Genome-wide identification, characterization and expression profiling of TLR family genes in Chromileptes altivelis.

Toll-like receptors (TLRs) represent a prominent category of pattern recognition receptors that have been extensively investigated for their pivotal role in combating pathogen incursions. Despite this, there has been a notable absence of comprehensive identification and exploration of the immune response associated with the TLR family genes in C. altivelis. This study successfully identified and named fourteen genes as Catlr1-1, Catlr1-2, Catlr2-1, Catlr2-2, Catlr3, Catlr5, Catlr7, Catlr8, Catlr9, Catlr13-1, Catlr13-2, Catlr18, Catlr21, and Catlr22. A series of bioinformatic analysis were performed, encompassing analysis of protein properties, examination of gene structures, evolutionary assessments, and prediction of protein tertiary structures. The expression patterns of Catlr genes were analyzed in five immune tissues: liver, spleen, kidney, gill, and intestine, in both healthy and bacterial stimulated-fish. The results showed that different tissue and different genes showed differed expression patterns after V. harveyi infection, indicating the involvement of all Catlr members in mounting immune responses following infection in various tissues. Additionally, histological evaluations of immune tissues unveiled varying levels of damage. In conclusion, this investigation into the TLR gene family offers novel information that contribute to a more profound comprehension of the immune response mechanisms in C. altivelis.

A Well-Established Gut Microbiota Enhances the Efficiency of Nutrient Metabolism and Improves the Growth Performance of Trachinotus ovatus.

The gut microbiota has become an essential component of the host organism and plays a crucial role in the host immune system, metabolism, and physiology. Nevertheless, our comprehension of how the fish gut microbiota contributes to enhancing nutrient utilization in the diet and improving host growth performance remains unclear. In this study, we employed a comprehensive analysis of the microbiome, metabolome, and transcriptome to analyze intestines of the normal control group and the antibiotic-treated model group of T. ovatus to investigate how the gut microbiota enhances fish growth performance and uncover the underlying mechanisms. First, we found that the growth performance of the control group was significantly higher than that of the antibiotic-treated model under the same feeding conditions. Subsequent multiomics analyses showed that the gut microbiota can improve its own composition by mediating the colonization of some probiotics represented by Lactobacillus in the intestine, improving host metabolic efficiency with proteins and lipids, and also influencing the expression of genes in signaling pathways related to cell proliferation, which together contribute to the improved growth performance of T. ovatus. Our results demonstrated the important contribution of gut microbiota and its underlying molecular mechanisms on the growth performance of T. ovatus.

Evaluation of belimumab in treatment of Chinese childhood-onset systemic lupus erythematosus: a prospective analysis from multicenter study.

OBJECTIVE: The aim of this study is to identify whether low lupus disease activity status (LLDAS) and clinical remission (CR) of belimumab plus standard of care (SoC) therapy are achievable goals in childhood-onset SLE (cSLE). METHODS: This multicentre, one arm pre-post intervention study was conducted at 15 centers in China. The primary end point was to describe the proportion of patients who achieved LLDAS and CR after 3, 6, and 12 months after treatment with belimumab plus SoC therapy. A multiple regression model was used to impute missing data. A Poisson regression model was used to calculate the effect of belimumab treatment on the reduced risk of serious diseases and the incidence of new damage. RESULT: 193 (92.2% female) with active cSLE from 15 centers were included. At 3, 6 and 12 months, the proportion of LLDAS (CR) was 12.4% (1.0%), 25.6% (4.5%) and 70.3% (29.7%), respectively. The mean SELENA-SLEDAI score decreased from 11.0 at baseline to 3.7, 2.9 and 1.7 at 3, 6, and 12 months. At baseline, all patients received steroids at a mean (SD) prednisone equivalent dose of 31.0 (18.2) mg/day, which decreased to 19.4 (10.8) mg/day at month 3, 12.6 (7.2) mg/day at month 6 and 6.7 (5.3) mg/day at month 12. The symptoms and immunological indicators were also significantly improved. CONCLUSION: This is the first and largest sample size prospective clinical intervention study of cSLE patients treated with belimumab in China. LLDAS and CR were attainable treat-to-target of belimumab plus SoC therapy in cSLE.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

The removal of selenite and cadmium by immobilized biospheres: Efficiency, mechanisms and bacterial community.

