phosphate buffer saline (PBS) before removing the spinal cords. The total RNA from spinal cords was extracted using TRIzol reagent . Genomic DNA was removed by using DNase I. The 1% agarose gels were used to monitor RNA degradation and contamination. NanoPhotometer® spectrophotometer  was used to check RNA purity. Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer  was used to measure RNA concentration. RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system  was used to assess RNA integrity.Spinal cords  of sham, acute, subacute, and chronic phases were harvested at 6 days, 1 day, 6 days, and 28 days after injury, respectively  was used to generate sequencing libraries. For each sample, the index codes were added to attribute sequences. The poly-T oligo-attached magnetic beads were used to purify mRNA from total RNA. In NEBNext First Strand Synthesis Reaction Buffer (5×), the mRNA was fragmented by divalent cations under elevated temperature. M-MuLV Reverse Transcriptase (RNase H) and random hexamer primers were used to synthesize the first strand cDNA. RNase H and DNA polymerase I were subsequently used to synthesize the second strand cDNA. The exonuclease/polymerase activities converted remaining overhangs into blunt ends. To prepare for hybridization, the 3′ ends of DNA fragments were adenylated and ligated to NEBNext Adaptor with hairpin loop structure. AMPure XP system  was used to purify the library fragments to select cDNA fragments of preferentially 150–200 bp. Before PCR, size-selected, adaptor-ligated cDNAs were treated with 3 μl USER Enzyme  at 37 °C for 15 min followed by 95 °C for 5 min. Index (X) Primer, Universal PCR primers and Phusion High-Fidelity DNA polymerase were used to perform PCR. Finally, AMPure XP system was used to purify PCR products, and the Agilent Bioanalyzer 2100 system was used to assess library quality.TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was used to cluster the index-coded samples on a cBot Cluster Generation System by Novogene  according to the manufacturer’s instructions. Finally, the 125 bp/150 bp paired-end reads were generated and sequenced on an Illumina Hiseq platform.To obtain high-quality clean data for downstream analyses, the low quality reads and reads containing adapters or ploy-N in raw data were removed and the Q20, Q30 and GC content of those clean data were also calculated.From genome website, the files of reference genome and gene model annotation were downloaded. Bowtie v2.2.3 was used to build the reference genome index. TopHat v2.0.12 was used to align the paired-end clean reads to the reference genome.The reads numbers mapped to each gene were counted by HTSeq v0.6.1. Fragments Per Kilobase of transcript sequence per millions base pairs sequenced (FPKM) was calculated. FPKM is the most commonly used method for evaluating the sequencing depth and gene length of the reads and estimating the levels of gene expression .P-values obtained from DESeq analysis. The adjusted P-value < 0.05 was adopted as the standard for judging statistically significant differences in gene expression.Differential expression of four groups  was analyzed using the DESeq R package (1.18.0). To control the false discovery rate, Benjamini and Hochberg’s approach was used to adjust the P-value < 0.05 was adopted as the standard for judging statistically significant enrichment of differentially expressed genes. KEGG enrichment analysis of DEGs was implemented by KOBAS software.The GOseq R package was used to perform GO enrichment analysis of DEGs. A corrected ΔΔCt method [To validate RNA-Seq results, 13 selected DEGs were verified by qRT-PCR as described [t method  was usedrd and 6th days, the mean BBB scores of the six surviving injured rats were 1 ± 0 and 5.33 ± 1.53, respectively. After 6 days, with only three injured rats remaining, BBB scores had improved substantially. The mean scores at 10 days post-injury (dpi), 14 dpi, 21 dpi and 28 dpi were 8.33 ± 1.53, 9.33 ± 1.53, 10.67 ± 0.58, and 11.67 ± 0.58, respectively. According to our previous experience, this was a moderate level of impairment.To observe the level of impairment produced by SCI, BBB scores were evaluated. As shown in Fig. In this study, we established 12 cDNA libraries with the following designations. Libraries with the A designation were from the sham-operated spinal cords and libraries B1, B2 and B3 were from injured spinal cords at 1 dpi, 6 dpi and 28 dpi, respectively, which represented of sham and three different pathological stages of SCI . RNA-Seq generated 41,724,966 to 58,619,478 raw reads for each library. After filtering the low-quality reads, the average number of clean reads was 42,505,205 (87.73%), 43,520,840 (82.37%), 43,336,187 (91.37%), and 42,483,112 (89.70%) for the A, B, C and D groups, respectively  was performed. As shown in Fig. , and 3 