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.

SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.

Background: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants. Methods: Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature. Results: RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/µL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/µL. Conclusions: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

Circular RNA circCCNB1 inhibits the migration and invasion of nasopharyngeal carcinoma through binding and stabilizing TJP1 mRNA.

Nasopharyngeal carcinoma (NPC) is a malignant tumor that usually occurs in people from Southeast Asia and Southern China. NPC is prone to migration and invasion, leading to poor prognosis. A large number of circular RNAs (circRNAs) exacerbate the process of metastasis in NPC; however, their underlying mechanisms remain unclear. We found that the circular RNA circCCNB1, encoded by the oncogene CCNB1, was downregulated in NPC biopsies and cell lines. In vitro assays show that circCCNB1 inhibits NPC cell migration and invasion. Moreover, circCCNB1 induces a protein, nuclear factor 90 (NF90), to bind and prolong the half-life of tight junction protein 1 (TJP1) mRNA. Upregulation of TJP1 enhances tight junctions between cancer cells and inhibits NPC cell migration and invasion. This study reveals a novel biological function of circCCNB1 in the migration and invasion of NPC by enhancing the tight junctions of cancer cells by binding to NF90 proteins and TJP1 mRNA, and may provide a potential therapeutic target for NPC.

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.

N6-methyladenosine-dependent signalling in cancer progression and insights into cancer therapies.

The N6-methyladenosine (m6A) modification is a dynamic and reversible epigenetic modification, which is co-transcriptionally deposited by a methyltransferase complex, removed by a demethylase, and recognized by reader proteins. Mechanistically, m6A modification regulates the expression levels of mRNA and nocoding RNA by modulating the fate of modified RNA molecules, such as RNA splicing, nuclear transport, translation, and stability. Several studies have shown that m6A modification is dysregulated in the progression of multiple diseases, especially human tumors. We emphasized that the dysregulation of m6A modification affects different signal transduction pathways and involves in the biological processes underlying tumor cell proliferation, apoptosis, invasion and migration, and metabolic reprogramming, and discuss the effects on different cancer treatment.

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.

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.

Identification and initial functional characterization of lysosomal integral membrane protein type 2 (LIMP-2) in turbot (Scophthalmus maximus L.).

The immune system protects organism from external pathogens, this progress starts with the pathogen recognition by pattern recognition receptors (PRRs). As a group of PRRs, the class B scavenger receptors showed important roles in phagocytosis. Among three class B scavenger receptors, lysosomal integral membrane protein type 2 (LIMP-2) was reported to present in the limiting membranes of lysosomes and late endosomes, but its immune roles in teleost species are still limited in handful species. Here, we characterized LIMP-2 gene in turbot, and its expression patterns in mucosal barriers following different bacterial infection, as well as ligand binding activities to different microbial ligands and agglutination assay with different bacteria. In our results, one SmLIMP2 gene was identified with a 1,593 bp open reading frame (ORF). The multiple species comparison and phylogenetic analysis showed the closest relationship to Paralichthys olivaceus, the genomic structure analysis and syntenic analysis revealed the conservation of LIMP-2 during evolution. In tissue distribution analysis, SmLIMP-2 was expressed in all the examined turbot tissues, with the highest expression level in brain, and the lowest expression level in liver. In addition, SmLIMP-2 was significantly up-regulated in all the mucosal tissues (skin, gill and intestine) following Gram-negative bacteria Vibrio anguillarum infection, and was only up-regulated in gill following Gram-positive bacteria Streptococcus iniae challenge. Finally, the rSmLIMP-2 showed strong binding ability to all the examined microbial ligands, and strong agglutination with Escherichia coli, Staphylococcus aureus and V. anguillarum. Taken together, our results suggested SmLIMP-2 played important roles in fish immune response to bacterial infection. However, further functional studies should be carried out to better characterize its detailed roles in teleost immunity.

Dynamics of MiRNA Transcriptome in Turbot (Scophthalmus maximus L.) Intestine Following Vibrio anguillarum Infection.

MicroRNAs (miRNAs) are a group of small non-coding RNAs, which could bind to the 3'-untranslated regions of their target mRNAs to regulate gene expression in various biological processes, including immune-regulated signaling pathways. Turbot (Scophthalmus maximus L.), an important commercial fish species in China, has been suffering with Vibrio anguillarum infection resulted in dramatic economic loss. Therefore, we investigated the expression profiles of miRNAs, as well as the immune-related miRNA-mRNA pairs in turbot intestine at 1 h, 4 h, and 12 h following V. anguillarum infection. As a result, 266 predicted novel miRNAs and 283 conserved miRNAs belonging to 92 miRNA families were detected. A total of 44 miRNAs were differentially expressed in the intestine following V. anguillarum infection. Following prediction, the potential target genes of differentially expressed miRNAs were grouped into a wide range of functional categories, including immune defense/evasion, inflammatory responses, RIG-I signaling pathway, and Toll-like receptor signaling pathway. Moreover, we selected 15 differentially expressed immune genes and their related differentially expressed miRNAs to construct an interaction network for V. anguillarum infection in turbot. These results suggested that in teleost, as in higher vertebrates, miRNAs prominently contribute to immune responses, protecting the host against infection. In addition, this is the first report of comprehensive identification of turbot miRNAs being differentially regulated in the intestine related to V. anguillarum infection. Our results provided an opportunity for further understanding of the molecular mechanisms of miRNA regulation in turbot host-pathogen interactions.

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 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.

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.

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.

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.

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.

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.