In this study, a complex bacterial consortium was enriched from a typical Pb-Zn mine area and immobilized by sodium alginate to form biospheres, which were used for treatment of selenite (Se(IV))- and cadmium (Cd(II))-containing wastewater without external carbon source. Batch experiments showed that the maximum Se(IV) removal efficiency was 92.36% under the optimal conditions of an initial pH of 5, dosage of 5 g/L, initial Se(IV) concentration of 7.9 mg/L and reaction time of 168 h. Subsequently, more than 99% of 11.2 mg/L Cd(II) was removed by the biospheres within 10 h. Physicochemical characterization showed that reduction and adsorption were the main mechanisms for Se(IV) and Cd(II) removal, respectively. During the removal process, selenium and CdSe nanoparticles were formed. Bacterial community analysis showed the dominant bacterial genera changed after treatment of Se(IV)- and Cd(II)-containing wastewater. Additionally, 16S rRNA gene function prediction results showed that amino acid transport, carbohydrate transport, ion transport and metabolism were the dominant gene functions. The present study provides a potential way for the biological treatment of Se(IV)- and Cd(II)-containing wastewater using immobilized biospheres without external carbon source in short-term.

Intensive masculinization caused by chronic heat stress in juvenile Cynoglossus semilaevis: Growth performance, gonadal histology and gene responses.

The sea temperature has been observed to chronically increase during the past decades, leaving unpredictable influences to the marine biological resources. Thus, it is of vital significance to study the biological responses of ocean inhabited organisms with the artificially stimulated heat stress environment. Cynoglossus semilaevis provides us with an ideal model to study the influence of chronic heat stress on the sexual differentiation in marine teleosts for its genetic sex determination (GSD) + environmental effected (EE) sex determination system. In this study, the comparative experiment was conducted employing heated seawater (HT group) and ambient seawater (CT group) to cultivate juvenile C. semilaevis respectively. Significant differences were exhibited in growth performance and a delayed germ cell development effect was found in pseudomales formed under chronic heat stress. Using transcriptome analysis, the transcription profile of 55 days post fertilization (dpf) and 100 dpf juveniles' gonads were studied. A total of 47 libraries were constructed with an average mapping rate of 94.63% after assembling. GO and KEGG enrichment were proceeded using DEGs screened out between (1) pseudomale gonads at 55 dpf and 100 dpf in HT and CT group (2) pseudomale and female gonads at 55 dpf and 100 dpf in HT and CT group. Terms and pathways involved in steroid stimulation, reproduction ability, germ cell proliferation et al. were shed light on. The expression pattern of 29 DEGs including amh, hsp90b1, pgr et al. were also provided to supplement the results of functional enrichment. Weighted gene co-expression networks analysis (WGCNA) was constructed and hspb8-like, histone H2A.V were exhibited to play vital roles in the heat-induced masculinization. Our findings facilitate the understanding for transcriptional variations in intensive masculinization cause by chronic heat stress of C. semilaevis and provide referable study of the influences on the teleosts in elevated sea temperature.

Deficiency in the frequency and function of Tr1 cells in IgAV and the possible role of IL-27.

OBJECTIVE: Type 1 regulatory T (Tr1) cells are involved in the pathogenesis of numerous immune-mediated diseases. However, little is known about whether and how Tr1 cells affect the development of IgA vasculitis (IgAV). We aimed to investigate this question in IgAV patients. METHODS: . Tr1 cells in peripheral blood and kidney tissue of IgAV patients were analysed by multi-parametric flow cytometry and immunofluorescence techniques. An in vitro assay of suppression of T cell proliferation and cytokine release was performed to evaluate the function of Tr1 cells. Real-time PCR and cell stimulation in vitro were used to explore the roles of IL-27 and early growth response gene 2 (EGR2). RESULTS: The frequency of Tr1 cells was decreased in peripheral blood but increased in kidney tissue from IgAV patients. A defective suppressive function of Tr1 cells in IgAV was observed. The frequency of Tr1 cells and the cytokines secreted by them were up-regulated in the presence of recombinant IL-27 in vitro. Moreover, IL-27 also increased the expression of EGR2. Furthermore, lower frequency of Tr1 cells during remission had a higher recurrence rate. CONCLUSION: Tr1 cells are involved in the pathogenesis of IgAV. The low IL-27 in IgAV is responsible for impaired frequency and function of Tr1 cells, and EGR2 may be the specific transcription factor involved in the progression. Tr1 may be a risk factor for IgAV recurrence.

