Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

Contrasting responses of cuticular bacteria of Pardosa pseudoannulata under cadmium stress.

Although research into how spiders respond to cadmium (Cd)-induced toxicity is ongoing, little is known about the effects of Cd contamination on the exogenous microorganisms of spiders. The current study used 16 S rRNA gene sequencing to evaluate the richness and structure of external bacterial communities in the wolf spider Pardosa pseudoannulata under long- and short-term Cd stress. Our results showed that Proteobacteria and Acidibacter were the dominating bacterial phylum and genus. The alpha diversity analysis showed that the high background of Cd concentration (Cd) reduced bacterial alpha diversity, and short-term Cd (SCd) stress elevated bacterial richness and diversity. Hub bacterial genera, including Stenotrophobacter, Hymenobacter, Chitinophaga, and Bryobacter, were identified by co-occurrence network analysis and showed high connectance with other bacterial genera. Further investigation revealed 15 and 14 bacterial taxa that were classified distinctively under SCd and Cd stresses. Interestingly, functional prediction analysis showed that Cd stress enhanced some crucial pathways involved in specialized functions, including manganese oxidation and aromatic compound degradation. Random forest and correlation analyses found that the spider's molting time was the dominant driver affecting bacterial phyla (i.e., Proteobacteria and Planctomycetes) and genera (e.g., Acidibacter, Reyranella, and Haliangium). Collectively, this comprehensive analysis creates new perspectives to investigate the divergent responses of microbial communities in the spiders' external habitat under Cd stress, and provides valuable viewpoints for Cd pollution evaluation and control.

Characterization, evolution and expression analysis of Toll-like receptor 7 (TLR7) in turbot (Scophthalmus maximus L.).

The pattern recognition receptors (PRRs) can recognize the conserved molecular structures of pathogens to active the innate immune responses, and subsequently induce the antigen-specific adaptive immune responses for the clearance of infected pathogen. Among the PRRs, Toll-like receptors (TLRs) are the first and best characterized PRRs across all the species. Among the TLR members, TLR7 showed significant conservation across the vertebrates, with the lowest rate of evolution for its LRR domains from primates to fishes. In the current study, one TLR7 (SmTLR7) gene was captured in turbot, with a 3144 bp open reading frame (ORF), that encoding 1047 amino acid residues. Following multiple sequence comparison, SmTLR7 was found to have the highest similarity and identity both to Paralichthys olivaceus with 91.9% and 85.9%, respectively. In phylogenetic analysis, SmTLR7 was firstly clustered with Japanese flounder, and then clustered with fugu, rainbow trout, and zebrafish. In addition, SmTLR7 was widely expressed in all the examined tissues with the highest expression level in spleen, followed by skin, while the lowest expression level was detected in blood. Following both Edwardsiella tarda and Vibrio anguillarum challenge, SmTLR7 was significantly down-regulated in gill and intestine, and up-regulated in skin. Moreover, SmTLR7 was significantly up-regulated in head kidney macrophages following LPS, LTA, PGN and polyI:C stimulation, as well as showed the strongest binding ability to LPS, followed by PGN, LTA, and polyI:C in a dose-dependent manner. Finally, following RNAi of SmTLR7, MyD88 and IL-1ß were slightly up-regulated, while TRAF6 and IL-8 were significantly down-regulated. The characterization of TLR7 can expand our understanding of the PRRs in teleost fishes, and eventually aid the exploration of interactions between host and pathogen.

Characterization of toll-like receptor 1 (TLR1) in turbot (Scophthalmus maximus L.).

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus, and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species.

Galectins in turbot (Scophthalmus maximus L.): Characterization and expression profiling in mucosal tissues.

