Identification and functional analysis of lysophosphatidic acid phosphatase type 6 (ACP6) gene in golden pompano (Trachinotusovatus).

Golden pompano (Trachinotus ovatus), a marine farmed fish, is economically valuable in China. Lysophosphatidic acid phosphatase type 6 (ACP6) is a type of histidine acid phosphatase and plays an important role in regulating host inflammatory responses and anti-cancer effects in mammals. However, its function in teleost remains unknown. The present study aimed to investigate ACP6 function in golden pompano. ACP6 from golden pompano was identified, cloned, and named TroACP6. The open reading frame of TroACP6 was 1275 bp in length, encoding 424 amino acids. The TroACP6 protein shared high sequence identity (43.32%-90.57 %) with the ACP6 of other species. It contained a histidine phosphatase domain with the active site motif "RHGART" and the catalytic dipeptide HD (histidine and aspartate). Meanwhile, TroACP6 mRNA was widely distributed in the various tissues of healthy golden pompano, with the maximum expression in the head kidney. The function of TroACP6 was analyzed both in vitro and in vivo, and the results revealed that the purified recombinant TroACP6 protein exhibited optimum phosphatase activity at pH 6.0 and 50 °C in vitro. Meanwhile, upon Edwardsiella tarda challenge, TroACP6 expression in tissues increased significantly in vivo. In addition, TroACP6 overexpression enhanced the respiratory burst activity and superoxide dismutase activity of head kidney macrophages in vivo. Furthermore, the overexpression and knockdown of TroACP6 in vivo had a significant effect on bacterial infection. In summary, the study findings indicate that TroACP6 in golden pompano is involved in host defense against bacterial infection.

Construction and analysis of the immune effect of two different vaccine types based on Vibrio harveyi VgrG.

Vibrio harveyi poses a significant threat to fish and invertebrates in mariculture, resulting in substantial financial repercussions for the aquaculture sector. Valine-glycine repeat protein G (VgrG) is essential for the type VI secretion system's (T6SS) assembly and secretion. VgrG from V. harveyi QT520 was cloned and analyzed in this study. The localization of VgrG was determined by Western blot, which revealed that it was located in the cytoplasm, secreted extracellularly, and attached to the membrane. The effectiveness of two vaccinations against V. harveyi infection-a subunit vaccine (rVgrG) and a DNA vaccine (pCNVgrG) prepared with VgrG was evaluated. The findings indicated that both vaccines provided a degree of protection against V. harveyi challenge. At 4 weeks post-vaccination (p.v.), the rVgrG and pCNVgrG exhibited relative percent survival rates (RPS) of 71.43% and 76.19%, respectively. At 8 weeks p.v., the RPS for rVgrG and pCNVgrG were 68.21% and 72.71%, respectively. While both rVgrG and pCNVgrG elicited serum antibody production, the subunit vaccinated fish demonstrated significantly higher levels of serum anti-VgrG specific antibodies than the DNA vaccine group. The result of qRT-PCR demonstrated that the expression of major histocompatibility complex (MHC) class Iα, tumor necrosis factor-alpha (TNF-α), interferon γ (IFNγ), and cluster of differentiation 4 (CD4) were up-regulated by both rVgrG and pCNVgrG. Fish vaccinated with rVgrG and pCNVgrG exhibited increased activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme. These findings suggest that VgrG from V. harveyi holds potential for application in vaccination.

Intestinal microbiota and gene expression alterations in leopard coral grouper (Plectropomus leopardus) under enteritis.