Association between alcohol intake and the risk of systemic lupus erythematosus: A systematic review and meta-analysis.

OBJECTIVES: Previous studies have reported inconsistent results on the relationship between alcohol intake and the risk of systemic lupus erythematosus (SLE). Therefore, we conducted a systematic review and meta-analysis to illustrate the potential role of alcohol intake on the progression of SLE. METHODS: An electronic search of the PubMed, EmBase, and the Cochrane library databases was conducted from their inception up to March 2020. Observational studies that investigated the role of alcohol intake on the risk of SLE were eligible for inclusion in this study. The pooled odds ratio (OR) with 95% confidence interval (CI) was calculated as an effect estimate using the random-effects model. RESULTS: Seven case-control studies (n = 3, 251) and three cohort studies (n = 322, 479) were selected for the final meta-analysis. Mild (OR: 0.85; 95% CI: 0.53-1.38; p = 0.515) or heavy (OR: 0.63; 95% CI: 0.37-1.09; p = 0.102) alcohol intake were not associated with the risk of SLE, while moderate alcohol intake could protect against the risk of SLE (OR: 0.71; 95% CI: 0.55-0.93; p = 0.012). Sensitivity analysis suggested that heavy alcohol intake was associated with a reduced risk of SLE (OR: 0.47; 95% CI: 0.32-0.67; p < 0.001). CONCLUSIONS: This study found that moderate alcohol intake could protect against the risk of SLE, while mild or heavy alcohol intake did not significantly affect the risk of SLE.

Combined DFT and kinetic Monte Carlo study of a bridging-spillover mechanism on fluorine-decorated graphene.

In this work, combining first-principles calculations with kinetic Monte Carlo (KMC) simulations, we constructed an irregular carbon bridge on the graphene surface and explored the process of H migration from the Pt catalyst to carbon bridge, and further migration to the graphene surface. The calculated reaction diagrams show that the hydrogen atoms can easily migrate from the Pt cluster to the carbon bridge with a low barrier of 0.22-0.86 eV, and KMC simulations indicate that the migration reactions can take place at intermediate temperatures (91.9-329.5 K). Our research clarified the role of the carbon bridge: (1) the close combination of Pt clusters and carbon bridges reduces H2 adsorption enthalpy, which facilitates the spillover of H atoms from the Pt cluster to the carbon bridges and (2) the unsaturated carbon atoms on the carbon bridges have radical character and tend to bind radical H atoms. The subsequent study shows that the F atoms decorated on graphene can greatly reduce the migration barrier of H atoms from the carbon bridge to graphene. With F atoms decorated, the carbon atoms are in an electron-deficient state, which have a strong ability to bind the hydrogen atoms, and it promotes the migration of H atoms to the graphene surface. The migration barrier and reaction temperature are reduced to 0.72 eV and 279 K, respectively.

Genome-wide identification of nonvisual opsin family reveals amplification of RPE-retinal G protein receptor gene (RGR) and offers novel insights into functions of RGR(s) in Paralichthys olivaceus (Paralichthyidae, Teleostei).

Opsins play important roles in the image-forming visual pathways and numerous biological systems such as the biological clock and circadian rhythm. However, the nonvisual opsins involved in nonimage forming process are not clear to date. The aim of this study was to characterize nonvisual opsins in Paralichthys olivaceus. A total of 24 nonvisual opsin genes were identified. Expressions of these genes in eye, brain, heart, testis, and fin were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Testis contained a surprisingly large number of nonvisual opsins including Opn4m2, Tmt2a, Tmt3b, Opn3, RRH, Opn7a, and Opn7b. Syntenic and phylogenetic analyses confirmed that the RGRa and RGRb originated from the teleost-specific genome duplication (TSGD). qRT-PCR results demonstrated high RGRa and RGRb expression in the eye, while the expression levels in the brain, heart, testis, and fin were relatively weak. In situ hybridization results presented here revealed the presence of both RGRa and RGRb mRNA-positive signals in the ganglion cell layer but absence in the intracellular compartment of retinal pigment epithelium (RPE) and Müller glial cells. Therefore, we hypothesized that RGRa and RGRb had undergone subfunctionalization in P. olivaceus after TSGD. In conclusion, this study provides novel insights into the evolutionary fates of the RGR genes, still, further studies need to be done to explore the mechanism about the lack of RGR genes' expression in RPE.