Galectins, a family of evolutionary conserved ß-galactoside-binding proteins, have been characterized in a wide range of species. Many reports have indicated vital roles of galectins in innate immunity, especially in the mucosal tissues against infection. However, the systematic identification of galectin gene family is still lacking in teleost. Here, we characterized the galectin gene family and investigated their expression profiles post bacterial challenge in turbot (Scophthalmus maximus L.). In this study, a total of 13 galectin genes were characterized in turbot, phylogenetic analyses revealed their strong relationships to half smooth tongue sole and puffer fish, and syntenic analyses confirmed the orthology suggested by the phylogenetic analysis. In addition, the copy number of galectin genes is similar across a broad spectrum of species from fish to amphibians, birds, and mammals, ranging from 8 to 16 genes. Furthermore, the galectin genes were widely expressed in all the examined turbot tissues, and most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after Vibrio anguillarum infection in the intestine, gill and skin, suggesting that galectins were involved in the mucosal immune response to V. anguillarum infection in turbot. In addition, subcellular localization analysis showed lgals3a was distributed in the cytoplasm and nucleus. However, the knowledge of galectins are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Characterization of a novel lncRNA (SETD3-OT) in turbot (Scophthalmus maximus L.).

LncRNAs have been demonstrated to play pivotal roles in virous biological processes, especially the gene expression regulation, including transcriptional regulation, posttranscriptional control and epigenetic processes. However, most of the current studies of lncRNAs are still limited in mammalian species, the investigations of functional roles of lncRNAs in teleost species are still lacking. In current study, we identified a novel lncRNA (SETD3-OT) in turbot, with 2,504 bp full-length obtained by 5' and 3' RACE, located in turbot chromosome 17, ranged from 20,933,835 to 20,936,302 bp. In addition, 8 neighboring genes of SETD3-OT were identified within 100 kbp in genome location. From the annotation of the neighboring adjacent genes, SETD3-OT might involve in regulation of cell apoptosis and cycle, the immune cell development, and the immune response against infection, and its expression pattern is similar to majority of the neighboring genes following Aeromonas salmonicida challenge. Intriguingly, SETD3-OT showed significant high expression levels in mucosal surfaces (intestine, gill and skin), and was dramatically down-regulated in these mucosal tissues following Vibrio anguillarum challenge, especially in gill and skin. In addition, SETD3-OT was distributed in nucleus, it might regulate the neighboring genes in cis or in trans. Taken together, our results provide insights for lncRNA in fish innate immunity, further studies should be conduct to explore the detailed molecular mechanism of the gene regulation between SETD3-OT and its neighboring genes.

Expression profiling and functional characterization of galectin-3 of turbot (Scophthalmus maximus L.) in host mucosal immunity.

Galectins, a family of evolutionary conserved ß-galactoside-binding proteins, have been characterized in a wide range of species. Galectin-3 is the only member in the chimera type, which is a monomeric lectin with one CRD domain. A growing body of evidence have indicated vital roles of galectin-3 in innate immune responses against infection. Here, one galectin-3 gene was captured in turbot (SmLgals3) with a 1203 bp open reading frame (ORF). In comparison to other species, SmLgals3 showed the highest similarity and identity to large yellow croaker and medaka, respectively. The genomic structure analysis showed that SmLgals3 had 5 exons similar to other vertebrate species. The syntenic analysis revealed that galectin-3 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-3 region during vertebrate evolution. Subsequently, SmLgals3 was widely expressed in all the examined tissues, with the highest expression level in brain and the lowest expression level in skin. In addition, SmLgals3 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals3 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals3 played vital roles in fish innate immune responses against infection. However, the knowledge of SmLgals3 are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Identification and expression profiling analysis of microRNAs in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae infection.

MicroRNAs (miRNAs) play vital regulatory roles in various biological processes, including in immune responses. Nile tilapia (Oreochromis niloticus) is an important commercial fish species in China. To identify immune-related miRNAs of O. niloticus, 4 libraries from liver during S. agalactiae infection (0 h, 5 h, 50 h, and 7 d) were sequenced by high-throughput sequencing technology in tilapia. We obtained 10,703,531, 11,507,163, 11,180,179 and 13,408,414 clean reads per library, respectively. In our results, a total of 482 miRNAs were identified through bioinformatic analysis, including 220 conserved miRNAs and 262 putative novel miRNAs. Moreover, 21 (4.36%), 50 (10.37%), and 46 (9.54%) miRNAs were significantly differentially expressed at 5 h, 50 h and 7 d, respectively. In addition, 6939 target genes regulated by these differentially expressed miRNAs were predicted, and their functional annotations were predicted by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, which revealed that a majority of differentially expressed miRNAs were involved in apoptotic process, metabolic process, and immune responses. Finally, Real-time quantitative PCR experiments were performed for 7 miRNAs by stem-loop RT-PCR, and a general agreement was confirmed between the sequencing and RT-qPCR data. To our understanding, this is the first report of comprehensive identification of O. niloticus miRNAs being differentially regulated in liver related to S. agalactiae infection. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in O. niloticus host-pathogen interactions, and genetic resources for molecular assistant selection for disease resistant breeding program.