Enteritis poses a significant threat to fish farming, characterized by symptoms of intestinal and hepatic inflammation, physiological dysfunction, and dysbiosis. Focused on the leopard coral grouper (Plectropomus leopardus) with an enteritis outbreak on a South China Sea farm, our prior scrutiny did not find any abnormalities in feeding or conventional water quality factors, nor were any specific pathogen infections related to enteritis identified. This study further elucidates their intestinal flora alterations, host responses, and their interactions to uncover the underlying pathogenetic mechanisms and facilitate effective prevention and management strategies. Enteritis-affected fish exhibited substantial differences in intestinal flora compared to control fish (P = 0.001). Notably, norank_f_Alcaligenaceae, which has a negative impact on fish health, predominated in enteritis-affected fish (91.76 %), while the probiotic genus Lactococcus dominated in controls (93.90 %). Additionally, certain genera with pathogenesis potentials like Achromobacter, Sphingomonas, and Streptococcus were more abundant in diseased fish, whereas Enterococcus and Clostridium_sensu_stricto with probiotic potentials were enriched in control fish. At the transcriptomic level, strong inflammatory responses, accompanied by impaired metabolic functions, tissue damage, and iron death signaling activation were observed in the intestines and liver during enteritis. Furthermore, correlation analysis highlighted that potential pathogen groups were positively associated with inflammation and tissue damage genes while presenting negatively correlated with metabolic function-related genes. In conclusion, dysbiosis in the intestinal microbiome, particularly an aberrantly high abundance of Alcaligenaceae with pathogenic potential may be the main trigger for this enteritis outbreak. Alcaligenaceae alongside Achromobacter, Sphingomonas, and Streptococcus emerged as biomarkers for enteritis, whereas some species of Lactococcus, Clostridium_sensu_stricto, and Enterococcus showed promise as probiotics to alleviate enteritis symptoms. These findings enhance our understanding of enteritis pathogenesis, highlight intestinal microbiota shifts in leopard coral grouper, and propose biomarkers for monitoring, probiotic selection, and enteritis management.

Rash caused by lurasidone in old chinese patient with bipolar disorder: case-based review.

BACKGROUND: Rash is one of common adverse drug reaction and which have been reported in typical and atypical antipsychotics. Reports of lurasidone induced skin reactions are sparse. In this study, we report a case of rash caused by lurasidone. CASE PRESENTATION: A 63-year-old man with bipolar disorder (BD) who is treated by lurasidone. However, the patient presents a rash all over after lurasidone dose increasing from 40 mg/day to 60 mg/day. With the diagnosis of drug induced rash, lurasidone was discontinued, and the rash complete disappears within 2 weeks. In addition, all case reports about antipsychotics associated rash were reviewed by searching English and Chinese database including Pubmed, Embase, Cochrane Library, CNKI and Wanfang database. A total of 139 articles contained 172 patients were included in our study. The literature review and our case suggest that the cutaneous adverse events caused by antipsychotic drugs should not be ignored, particularly for the patient who was first use or at dose increasing of antipsychotic. CONCLUSIONS: In conclusion, we report a case of lurasidone related rash and review rash caused by antipsychotics. Psychiatrists should be alert to the possibility of the rash caused by antipsychotics, especially the patient was first use of antipsychotics or the antipsychotic dose was increasing.

Variable Time-Step Physics Engine with Continuous Compliance Contact Model for Optimal Robotic Grinding Trajectory Planning.

In the transition from virtual environments to real-world applications, the role of physics engines is crucial for accurately emulating and representing systems. To address the prevalent issue of inaccurate simulations, this paper introduces a novel physics engine uniquely designed with a compliant contact model designed for robotic grinding. It features continuous and variable time-step simulations, emphasizing accurate contact force calculations during object collision. Firstly, the engine derives dynamic equations considering spring stiffness, damping coefficients, coefficients of restitution, and external forces. This facilitates the effective determination of dynamic parameters such as contact force, acceleration, velocity, and position throughout penetration processes continuously. Secondly, the approach utilizes effective inertia in developing the contact model, which is designed for multi-jointed robots through pose transformation. The proposed physics engine effectively captures energy conversion in scenarios with convex contact surface shapes through the application of spring dampers during collisions. Finally, the reliability of the contact solver in the simulation was verified through bouncing ball experiments and robotic grinding experiments under different coefficients of restitution. These experiments effectively recorded the continuous variations in parameters, such as contact force, verifying the integral stability of the system. In summary, this article advances physics engine technology beyond current geometrically constrained contact solutions, enhancing the accuracy of simulations and modeling in virtual environments. This is particularly significant in scenarios wherein there are constant changes in the outside world, such as robotic grinding tasks.

Molecular characterization and functional analysis of tartrate-resistant acid phosphatase (ACP5) gene in red drum (Sciaenops ocellatus).

