Epinephelus coioides Sec3 promotes Singapore grouper iridovirus infection by negatively regulates immune response.

Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90% mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response.

Dual-specificity phosphatase 1 inhibits Singapore grouper iridovirus replication via regulating apoptosis in Epinephelus coioides.

The dual-specificity phosphatase (DUSP) family plays key roles in the maintenance of cellular homeostasis and apoptosis etc. In this study, the DUSP member DUSP1 of Epinephelus coioides was characterized: the length was 2371 bp including 281 bp 5' UTR, 911 bp 3' UTR, and a 1125 bp open reading frame encoding 374 amino acids. E. coioides DUSP1 has two conserved domains, a ROHD and DSPc along with a p38 MAPK phosphorylation site, localized at Ser308. E. coioides DUSP1 mRNA can be detected in all of the tissues examined, and the subcellular localization showed that DUSP1 was mainly distributed in the nucleus. Singapore grouper iridovirus (SGIV) infection could induce the differential expression of E. coioides DUSP1. Overexpression of DUSP1 could inhibit SGIV-induced cytopathic effect (CPE), the expressions of SGIV key genes, and the viral titers. Overexpression of DUSP1 could also regulate SGIV-induced apoptosis, and the expression of apoptosis-related factor caspase 3. The results would be helpful to further study the role of DUSP1 in viral infection.

Aberrant degree centrality profiles during rumination in major depressive disorder.

Rumination is closely linked to the onset and maintenance of major depressive disorder (MDD). Prior neuroimaging studies have identified the association between self-reported rumination trait and the functional coupling among a network of brain regions using resting-state functional magnetic resonance imaging (MRI). However, little is known about the underlying neural circuitry mechanism during active rumination in MDD. Degree centrality (DC) is a simple metric to denote network integration, which is critical for higher-order psychological processes such as rumination. During an MRI scan, individuals with MDD (N = 45) and healthy controls (HC, N = 46) completed a rumination state task. We examined the interaction effect between the group (MDD vs. HC) and condition (rumination vs. distraction) on vertex-wise DC. We further characterized the identified brain region's functional involvement with Neurosynth and BrainMap. Network-wise seed-based functional connectivity (FC) analysis was also conducted for the identified region of interest. Finally, exploratory correlation analysis was conducted between the identified region of interest's network FCs and self-reported in-scanner affect levels. We found that a left superior frontal gyrus (SFG) region, generally overlapped with the frontal eye field, showed a significant interaction effect. Further analysis revealed its involvement with executive functions. FCs between this region, the frontoparietal, and the dorsal attention network (DAN) also showed significant interaction effects. Furthermore, its FC to DAN during distraction showed a marginally significant negative association with in-scanner affect level at the baseline. Our results implicated an essential role of the left SFG in the rumination's underlying neural circuitry mechanism in MDD and provided novel evidence for the conceptualization of rumination in terms of impaired executive control.

Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Molecular cloning, inducible expression and function analysis of Epinephelus coioides Sec6 response to SGIV infection.

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

Identification and characterization of lncRNAs and the interaction of lncRNA-mRNA in Epinephelus coioides induced with Singapore grouper iridovirus infection.

Singapore grouper iridovirus (SGIV) is a highly pathogenic double-stranded DNA virus, and the fatality rate of SGIV-infected grouper is more than 90%. Up to now, there is no effective methods to control the disease. Long non-coding RNAs (lncRNAs) might play an important role in individual growth and development, immune regulation and other life processes. In this study, lncRNAs were identified in Epinephelus coioides, an important economic aquaculture marine fish in China and Southeast Asia, and the regulatory relationships of lncRNAs and mRNA response to SGIV infection were analyzed. A total of 11,678 lncRNAs were identified and classified from the spleen and GS (grouper spleen) cells. 105 differentially expressed lncRNAs (DElncRNAs) were detected during SGIV infection. The lncRNAs and the regulated mRNAs were analyzed using co-expression network, lncRNA target gene annotation and GO enrichment. At 24 and 48 h after SGIV infection, 118 and 339 lncRNA-mRNA pairs in GS cells were detected, and 728 and 688 differentially expressed lncRNA-mRNA pairs in spleen were obtained, respectively. GO and KEGG were used to predict the DE lncRNAs' target genes, and deduce the DE lncRNAs-affected signaling pathways. In GS cells, lncRNAs might participate in cell part, binding and catalytic activity; and lncRNAs might be involved in immune system process and transcription factor activity in spleen. These data demonstrated that lncRNAs could regulate the expression of immune-related genes response to viral infection, and providing a new insight into understanding the complexity of immune regulatory networks mediated by lncRNAs during viral infection in teleost fish.