Novel joint catalytic properties of Fe and N co-doped graphene for CO oxidation.

Using density functional theory, we have performed detailed calculations of the joint catalytic activity of graphene co-doped with Fe and N atoms. The Fe atom can be located on single vacancy graphene and acts as the active site. Due to the strong attraction of the Fe ion, the O-O bond length of the O2 molecule is elongated, which decreases the bonding energy between the O atoms. The energy barrier of CO oxidization is 0.84 eV. When N atoms are doped into the graphene, the interactions between the Fe ions and O2 molecules are stronger, and the O-O bond lengths are elongated further, which makes the desorption of the quasi-CO2 molecule easier. The energy barriers are reduced to 0.62 eV, 0.49 eV, and 0.33 eV for graphene doped with one, two and three N atoms, respectively. The O atom remaining on the Fe ion can form a CO2 molecule with an additional CO molecule. The produced CO2 molecule can be released with a small or even zero energy barrier by adsorbing an O2 molecule. The adsorbed O2 molecule is then involved in the next reaction process, and the material can be used as a recycled catalyst.

Two-dimensional hydrogen hydrates: structure and stability.

The structure and stability of two-dimensional hydrogen hydrate were investigated in this work using density functional theory. The results are in line with expectations that the occupied cages are more stable after their confinement between two parallel hydrophobic sheets. The four two-dimensional hydrogen hydrate crystals - BLHH-I, BLHH-II, BLHH-III and BLHH-IV - that we predicted were much more stable in a restricted environment than in a free environment, even close to or exceeding conventional hydrogen hydrates. Besides, we found that the stability of two-dimensional hydrates is inversely related to the increase in temperature. Our work highlights that two-dimensional hydrates provide a new research idea in the field of hydrogen storage.

Distinct variations of antibody secreting cells and memory B cells during the course of Kawasaki disease.

BACKGROUND: Both antibody secreting cells (ASCs) and memory B cells are essential for the maintenance of humoral immunity. To date, limit studies have focused on the two populations in Kawasaki disease (KD). To address the status of humoral immunity during KD, our current concentration is on the variations of ASCs and memory B cells, as well as their subsets in both acute and remission stages of KD. METHODS: ASCs were defined as the population with high expressions of CD27 and CD38 among CD3-CD20- lymphocytes. Based on the expression of surface marker CD138 and intracellular marker IgG, ASCs were further divided into two subsets. Memory B cells were characterized by the expressions of IgD, CD27 and IgM, upon which memory B cells were further categorized into CD27 + IgD- (switched memory, Sm), CD27-IgD- (Double negative, DN) and CD27 + IgD + IgM+ (marginal zone, MZ) B cells. Collectively, six populations were analyzed using flow cytometry. The blood samples were collected from KD patients in different stages and healthy controls. RESULTS: In the acute stage, the percentages of ASCs, CD138+ ASCs, and IgG+ ASCs were significantly increased. In contrast, the percentages of memory B cells including Sm and MZ B cells were significantly decreased. Correlation analysis found ASCs positively correlated with the level of serum IgM, whereas MZ B cells not only positively correlated with the level of serum IgG, IgA, and IgM, but also positively correlated with the level of serum complement C3 and C4 and negatively correlated with the value of C-reactive protein (CRP). In the remission stage, the percentages of IgG+ ASCs and MZ B cells were significantly reduced, whereas other subsets presented heterogeneous variations. CONCLUSIONS: Our study provided direct evidence that ASCs contributed to the pathogenesis of KD, and it was the first time to describe the variation of memory B cells in this disease. Among the subsets, only IgG+ ASCs presented a significant increase in the acute stage and decreased after IVIG administration, indicating the involvement of IgG+ ASCs in the inflammation of KD and also suggesting that IVIG played an inhibitory role in the expression of cytoplasmic IgG.

"On Water" Direct Organocatalytic Cyanoarylmethylation of Isatins for the Diastereoselective Synthesis of 3-Hydroxy-3-cyanomethyl Oxindoles.

An "on water" organocatalytic cyanoarylmethylation of aryl acetonitrile to isatins is developed, giving products in high yields and up to excellent diastereoselectivities. A remarkable enhancement of reaction rates and diastereoselectivities by water was observed under mild conditions. Moreover, this approach provides a highly efficient and environmentally benign access to thermodynamic 3-hydroxy-3-cyanomethyl oxindoles.