Tartrate-resistant acid phosphatase (ACP5) plays an important biological function in immune defense and is highly expressed in activated macrophages, osteoclasts and dendritic cells. In teleost, the functionality of ACP5 remains to be revealed. In this study, we cloned and identified SoACP5 from red drum (Sciaenops ocellatus) and analyzed its function in vivo and in vitro. The open reading frame of SoACP5 is 1002 bp in length, encoding 333 amino acids. SoACP5 shares high sequence identities (96.70%-49.25%) with ACP5 of other species. The SoACP5 mRNA was widely distributed in collected tissues of healthy red drum, and with the maximum in gills. The expression of SoACP5 increased significantly in vivo following challenge with Edwardsiella tarda. Moreover, the recombinant SoACP5 protein (rSoACP5) was purified with his-tag band resin columns, and confirmed to have phosphatase activity which was optimal at pH 5 and 55 °C. Various metal ions (K+, Zn2+, Mn2+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+) have different effects on phosphatase activity. rSoACP5 induced the cellular proliferation of peripheral blood leukocytes. The over-expression and knockdown of SoACP5 in vivo had a significant effect on bacterial proliferation. Furthermore, both of the antibacterial activity and phosphatase activity were decreased when the reducedSoACP5 was oxidized by H2O2. In summary, the present study indicated that SoACP5 is likely involved in host defense against bacterial infection in S. ocellatus.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1ß, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

TroTNFSF6, a tumor necrosis factor ligand superfamily member, promotes antibacterial immune response of golden pompano, Trachinotus ovatus.

Tumor necrosis factor ligand superfamily member 6 (TNFSF6), also known as FasL/CD95L, is essential for maintaining the body's immune homeostasis. However, the current reports on TNFSF6 in fish are relatively scarce. In the present study, we conducted functional analyses of a TNFSF6 (TroTNFSF6) from the teleost fish golden pompano (Trachinotus ovatus). TroTNFSF6 is composed of 228 amino acids and has a low similarity with other species (9.65%-58.79%). TroTNFSF6 was expressed in the 11 tissues tested and was significantly up-regulated after Edwardsiella tarda infection. In vivo, overexpression of TroTNFSF6 effectively stimulated the AKP and ACP activities, and reduced bacterial infection in fish tissues. Correspondingly, knockdown of TroTNFSF6 expression resulted in increasing bacterial dissemination and colonization in fish tissues. In vitro, recombinant TroTNFSF6 protein promoted the proliferation of T. ovatus head kidney lymphocytes (HKLs), and promoted the apoptosis of murine liver cancer cells (Hepa1-6). The results indicated that TroTNFSF6 plays an important role in the T. ovatus antibacterial immunity. These observations will facilitate the future in-depth study of teleost TNFSF6.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

Antibacterial Activity and Mechanisms of TroHepc2-22, a Derived Peptide of Hepcidin2 from Golden Pompano (Trachinotus ovatus).

Hepcidin, a cysteine-rich antimicrobial peptide, has a highly conserved gene structure in teleosts, and it plays an essential role in host immune response against various pathogenic bacteria. Nonetheless, few studies on the antibacterial mechanism of hepcidin in golden pompano (Trachinotus ovatus) have been reported. In this study, we synthesized a derived peptide, TroHepc2-22, from the mature peptide of T. ovatus hepcidin2. Our results showed that TroHepc2-22 has superior antibacterial abilities against both Gram-negative (Vibrio harveyi and Edwardsiella piscicida) and Gram-positive (Staphylococcus aureus and Streptococcus agalactiae) bacteria. Based on the results of a bacterial membrane depolarization assay and propidium iodide (PI) staining assay in vitro, TroHepc2-22 displayed antimicrobial activity by inducing the bacterial membrane depolarization and changing the bacterial membrane permeability. Scanning electron microscopy (SEM) visualization illustrated that TroHepc2-22 brought about membrane rupturing and the leakage of the cytoplasm for the bacteria. In addition, TroHepc2-22 was verified to have hydrolytic activity on bacterial genomic DNA in view of the results of the gel retardation assay. In terms of the in vivo assay, the bacterial loads of V. harveyi in the tested immune tissues (liver, spleen, and head kidney) were significantly reduced in T. ovatus, revealing that TroHepc2-22 significantly enhanced the resistance against V. harveyi infection. Furthermore, the expressions of immune-related genes, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1-ß (IL-1ß), IL-6, Toll-like receptor 1 (TLR1), and myeloid differentiation factor 88 (MyD88) were significantly increased, indicating that TroHepc2-22 might regulate inflammatory cytokines and activate immune-related signaling pathways. To summarize, TroHepc2-22 possesses appreciable antimicrobial activity and plays a vital role in resisting bacterial infection. The observation of our present study unveils the excellent application prospect of hepcidin as a substitute for antibiotics to resist pathogenic microorganisms in teleosts.

Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin.

NK-lysin, a homologue of granulysin among human, is predominantly found in natural killer cells and cytotoxic T-lymphocytes, which plays a pivotal part in innate immune responses against diverse pathogenic bacteria. Nonetheless, in teleosts, the research on antimicrobial activity and mechanisms of NK-lysin are seldom reported. In this study, we determined the antimicrobial activity of the truncated peptide TroNKL-27 that derived from golden pompano (Trachinotus ovatus) NK-lysin, and investigated its antimicrobial mechanisms. The results showed that TroNKL-27 had considerable antimicrobial potency against both Gram-positive (Staphylococcus aureus, Streptococcus agalactiae) and Gram-negative bacteria (Vibrio harveyi, V. alginolyticus, Escherichia coli, Edwardsiella tarda). Cytoplasmic membrane depolarization and propidium iodide (PI) uptake assay manifested that TroNKL-27 could induce the bacterial membrane depolarization and change its membrane permeability, respectively. In the light of scanning electron microscopy (SEM) observation, TroNKL-27 was capable of altering morphological structures of bacteria and leading to leakage of cellular contents. Moreover, the results of gel retardation assay indicated TroNKL-27 had the ability to induce the degradation of bacterial genomic DNA. As regards in vivo assay, TroNKL-27 could reduce the replication of V. harveyi in tissues of golden pompano, protect the tissue from pathological changes. Moreover, TroNKL-27 in vivo could significantly increase the expression of the immune genes (such as IL1ß, TNFα, IFN-γ, C3 and Mx) in presence or absence of V. harveyi infection. All of these results suggest that TroNKL-27 is a novel antimicrobial peptide possessing antibacterial and immunoregulatory function in vivo and in vitro, and the observed effects of TroNKL-27 will lay a solid foundation for the development of new antimicrobial agents used in aquaculture.

CC chemokine 1 protein from Cromileptes altivelis (CaCC1) promotes antimicrobial immune defense.

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%-90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.

Bactericidal permeability-increasing protein/LPS-binding protein (BPI/LBP) enhances resistance of golden pompano Trachinotus ovatus against bacterial infection.

Antimicrobial peptides are crucial components of innate immunity against microbial invasions. As a kind of antimicrobial peptides, bactericidal permeability-increasing protein (BPI)/lipopolysaccharide-binding protein (LBP) play vital roles in defending the host against gram-negative bacteria. In the current study, a novel BPI/LBP from Trachinotus ovatus (TroBPI/LBP) was characterized. The full length of TroBPI/LBP cDNA sequence is 1434 bp, which contained 477 amino acids. Multiple amino acid alignments of TroBPI/LBP shows 34.07%-84.49% identity with other fish BPI/LBP. Similar to other BPI/LBP, TroBPI/LBP also possesses an N-terminal signal peptide, a BPI/LBP/CETP N-terminal domain, and a BPI/LBP/CETP C-terminal domain. In vitro, the recombinant protein of TroBPI/LBP showed effective bacterial depression activity and binding activity to gram-negative bacteria. In vivo, TroBPI/LBP was constitutively expressed in tested tissues, and the highest expression level was in liver. Following Vibrio alginolyticus stimulation, the mRNA expression of TroBPI/LBP was significantly upregulated in immune-related tissues, and peaked at 12 h post-infection, which confirmed that TroBPI/LBP was highly sensitive to V. alginolyticus stimuli. Furthermore, functional analyses showed that the overexpression of TroBPI/LBP could enhance the ability of fish to against V. alginolyticus infection, and the knockdown of TroBPI/LBP significantly diminished bacterial clearance capacity post-infection. Therefore, these results suggest that TroBPI/LBP may play an important role in host defense against bacterial infection.

Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus).

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys251, His397, and Asn419). It shares high homology (69.47%-90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn2+ and Ca2+ but inhibited by Fe2+ and Cu2+. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1ß), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

An established kidney cell line from humpback grouper (Cromileptes altivelis) and its susceptibility to bacteria and heavy metals.