wRPKM was used to estimate the level of gene expression, and DEGSeq was used to examine the differential gene expression profile. Comparing the SCI and sham animals, we observed 1,979 DEGs at 1 dpi, with 1,223 upregulated and 574 downregulated genes , 35 significant GO terms were detected in the upregulated genes , 33 significant GO terms were detected in the up-regulated genes , 31 significant GO terms were detected in the up-regulated genes , ribosome, TNF signaling pathway, proteoglycans in cancer, malaria, and staphylococcus aureus infection were enriched pathways in all DEGs; Of the 13 enriched pathways in upregulated DEGs, the top ten were ribosome, TNF signaling pathway, malaria, salmonella infection, leishmaniasis, HIF-1 signaling pathway, proteoglycans in cancer, leukocyte transendothelial migration, Chagas disease (American trypanosomiasis), and staphylococcus aureus infection; the 2 enriched pathways in downregulated DEGs were circadian entrainment and axon guidance , glutamatergic synapse, circadian entrainment, GABAergic synapse and retrograde endocannabinoid signaling were pathways enriched in all DEGs. The top ten pathways of the 27 enriched pathways in upregulated DEGs were ribosome, osteoclast differentiation, lysosome, NF-kappa B signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, Chagas disease (American trypanosomiasis), pertussis and salmonella infection. The top ten pathways of the 37 pathways enriched in downregulated DEGs were glutamatergic synapse, circadian entrainment, GABAergic synapse, retrograde endocannabinoid signaling, calcium signaling pathway, dopaminergic synapse, neuroactive ligand-receptor interaction, adrenergic signaling in cardiomyocytes, insulin secretion and nicotine addiction , 39 pathways were enriched in all DEGs; the top ten were ribosome, circadian entrainment, glutamatergic synapse, GABAergic synapse, retrograde endocannabinoid signaling, salivary secretion, platelet activation, cAMP signaling pathway, nicotine addiction and lysosome; In the 21 pathways enriched in upregulated DEGs, the top ten were ribosome, lysosome, hematopoietic cell lineage, staphylococcus aureus infection, osteoclast differentiation, tuberculosis, leishmaniasis, Fc gamma R-mediated phagocytosis, phagosome and NF-kappa B signaling pathway. Of the 25 pathways enriched in downregulated DEGs, the top ten were circadian entrainment, cAMP signaling pathway, glutamatergic synapse, nicotine addiction, calcium signaling pathway, GABAergic synapse, retrograde endocannabinoid signaling, adrenergic signaling in cardiomyocytes, axon guidance and neuroactive ligand-receptor interaction , hexokinase-3 (Hk3) and triggering receptor expressed on myeloid cells 2 (Trem2) had not previously been reported in SCI. Plau is a serine protease involved in migration and proliferation of some tumor cells and degradation of the extracellular matrix . Hk3, anIn the chronic phase, from the ten most upregulated genes, we found that tumor necrosis factor alpha-induced protein 6 (Tnfaip6), neutrophil cytosol factor 1 (Ncf1), ankyrin repeat and suppressor of cytokine signaling box protein 15 (Asb15), cupredoxins (Cp), tartrate-resistant acid phosphatase 5 (Acp5) and C-type lectin domain family 4, member A3 (Clec4a3) had not previously been reported in SCI.Ncf1 is a cytosolic subunit of neutrophil NADPH oxidase which can be activated to produce superoxide anion. Ncf1 and NADPH oxidase 2 complex-derived reactive oxygen species are important regulators of several chronic inflammatory disorders, such as multiple sclerosis, gout, psoriasis and psoriatic arthritis, rheumatoid arthritis, and lupus . AlthougTnfaip6, a secretory protein induced by tumor necrosis factor alpha, is involved in extracellular matrix stability and cell migration, and is important in the protease network associated with inflammation . Asb15, A second important point regarding our study is that the genes identified in SCI are involved in many signaling pathways. For example, the TNF , 58, 59,However, there were also some interesting new terms in SCI found by KEGG pathway analysis. For example, the ribosome term was significantly enriched in the whole pathological process. Ribosomes consist of complexes of proteins and RNAs and are complex molecular machines responsible for protein synthesis in living cells . TherefoIn summary, our study has not only characterized global changes in gene expression in injured rat spinal cords, but has also systematically identified the critical genes and signaling pathways in SCI pathology. Although the genes associated with damage and repair of spinal cord injury are still largely unknown, the RNA-Seq analysis presented in this study will expand our understanding of the complex molecular mechanisms involved and provide a foundation for future studies of tissue damage and repair after SCI.