Epinephelus coioides Hsp27 negatively regulates innate immune response and apoptosis induced by Singapore grouper iridovirus (SGIV) infection.

Heat shock proteins (Hsps) are important for maintaining protein homeostasis and cell survival. In this study, Hsp27 of Epinephelus coioides, an economically important marine fish in China and Southeast Asian countries, was characterized. E. coioides Hsp27 contains the consered ACD_HspB1_like domain and three p38 MAPK phosphorylation sites, located at Thr-13, Thr-60 and Ser-167. E. coioides Hsp27 was distributed in both the cytoplasm and nucleus, its mRNA was detected in all 14 tissues examined, and its expression was up-regulated after challenge with Singapore grouper iridovirus (SGIV), an important E. coioides pathogen. Over-expression of E. coioides Hsp27 significantly upregulated the expressions of the key SGIV genes (VP19, LITAF, MCP, and ICP18), downgraded the expressions of the E. coioides immune factors (IRF3, IRF7, ISG15, and TRAF6) and proinflammatory factors (TNF-α, IL-8), downgraded the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and substantially inhibited the cell apoptosis induced by SGIV infection. These data illustrated that E. coioides Hsp27 might be involved in SGIV infection by negatively regulating the innate immune response.

Epinephelus coioides PCSK9 affect the infection of SGIV by regulating the innate immune response.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1ß, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.

Characterization and function analysis of Epinephelus coioides Hsp40 response to Vibrio alginolyticus and SGIV infection.

Heat shock protein 40 (Hsp40), a member of Heat shock proteins (Hsps) family, plays a crucial role in regulation of cell proliferation, survival and apoptosis in mammals. In this study, Hsp40, EcHsp40, was identified from Epinephelus coioides, an economically important marine-cultured fish in China and Southeast Asian counties. The full length of EcHsp40 was 2236 bp in length containing a 1026 bp open reading frame (ORF) encoding 341 amino acids, with a molecular mass of 37.88 kDa and a theoretical pI of 9.09. EcHsp40 has two conserved domains DnaJ and DnaJ_C. EcHsp40 mRNA was detected in all tissues examined, and the expression was significantly up-regulated response to challenged with Vibrio alginolyticus or Singapore grouper iridovirus (SGIV), one of the important pathogens of marine fish. EcHsp40 was distributed in both the cytoplasm and nucleus, over-expression of EcHsp40 can inhibit the activity of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), significantly promote SGIV-induced apoptosis, intracellular caspase-3 activity and viral replication, suggesting that the EcHsp40 may play an important role in pathogenic stimulation.

The type 2 diabetes mellitus susceptibility gene CDKAL1 polymorphism is associated with depressive symptom in first-episode drug-naive schizophrenic patients.