A functional polymorphism in the promoter of RhoB is associated with susceptibility to Vibrio anguillarum in turbot (Scophthalmus maximus).

As an isoform of Rho family GTPases, RhoB plays a pivotal role in cytoskeletal organization, cell proliferation, apoptosis and immune response. However, the regulatory mechanisms of RhoB expression in aquatic animals are still unknown. In the present study, we first construct Vibrio anguillarum infection model in S. maximus, including susceptible and resistant individuals. Then the temporal expression of RhoB was detected after V. anguillarum challenge using qRT-PCR and found that RhoB transcripts were significantly induced in the liver, gill and blood despite of differential expression levels and responsive time points. In addition, the mRNA levels of RhoB in resistant individuals were significantly higher than in susceptible ones. The length of 2083 bp sequences of RhoB promoter was cloned and characterized. Moreover, DNA methylation of the RhoB promoter was measured by bisulfite sequencing (BSP) and hypo-methylated was detected in the CpG islands. Three SNPs (-1590, -1575 and -1449) and two haplotypes in the promoter region of RhoB were identified to be associated with V. anguillarum resistance in turbot by association analysis in group 17-R and 17-S. Deletion analysis indicated that these SNPs could negatively mediate the activity of RhoB promoter. Site-directed mutagenesis and qRT-PCR of individuals with different genotypes demonstrated that -1575 T/A polymorphism affected promoter activity. Further study showed that this mutation altered the binding site of the transcription factor CREB. Co-transfection of SmCREB and RhoB promoter was performed in HEK293T cells which confirmed the -1575 allelic differences on transcriptional activity, with the susceptibility allele showing reduced activity. Taken together, our findings implicate that losing of binding of CREB to SmRhoB promoter due to -1575T/A polymorphisms enhances SmRhoB expression in resistant turbot, which provide insights into the effect of SmRhoB expression in response to V. anguillarum infection.

Edwardsiella tarda-induced miR-7a functions as a suppressor in PI3K/AKT/GSK3ß signaling pathway by targeting insulin receptor substrate-2 (IRS2a and IRS2b) in Paralichthys olivaceus.

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF-κB signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3' untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3ß signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3ß signaling pathway via suppressing the IRS2a and IRS2b genes.

Spotted knifejaw (Oplegnathus punctatus) MyD88: Intracellular localization, signal transduction function and immune responses to bacterial infection.

Myeloid differentiation factor 88 (MyD88) links members of the toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of NF-κB as a "bridge" molecular in response to exogenous pathogen, but the function in spotted knifejaw (Oplegnathus. punctatus), a commercial fish in China, is still unknown. We present a functional analysis of spotted knifejaw MyD88 (OppMyD88) with a typical death domain (DD) at the N-terminus and a conservative Toll/IL-1R (TIR) domain at the C-terminus and suggest that MyD88 is important for the activation of TLR-mediated NF-κB with the synergy between domains. Subcellular localization showed that OppMyD88 was distributed in the cytoplasm in a condensed form. Tissues expression profiling analysis showed that OppMyD88 ubiquitously expressed in all tested tissues with the highest expression in the liver, as determined by real-time PCR. The expression of OppMyD88 significantly upregulated in the liver, spleen, kidney and gills within 120 h post Vibrio anguillarum infection. Moreover, we further confirmed that over-expressed OppMyD88 could also induce apoptosis. These results indicate that OppMyD88 might possess important roles in defense against microbial infection and other biological processes in spotted knifejaw similar to those in mammals, which will deepen our understandings in innate immunity of spotted knifejaw.

Whole-genome resequencing from bulked-segregant analysis reveals gene set based association analyses for the Vibrio anguillarum resistance of turbot (Scophthalmus maximus).