Humpback grouper (Cromileptes altivelis), one kind of commercial fish with considerable economic value, has been recognized as a promising candidate for mariculture. In the wake of the development of aquaculture industry, the breeding density of C. altivelis has increased gradually, which gave rise to the occurrence of various pathogenic diseases. In our research, we established a new kidney cell line (designated as CAK) from humpback grouper and evaluated its susceptibility to bacteria and heavy metals. The results of our study showed that the optimal growth temperature was 26 °C, and optimal medium was L-15 supplemented with 20% fetal bovine serum (FBS). The sequencing of 18S rRNA gene indicated that CAK cell line was derived from C. altivelis. Chromosome analysis showed that the number of chromosome in CAK was 48. After being transfected of pEGFP-N3 plasmid, high transfection efficiency of CAK was observed, suggesting the potential to be used for the study of foreign functional genes. Moreover, the bacterial susceptibility results revealed that CAK cells were sensitive to Vibrio harveyi and Edwardsiella tarda, especially V. harveyi. Meanwhile, three heavy metals (Hg, Cu, and Cd) had toxic effects on the CAK cells with a dose-dependent manner. To sum up, the CAK cell line might be an ideal tool in vitro for analyzing the function of exogenous genes, bacterial susceptibility, and toxicity assay of heavy metals.

An accidental discovery of mannan-oligosaccharide's protection effect against air exposure and its potential mechanism in hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀).

The original objective was to explore the potential benefiting effects of three prebiotics in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Therefore, three experimental diets (basal diet + 1% fructooligosaccharide, Diet F; basal diet + 1% inulin, Diet I; basal diet + 0.3% mannan-oligosaccharide, Diet M) and one basal diet (Diet C) were prepared and a feeding trial was conducted. However, at the end of the fourth week into the feeding experiment, a water-leaking accident occurred and fishes of all groups went through an unexpected air exposure event. Surprisingly, different prebiotic-supplemented groups showed significantly different air exposure tolerance: the mortality of M group was significantly lower (P ≤ 0.05) than all the other groups. Examination of antioxidant, non-specific immunity, and stress parameters revealed that comparing to control group, M group showed significantly increased catalase (CAT), acid phosphatase (ACP), and alkaline phosphatase (AKP) activities, decreased superoxide dismutase (SOD) activity, and similar cortisol level (P ≤ 0.05). Real-time PCR experiment revealed that M group significantly increased the expression of CAT, glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD) genes in head kidney (P ≤ 0.05). Overall, M exhibited the best anti-air exposure/antioxidative stress effects among the three prebiotics and could be considered a promising feed additive to relieve air exposure/oxidative stress in hybrid grouper culture.

Establishment of a new fish cell line from the brain of humpback grouper (Cromileptes altivelis) and its application in toxicology and bacterial susceptibility.

Cromileptes altivelis, humpback grouper, belongs to the family Epinephelidae and is one popular farmed fish species because of its high economic value and ornamental value. However, more and more diseases outbreaks have been reported with C. altivelis aquaculture. Today, a new brain cell line of C. altivelis (named CAB) was established and characterized. Our results showed that CAB cells were suitable for growth at 26 °C in L-15 medium supplemented with 15% fetal bovine serum (FBS). The results of 18S rRNA gene sequencing confirmed that CAB cell line was derived from C. altivelis. Moreover, chromosomal aneuploidy was observed in CAB cells, and the modal chromosome number of CAB cells was 48 by chromosome analysis. In addition, CAB cells could transfect pEGFP-N3 plasmid with high transfection efficiency, indicating that CAB cell line has the potential to investigate the function of exogenous genes in vitro. Furthermore, the bacterial susceptibility results suggested that CAB cells were susceptive to Vibrio harveyi and Edwardsiella tarda. And, heavy metals (Hg, Cd, and Cu) were toxic to the CAB cells, and the toxic effect was dose-dependent. In summary, the CAB cell line could be a powerful tool in vitro to study functional genes and has the potential application in bacterial susceptibility and toxicology.

Molecular characterization and antibacterial immunity functional analysis of liver-expressed antimicrobial peptide 2 (LEAP-2) gene in golden pompano (Trachinotus ovatus).