BACKGROUND: Patients with schizophrenia have an increased prevalence of type 2 diabetes mellitus that has shown a significant association with the rs7754840 polymorphism in the gene encoding the cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1-like 1 (CDKAL1). OBJECTIVE: To examine whether this polymorphism was involved in the susceptibility in first-episode drug-naive schizophrenic patients (FDSP), and further influenced their clinical symptoms. METHODS: This polymorphism was genotyped in 239 FDSP and 368 healthy controls. The clinical symptoms in FDSP were assessed using the Positive and Negative Syndrome Scale (PANSS) five-factor models. RESULTS: There was no significant difference in the allelic and genotypic frequencies of this polymorphism between two groups (both p > 0.05) after adjusting for covariates. However, the PANSS depressive score significantly differed by genotype in FDSP after adjusting for covariates (F = 5.25, p = 0.006). This significant difference also persisted after Bonferroni correction (p < 0.05). FDSP with C/C genotype had significantly higher PANSS depressive score than those with C/G genotype (p = 0.007) and those with G/G genotype (p = 0.005). Moreover, further stepwise multivariate regression analysis showed the significant association between the rs7754840 polymorphism and PANSS depressive score in FDSP (ß = -1.07, t = -2.75, p = 0.007). CONCLUSIONS: Our findings demonstrated that although the CDKAL1 rs7754840 polymorphism did not contribute to the susceptibility to FDSP, it might be implicated in depressive symptoms in this patient group.

Molecular characterization and function analysis of Epinephelus coioides Hsp22 response to SGIV and Vribro alginolyticus infection.

Heat shock protein 22 (Hsp22) is an important regulatory factor response to various stresses in mammals. In this study, the full length cDNA of Epinephelus coioides Hsp22, which was 1680bp in length, with a 289 bp 5' UTR, a 725 bp 3'UTR, and a 666 bp open reading frame encoding 221 amino acids, was obtained. E. coioides Hsp22 contains a highly conserved α-crystallin domain. E. coioides Hsp22 mRNA was detected in all tissues examined by quantitative real-time PCR, with the highest expression in blood, followed by the spleen, skin, gill, head kidney, muscle, heart, liver, trunk kidney, stomach, pyloric caeca, intestine, brain and thymus. The expression patterns of E. coioides Hsp22 response to infection with Singapore grouper iridovirus (SGIV) and Vribro alginolyticus, the important pathogens of E. coioides, were studied. The expression levels of the gene were up-regulated in the tissues examined. Subcellular localization analysis demonstrated that E. coioides Hsp22 was distributed in both the cytoplasm and nucleus. In addition, E. coioides Hsp22 significantly inhibited the SGIV-induced cell apoptosis. In summary, the E. coioides Hsp22 might play a critical role in pathogenic stimulation.

Temperature-sensitive bone mesenchymal stem cells combined with mild hypothermia reduces neurological deficit in rats of severe traumatic brain injury.

BACKGROUND: To explore the combined influences of temperature-sensitive bone mesenchymal stem cells (tsBMSCs) and mild hypothermia (MH) on neurological function and glucose metabolism in rats with severe traumatic brain injury (TBI). METHODS: SD rats were randomly divided into sham, TBI, TBI + MH, TBI + BMSCs and TBI + MH +tsBMSCs groups. Then, the brain water content, serum-specific proteins (S100ß, NSE, LDH, and CK), and blood glucose at different time points were measured. Furthermore, GLUT-3 expression was detected by Western blotting, and apoptotic rate was determined by TUNEL staining. RESULTS: After TBI rat establishment, the brain injury resulted in significant increases in mNSS scores and brain water content, and upregulations in serum levels of S100ß, NSE, LDH and CK, and blood glucose, with the elevated cell apoptotic rate in the injured cortex. However, these changes were reversed by MH alone, BMSCs alone, or combination treatment of MH and tsBMSCs in varying degrees, and the combination treatment was superior to the treatment with BMSCs or MH alone. CONCLUSION: Combination therapy of tsBMSCs and MH can reduce the neuronal apoptosis in severe TBI rats, with the suppression of serum biomarkers and hyperglycemia, contributing to the recovery of neurological functions. ABBREVIATIONS: tsBMSCs: temperature-sensitive bone mesenchymal stem cells; MH: mild hypothermia; TBI: traumatic brain injury; mNSS: modified Neurological Severity Score.

Characterization and functional analysis of grouper (Epinephelus coioides) MEK1 and MEK2.