Expression profiling and microbial ligand binding analysis of galectin-4 in turbot (Scophthalmus maximus L.).

Galectins are a family of galactoside-binding proteins with an affinity for ß-galactosides, involved in mediating fundamental processes including development, inflammation, cell migration and apoptosis. Galectin-4 is a member of tendem-repeat galectins, plays vital roles in intestinal epithelial barrier. Here, one galectin-4 gene was captured in turbot (SmLgals4) contains a 1197 bp open reading frame (ORF). In comparison to other species, SmLgals4 showed the highest similarity and identity both to large yellow croaker. The genomic structure analysis showed that SmLgals4 had conserved exons in the CRD domains compared to other vertebrate species. The syntenic analysis revealed that galectin-4 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-4 region during vertebrate evolution. Subsequently, SmLgals4 was widely expressed in all the examined tissues, with the highest expression level in intestine and the lowest expression level in skin. In addition, SmLgals4 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals4 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals4 plays vital roles in fish intestinal immune responses against infection, but the detailed roles of galectin-4 in teleost are still lacking, further studies are needed to be carried out to characterize whether galectin-4 plays similar roles in teleost intestinal immunity.

Characterization, expression profiling and functional characterization of cathepsin Z (CTSZ) in turbot (Scophthalmus maximus L.).

Cathepsin Z (CTSZ) is a lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. In this study, we reported the identification of SmCTSZ, a CTSZ homolog from turbot (Scophthalmus maximus L.). SmCTSZ was 317 residues in length and contains a Pept-C1 domain. In multiple species comparison, SmCTSZ shared 65-93% overall sequence identities with the CTSZ counterparts from human, rat, and several fish species. In the phylogenetic analysis, SmCTSZ showed the closest relationship to Cynoglossus semilaevis. The syntenic analysis revealed the similar neighboring genes of CTSZ across all the selected species, which suggested the synteny encompassing CTSZ region during vertebrate evolution. Subsequently, SmCTSZ was constitutively expressed in various tissues, with the lowest and highest levels in brain and intestine respectively. In addition, SmCTSZ was significantly up-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSZ showed strong binding ability to all the examined microbial ligands, and the agglutination effect to different bacteria. Taken together, these results indicated SmCTSZ could play important roles in mucosal immune response in the event of bacterial infection in teleost. However, the knowledge of CTSZ are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Characterization and initial functional analysis of cathepsin K in turbot (Scophthalmus maximus L.).

Cathepsins are the best-known group of proteases in lysosomes, playing a significant role in immune responses. Cathepsin K (CTSK) is abundantly and selectively expressed in osteoclasts, dendritic cells and monocyte-derived macrophages, where it is involved in ECM degradation and bone remodeling. A growing body of evidences have indicated the vital roles of cathepsin K in innate immune responses. Here, one CTSK gene was captured in turbot (SmCTSK) with a 993 bp open reading frame (ORF). The genomic structure analysis showed that SmCTSK had 7 exons similar to other vertebrate species. The syntenic analysis revealed that CTSK had the same neighboring genes across all the selected species, which suggested the synteny encompassing CTSK region was conserved during vertebrate evolution. Subsequently, SmCTSK was widely expressed in all the examined tissues, with the highest expression level in spleen and the lowest expression level in liver. In addition, SmCTSK was significantly down-regulated in intestine following Gram-negative bacteria Vibrio anguillarum immersion challenge, but up-regulated in three tissues (gill, skin and intestine) following Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSK showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmCTSK played vital roles in fish innate immune responses against infection. However, the knowledge of SmCTSK is still limited in teleost species, further studies should be carried out to better characterize its comprehensive roles in teleost mucosal immunity.