Many achievements have been made to develop quantitative trait loci (QTLs) and gene-associated single nucleotide polymorphisms (SNPs) to facilitate practical marker-assisted selection (MAS) in aquatic animals. However, the systematic studies of SNPs associated with extreme threshold traits were poor in populations lacking of parental genomic information. Coupling next generation sequencing with bulked segregant analysis (BSA) should allow identification of numerous associated SNPs with extreme phenotypes. In the present study, using combination of SNP frequency difference and Euclidean distance, we conducted linkage analysis of SNPs located in genes involved in immune responses, and identified markers associated with Vibrio anguillarum resistance in turbot (Scophthalmus maximus). A total of 221 SNPs was found as candidate SNPs between resistant and susceptible individuals. Among these SNPs, 35 loci located in immune related genes were genotyped in verification population and 7 of them showed significant association with V. anguillarum resistance in both alleles and genotypes (P < 0.05). Among these 7 genes, PIK3CA-like, CYLD, VCAM1, RhoB and RhoGEF are involved in PI3K/Akt/mTOR pathway and NF-κB pathway, which influence the efficiency of bacteria entering the host and inflammation. SNP-SNP interaction analysis was performed by generalized multifactor dimensionality reduction (GMDR). The combination of SNP loci in RhoB, PIK3CA-like and ADCY3 showed a significant effect on V. anguillarum resistance with the verification rate in the sequencing population up to 70.8%. Taken all, our findings demonstrated the feasibility of BSA-seq approach in identifying genes responsible for the extreme phenotypes and will aid in performing MAS in turbot.

Functional differentiation of three phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) in response to Vibrio anguillarum infection in turbot (Scophthalmus maximus).

PIK3CA has been extensively investigated from its molecular mechanism perspective and association with its mutations in different types of cancers. However, little has been reported regarding the pathological significance of PIK3CA expression in teleost. Here, in our present study, three PIK3CA genes termed SmPIK3CAa, SmPIK3CAb and SmPIK3CA-like were firstly identified in the genome of turbot S. maximus. Although these three genes located in different chromosomes, all of them share the same five domains. Phylogenetic and synteny analysis indicated that SmPIK3CAa, SmPIK3CAb and SmPIK3CA-like were three paralogs that may originate from duplication of the same ancestral PIK3CA gene. Subcellular localization analysis confirmed the cytoplasm distribution of these three paralogs. All three SmPIK3CA were ubiquitously expressed in examined tissues in turbot, with the higher expression levels in immune-related tissues such as blood, spleen, kidney, gills and intestines. Upon Vibrio anguillarum challenge, SmPIK3CAa and SmPIK3CA-like transcripts were significantly induced in spleen, intestine and blood despite of differential expression levels and responsive time points. Additionally, individuals in resistant group showed significantly higher expression level of both two genes than in the susceptible group. Moreover, four SNPs (102, 2530, 3027 and 3060) and one haplotype (Hap2) located in exon region of SmPIK3CA-like were identified and confirmed to be associated with V. anguillarum resistance in turbot by association analysis in different populations. Taken together, these results suggested that functional differentiation occurred in three SmPIK3CA paralogs with Vibrio anguillarum resistance and SmPIK3CAa and SmPIK3CA-like probable play potential roles in innate immune response to pathogenic invasions in turbot.

A novel C-type lectin from spotted knifejaw, Oplegnathus punctatus possesses antibacterial and anti-inflammatory activity.

C-type lectin is a type of carbohydrate-binding protein and plays significant roles in innate immune response against pathogen infection. To date, thousands of C-type lectin had been identified in teleost. In the present study, we isolated a novel isoform of C-type lectin (OppCTL) from spotted knifejaw (Oplegnathus punctatus). The OppCTL encoded a typical Ca2+-dependent carbohydrate-binding protein, and was mainly expressed in liver in a tissue specific fashion. The expression of OppCTL was significantly up-regulated following Vibrio anguillarum infection in vivo, suggesting involvement in immune response. Hemagglutination analysis showed that the recombinant OppCTL (rOppCTL) could agglutinate erythrocyte from Mus musculus, Oplegnathus punctatus, Sebastes schlegelii and Paralichthys olivaceus. The rOppCTL could bind and agglutinate all tested bacteria. The rOppCTL possessed capacities of calcium-dependent agglutination to all tested bacteria. Sugar binding assay revealed that rOppCTL could also bind to the glycoconjugates of the bacterial surface, including lipopolysaccharide and peptidoglycan. Interestingly, Dual-luciferase analysis revealed that OppCTL could inhibit the activity of NF-κB in HEK-293T cells after OppCTL overexpression. Taken together, these results indicate that OppCTL has immune activity capable of defending invading pathogens and possesses potential immunoregulatory activity, enriching our understanding of the function of C-type lectin.