MEK dual-specificity protein kinases are a group of mitogen-activated protein kinase kinases, which act as an integration point by transferring extracellular signals to the nucleus. To investigate the function of MEK in teleost fish, we cloned MEK1 and MEK2 cDNA sequences from the orange-spotted grouper (Epinephelus coioides). EcMEK1 and EcMEK2 shared 80% amino acid identity with each other. EcMEK1 had 89-99% amino acid identity with teleosts or mammals, whereas EcMEK2 shared 85-97% amino acid identity. The exon structures of the grouper MEK1/2 genes were conserved with zebrafish and human MEK1/2. Tissue distribution analysis showed that EcMEK1 and EcMEK2 had a similar expression pattern in grouper tissues and was mainly transcribe in systemic immune organs. Both EcMEK1 and EcMEK2 were distributed throughout the cytoplasm of transfected GS or HEK293T cells. Overexpression of EcMEK1 or EcMEK2 activated Activator protein 1 dependent luciferase. The phosphorylation levels of EcMEK1/2 and EcERK1/2 were significantly increased in head kidney leukocytes by stimulation with PMA treatment. The grouper MEK1/2-ERK1/2 axis was activated in Cryptocaryon irritans infection and showed an enhanced phosphorylation after immunization.

Effects of antimicrobial peptides on serum biochemical parameters, antioxidant activity and non-specific immune responses in Epinephelus coioides.

Antimicrobial peptides (AMPs) are small proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens (bacteria, fungi and viruses). In this study, the effects of AMPs from Bacillus subtilis on Epinephelus coioides were examined. E. coioides were fed with diets containing AMPs (0, 100, 200, 400 or 800 mg/kg) for four weeks. Results showed that the levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU) and lipopolysaccharide (LPS) in the serum of E. coioides changed than those of the control group; compared to the control group, the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and lysozyme (LZM) levels in E. coioides fed with different dosages AMP diets were also different; in addition, the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1-beta (IL-1ß), and heat shock protein 90 (Hsp90) in the tissues of E. coioides were measured, the three genes in the tissues examined were significantly upregulated. The results demonstrated that diets containing AMPs can enhance the antioxidant capacity and innate immune ability of E. coioides, indicating that AMPs might be a potential alternative to antibiotics in E. coioides.

A novel MKK gene (EcMKK6) in Epinephelus coioides: Identification, characterization and its response to Vibrio alginolyticus and SGIV infection.

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.

miR-30 Family Reduction Maintains Self-Renewal and Promotes Tumorigenesis in NSCLC-Initiating Cells by Targeting Oncogene TM4SF1.

Increasing evidence indicates that tumor-initiating cells (TICs) are responsible for the occurrence, development, recurrence, and development of the drug resistance of cancer. MicroRNA (miRNA) plays a significant functional role by directly regulating targets of TIC-triggered non-small-cell lung cancer (NSCLC), but little is known about the function of the miR-30 family in TICs. In this study, we found the miR-30 family to be downregulated during the spheroid formation of NSCLC cells, and patients with lower miR-30a/c expression had shorter overall survival (OS) and progression-free survival (PFS). Moreover, transmembrane 4 super family member 1 (TM4SF1) was confirmed to be a direct target of miR-30a/c. Concomitant low expression of miR-30a/c and high expression of TM4SF1 correlated with a shorter median OS and PFS in NSCLC patients. miR-30a/c significantly inhibited stem-like characteristics in vitro and in vivo via suppression of its target gene TM4SF1, and then it inhibited the activity of the mTOR/AKT-signaling pathway. Thus, our data provide the first evidence that TM4SF1 is a direct target of miR-30a/c and miR-30a/c inhibits the stemness and proliferation of NSCLC cells by targeting TM4SF1, suggesting that miR-30a/c and TM4SF1 may be useful as tumor biomarkers for the diagnosis and treatment of NSCLC patients.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK.

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%-92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14-21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.

Identification and functional analysis of grouper (Epinephelus coioides) B-cell linker protein BLNK.