Expression profiling and microbial ligand binding analysis of high-mobility group box-1 (HMGB1) in turbot (Scophthalmus maximus L.).

High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, was involved in nucleosome formation and transcriptional regulation, and could also act as an extracellular cytokine to trigger inflammation and immune responses. In this study, we identified a HMGB1 gene in turbot (Scophthalmus maximus L.). The full-length SaHMGB1 cDNA includes an open reading frame of 615 bp which encoded a 204 amino acid polypeptide with an estimated molecular mass of 23.19 kDa. SaHMGB1 was closely related to several fish HMGB1 and shared 74.4% overall identity with human. In addition, phylogenetic analyses revealed SaHMGB1 showed the closest relationship to Larimichthys crocea. Furthermore, QPCR analysis showed that SaHMGB1 was expressed in all examined tissues with abundant expression levels in brain, gill, intestine, and head kidney, and showed different expression patterns following different bacterial challenge. The significant quick regulation of SaHMGB1 in mucosal surfaces against infection suggest that HMGB1 might play critical roles in mucosal immunity against bacterial challenge. Finally, the in vitro binding assay showed that SaHMGB1 had strong binding ability to LPS, LTA, and PGN. Functional studies should further characterize HMGB1 function to understand the importance of the integrity of the mucosal barriers against infection, and to facilitate selection of the disease resistant family/strain in turbot.

l-rhamnose-binding lectins (RBLs) in turbot (Scophthalmus maximus L.): Characterization and expression profiling in mucosal tissues.

Rhamnose-binding lectin (RBL) were mostly identified from egg cortex and ovary cells from vertebrates and invertebrates, with the specific binding activities to l-rhamnose or d-galactose. Previously, we found that a RBL gene was dramatically down-regulated (-11.90 fold at 1 h, -48.95 fold at 4 h, -905.94 fold at 12 h) in the intestine of turbot following Vibrio anguillarum challenge using RNA-seq expression analysis. In this regard, we sought here to identify RBLs in turbot, as well as the analysis of genomic structure, phylogenetic relationships, basal tissue distribution and the expression patterns following different bacteria challenge in mucosal tissues. In this study, two RBLs were captured in turbot with two conserved type 5 CRD5s, which were belong to type IIIc RBL. In phylogenetic tree analysis, turbot RBLs were clustered with tilapia, European sea bass and snakehead. In addition, in comparison of genomic architecture of turbot RBLs with the available published RBL genes revealed a high degree of conservation in the exon/intron organization among the teleost species. Moreover, both RBLs were significantly up-regulated in mucosal tissues following V. anguillarum and Streptococcus iniae challenge, indicated their critical roles in turbot mucosal immunity. Further studies are needed to expand functional characterization of detailed mechanisms of RBLs in fish innate immunity.

The involvement of cathepsin F gene (CTSF) in turbot (Scophthalmus maximus L.) mucosal immunity.

Cathepsin F (CTSF) is a recently described papain-like cysteine protease and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. CTSF likely plays a regulatory role in processing the invariant chain which is associated with the major histocompatibility complex (MHC) class II. In this regard, we identified the CTSF gene of turbot as well as its protein structure, phylogenetic relationships, and expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. We also determined the expression patterns of CTSF in mucosal tissues after vaccinated with the formalin-inactivated V. vulnificus whole-cell vaccine. Briefly, turbot CTSF gene showed the closest relationship with that of Paralichthys olivaceus in phylogenetic analysis. And CTSF was ubiquitously expressed in all tested tissues with the highest expression level in gill. In addition, CTSF gene showed different expression patterns following different bacterial challenge. The significant quick regulation of CTSF in mucosal surfaces against infection indicated its roles in mucosal immunity. Functional studies should further characterize avail utilization of CTSF function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infection and to facilitate selection of the disease resistant family/strain in turbot.