B-cell linker protein (BLNK) is an adaptor protein that plays a crucial role in the B cell antigen receptor (BCR) signal pathway. To investigate the function of BLNK in teleost fish, we cloned a BLNK ortholog gene from the orange-spotted grouper (Epinephelus coioides). Homology analysis showed that the grouper BLNK (EcBLNK) had a 34%-77% amino acid identity in comparison to other vertebrates and shared the highest amino acid identity with BLNK from the Asian seabass Lates calcarifer. EcBLNK comprises an N-terminal SAM domain and a C-terminal B-cell linker SH2 domain. Ten tyrosine residues were well conserved between teleost fish and mammals. Tissue distribution analysis showed that EcBLNK was expressed mainly in immune organs and expression was at the highest level in head kidney. Co-localization of EcBLNK and EcCD79a was observed in transfected HEK293T cells. Overexpression of EcBLNK did not activate nuclear factor kappa-light-chain-enhancer of activated B cells. The protein level of EcBLNK in grouper head kidney leukocytes was increased by stimulation with lipopolysaccharide. In groupers infected with Cryptocaryon irritans, EcBLNK was regulated in the infected sites and the systemic organ which suggests that EcBLNK was activated in the immune response to parasite infection.

Characterization and expression patterns of ERK1 and ERK2 from Epinephelus coioides against Cryptocaryon irritans infection.

Mitogen-activated protein kinases (MAPKs), a group of serine-threonine protein kinases, play a crucial role in immunoreaction response to extra environmental stresses. In this study, two novel MAPKs, Ec-ERK1 and Ec-ERK2, were identified from Epinephelus coioides. Both Ec-ERK1 and Ec-ERK2 sequences contain a highly conserved Thr-Glu-Tyr (TEY) motif, an HRD domain, and an ATP binding loop containing GXGXXG. An analysis of phylogenetic relationships demonstrated that ERK amino acid sequences were conserved between different species indicating that the functions may be similar. Ec-ERK1 and Ec-ERK2 mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of these two genes in E. coioides were also detected against Cryptocaryon irritans infection, which is capable of killing large numbers of fish in a short time and has a serious impact on aquaculture. The expression was up-regulated in most of the tissues examined, with the highest expressions of Ec-ERK1 (3.9 times) occurring in the head kidney and Ec-ERK2 (3.5 times) occurring in the spleen. There was no significant correlation between the expression of Ec-ERK1/Ec-ERK2 and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-ERK1/ERK2 were conserved, Ec-ERK1/ERK2 showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly varied post C. irritans infection, suggesting Ec-ERK1/ERK2 may play important roles in these tissues during pathogen-caused inflammation.

Two novel p38 MAPKs identified from Epinephelus coioides and their expression pattern in response to Cryptocaryon irritans infection.

P38 mitogen-activated protein kinases (MAPKs) are one of the most important central regulatory proteins response to extra environmental stresses. In this study, two novel p38 MAPKs, Ec-P38γ and Ec-P38δ, were identified from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. Both of Ec-p38γ and Ec-p38δ sequences contain a serine/threonine protein kinase (S_TKc) domain and a highly conserved Thr-Gly-Tyr (TGY) motif. Analysis of phylogenetic relationships illustrated that p38 amino acid sequences were conserved between different species indicating that the functions may be similar. The four subtypes of p38 (α, ß, γ, and δ) mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of the four Ec-p38 subtypes in E. coioides were also detected response to Cryptocaryon irritans infection, one of the most important protozoan pathogens of marine fish. The expression of four p38 subtypes was up-regulated in the tissues examined, with the highest expressions of Ec-p38α (5.2 times) and Ec-p38δ (4.2 times) occurring in the skin, while Ec-p38ß (24.8 times) and γ (16.6 times) occurred in the spleen. There was no significantly correlation between the expression of Ec-p38γ/Ec-p38δ and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-p38γ and Ec-p38δ were conserved, the p38 subtypes showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly up-regulated post C. irritans infection, suggesting these p38 MAPKs may play important roles in these tissues during pathogen-caused inflammation.