Identification, characterization and expression analysis of TLR5 in the mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

TLRs (Toll-like receptors) are very important pathogen pattern recognition receptors, which control the host immune responses against pathogens through recognition of molecular patterns specific to microorganisms. In this regard, investigation of the turbot TLRs could help to understand the immune responses for pathogen recognition. Here, transcripts of two TLR5 (TLR5a and TLR5b) were captured, and their protein structures were also predicted. Meanwhile, we characterized their expression patterns with emphasis on mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot TLR5 genes showed the closest relationship to Paralichthys olivaceus. These two TLR5 genes were ubiquitously expressed in healthy tissues although expression levels varied among the tested tissues. In addition, the two copies of turbot TLR5 showed different expression patterns after bacterial infections. After Vibrio anguillarum infection, TLR5a was generally up-regulated in intestine and skin while down-regulated in gill, while TLR5b showed a general down-regulation in mucosal tissues. After Streptococcus iniae infection, the TLR5a was down-regulated at 2 h while generally up-regulated after 4 h in mucosal tissues. Interestingly, the TLR5b was up-regulated in intestine while down-regulated in skin and gill after Streptococcus iniae infection. These findings suggested a possible irreplaceable role of TLR5 in the immune responses to the infections of a broad range of pathogens that include Gram-negative and Gram-positive bacteria. Future studies should apply the bacteriological and immune-histochemical techniques to study the main sites on the mucosal tissue for bacteria entry and identify the ligand specificity of the turbot TLRs after challenge.

Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

The mucosal surfaces are important for teleost as they are directly and continuously exposed to pathogen-rich aquatic environments. Scrutinization and recognition of the attached pathogens is the first crucial step of mucosal immunity initiation. Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a large group of intracellular pathogen recognition receptors (PRRs) which play key roles in pathogen recognition and subsequent immune signaling pathways activation. In this study, we identified two NLRC3 genes (NLRC3a and NLRC3b), a subfamily of NLRs from turbot, and profiled their expression patterns in mucosal tissues following bacterial challenge. NLRC3a transcript contains an open reading frame (ORF) of 3405 bp that encodes a putative peptide of 1134 amino acids. While NLRC3b has an ORF of 3114 bp encoding 1037 amino acids. A caspase recruitment domain (CARD) at N-terminus characterized turbot NLRC3a, while NLRC3b seems to be unique to teleost, containing a fish specific NACHT associated (FISNA) domain and an extra B30.2 (PRY/SPRY) domain at C-terminus. In addition, NLRC3a and NLRC3b were detected in all the examined tissues, with the highest expression levels in kidney and blood, respectively. After bacteria challenge, expression levels of turbot NLRC3 genes were strongly induced in intestine rather than in skin and gill, while NLRC3a had relatively higher expression level than that of NLRC3b. Taken together, NLRC3 genes found in this study were the first NLR members identified in turbot. The different expression signatures of NLRC3a and NLRC3b in mucosal tissues following two bacterial infections indicated they probably have important roles in early response to bacterial infection in the first line of host defense system.

The expression signatures of neuronal nitric oxide synthase (NOS1) in turbot (Scophthalmus maximus L.) mucosal surfaces against bacterial challenge.

The mucosal surfaces constitute the first immune barrier of host defense and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes. It has been long hypothesized that observed difference of disease resistance among different fish strains and species are strongly correlated to the activities of the immune actors in mucosal surfaces. Particularly, neuronal NOS (nNOS or NOS1) is a constitutively expressed gene that catalyzes the oxidation of l-arginine and water to nitric oxide (NO), which is known as a potent host defence effector in immune system with antimicrobial activity. Moreover, NOS1 was detected to be expressed in fish mucosal surfaces, but its activities in mucosal immune responses were always overlooked. In this regard, we identified the NOS1 of turbot and characterized its expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. The results showed that the NOS1 gene had a 4389 bp open reading frame (ORF) that encoded 1462 amino acids. Phylogenetic analysis showed the turbot NOS1 had the strongest relationship to Larimichthys crocea. And the syntenic analysis revealed the similar neighboring genes associated with turbot NOS1, compared with other teleost and mammals. In addition, NOS1 was widely expressed in all examined tissues with the highest expression level in brain, followed by intestine and gill. Finally, the NOS1 showed a general trend of up-regulation in mucosal tissues following both bacterial challenge, with the highest up-regulation in intestine. The significant quick induction of NOS1 in mucosal surfaces against infection indicated its key roles to prevent pathogen attachment and entry in mucosal immunity. More functional studies are needed to conduct in teleost to better understand the roles of NOS1 in maintaining the integrity of the mucosal barriers against infection.

Identification and expression analysis of TLR2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of the bacteria, play key roles in the mucosal surfaces for pathogen recognition and activation of immune signaling pathways. However, our understanding of the PRRs and their activities in mucosal surfaces in the critical early time points during pathogen infection is still limited. Towards to this end, here, we sought to identify the Toll-like receptor 2 (TLR2) in turbot as well as its expression profiles in mucosal barriers following bacterial infection in the early time points. The full-length TLR2 transcript consists of open reading frame (ORF) of 2451 bp encoding the putative peptide of 816 amino acids. The phylogenetic analysis revealed the turbot TLR2 showed the closest relationship to Paralichthys olivaceus. The TLR2 mRNA expression could be detected in all examined tissues, with the most abundant expression level in liver, and the lowest expression level in skin. In addition, TLR2 showed different expression patterns following Vibrio anguillarum and Streptococcus iniae infection, but was up-regulated following both challenge, especially post S. iniae challenge. Characterization of TLR2 will probably contribute to understanding of a number of infectious diseases and broaden the knowledge of interactions between host and pathogen, which will eventually help in the development of novel intervention strategies for farming turbot.

Identification and expression analysis of toll-like receptor genes (TLR8 and TLR9) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

Mucosal immune system is one of the most important components in the innate immunity and constitutes the front line of host defense against infection, especially for teleost, which are living in the pathogen-rich aquatic environment. The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of bacteria, are considered as one of the most important component for pathogen recognition and immune signaling pathways activation in mucosal immunity. In this regard, we sought to identify TLR8 and TLR9 in turbot (Scophthalmus maximus), as well as their mucosal expression patterns following different bacterial infection in mucosal tissues for the first time. The full-length TLR8 transcript consists of an open reading frame (ORF) of 3108 bp encoding the putative peptide of 1035 amino acids. While the TLR9 was 6730 bp long, containing a 3168 bp ORF that encodes 1055 amino acids. The phylogenetic analysis revealed both TLR8 and TLR9 showed the closest relationship to large yellow croaker. Moreover, both TLR8 and TLR9 could be detected in all examined healthy turbot tissues, with the lowest expression level in liver and a relatively moderate expression pattern in healthy mucosal tissues. Distinct expression patterns of TLR8 and TLR9 were comparatively observed in the mucosal tissues (intestine, gill and skin) following Vibrio anguillarum and Streptococcus iniae infection, suggesting their different roles for mucosal immunity. Further functional studies are needed to better characterize TLR8 and TLR9 and their family members, to better understand the ligand specificity and to identify their roles in different mucosal tissues in protecting fish from the pathogenically hostile environment.

Identification and mucosal expression analysis of cathepsin B in channel catfish (Ictalurus punctatus) following bacterial challenge.

The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited. Cathepsin B, a lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and playing crucial roles for host immune defense against pathogen infection. In this regard, we identified the cathepsin B (ctsba) of channel catfish and investigated the expression patterns of the ctsba in mucosal tissues following Edwardsiella ictaluri and Flavobacterium columnare challenge. Here, catfish ctsba gene was widely expressed in all examined tissues with the lowest expression level in muscle, and the highest expression level in trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin. In addition, the phylogenetic analysis showed the catfish ctsba had the strongest relationship to zebrafish. Moreover, the ctsba showed a general trend of up-regulated in mucosal tissues following both Gram-negative bacterial challenge. Taken together, the increased expression of ctsba in mucosal surfaces indicated the protective function of ctsba against bacterial infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may play additional physiological and biological roles in catfish mucosal tissues.