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a member of the antimicrobial peptides family. Research has demonstrated that LEAP-2 contains a number of cations and plays a key role in the innate immune system of organism. In this study, we cloned and identified TroLEAP-2, from the golden pompano (Trachinotus ovatus), and analyzed its functions in vivo and in vitro. Results showed that TroLEAP-2 contains a 321 bp open reading frame (ORF) that encodes 106 putative amino acids with a molecular weight of 11.65 kDa. The mature TroLEAP-2 peptide possesses four conserved cysteine residues, which can form a core structure with two disulfide bonds between the cysteine residues in the relative 1-3 (Cys 77 and Cys 88) and 2-4 (Cys 83 and Cys 93) positions. It has a high amino acid sequence similarity (38.68%-83.02%) with the liver-expressed antimicrobial peptide -2 of other teleosts. Phylogenetic analysis showed that TroLEAP-2 clustered with the LEAP-2 of Paralichthys olivaceus and Miichthy milluy. TroLEAP-2 was most abundantly expressed in the liver, spleen, and kidney, and was significantly upregulated during Edwardsiella tarda and Streptococcus agalactiae infection. Purified recombinant TroLEAP-2 (rTroLEAP-2) could significantly inhibit the in vitro growth of E. tarda and S. agalactiae. Overexpression of TroLEAP-2 in vivo was shown to significantly reduce E. tarda and S. agalactiae colonization of tissues, whereas its knockdown resulted in an increase of bacteria in fish tissues. We also saw that TroLEAP-2 overexpression significantly improved macrophage activation in vivo. Moreover, TroLEAP-2 can induce the expression of nonspecific immune-related genes. These results showed that it might play a significant role in the innate immune system of golden pompano. In conclusion, our results indicate that TroLEAP-2 plays an important role in antibacterial immunity and provides a new avenue for protection against pathogenic infections in golden pompano.

Insulin-like growth factor binding protein 3 gene of golden pompano (TroIGFBP3) promotes antimicrobial immune defense.

Insulin-like growth factor binding protein 3 (IGFBP3), an important member of the IGFBP family, plays an important biological role in regulating cellular proliferation, differentiation, growth, apoptosis, and innate immunity. However, studies concerning IGFBP3 in teleosts are very limited and IGFBP3 function remains unclear. In this study, we conducted both in vivo and in vitro functional analyses of an IGFBP3 (TroIGFBP3) from the teleost fish golden pompano (Trachinotus ovatus). TroIGFBP3 is composed of 286 amino acid residues and shares a high amino acid sequence similarity (50.18%-93.71%) with other IGFBP3 sequences in humans and teleosts. TroIGFBP3 was widely distributed in various tissues, with the highest expression in the liver. TroIGFBP3 expression was significantly upregulated following Vibrio harveyi infection. The results of in vitro assays showed that TroIGFBP3 could stimulate macrophage activation and promote peripheral blood leukocytes (PBLs) proliferation. Meanwhile, TroIGFBP3 overexpression significantly inhibited bacterial infection in fish tissues, whereas TroIGFBP3 knockdown resulted in increased bacterial dissemination and colonization in golden pompano tissues in vivo. Furthermore, recombinant TroIGFBP3 could inhibit cellular proliferation and promote apoptosis of mouse tumor cells. Taken together, these results indicated that TroIGFBP3 plays a significant role in innate antibacterial immunity and provides a theoretical foundation for investigating the function of IGFBP3 in fish immune response.

Construction and analysis of the immune effect of Vibrio harveyi subunit vaccine and DNA vaccine encoding TssJ antigen.

Vibrio harveyi, a severe pathogen infects different kinds of sea animals, causes huge economic loss in aquaculture industry. In order to control the Vibriosis disease caused mainly by V. harveyi and other Vibrio spp., the best solution lies in developing corresponding efficient vaccines. In this study, we have cloned and analysed a putative antigen TssJ from the T6SS of V. harveyi, which has the potential as a vaccine against infection. The sequence analysis and western blotting experiments indicated that TssJ anchored in outer membrane and there were several antigenic determinants existed on its extracellular region. Two forms of universal vaccines, subunit vaccine and DNA vaccine, were developed based on TssJ and applied in Trachinotus ovatus. The results showed that both of the two vaccines could generate a moderate protection in fish against V. harveyi. The relative percentage survival (RPS) of subunit vaccine and DNA vaccine were 52.39% and 69.11%, respectively. Immunological analysis showed both subunit vaccine and DNA vaccine enhanced acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme activities. Specific serum antibodies against TssJ in the fish vaccinated with subunit vaccine was much higher than that in the DNA vaccine group. Several immune-related genes, i.e., IL10, C3, MHC Iα, MHC IIα, and IgM, were induced both by the two forms of vaccines. TNFα and Mx were only upregulated in the DNA vaccine group. However, the induction levels of these genes induced by DNA vaccine were higher than subunit vaccine. All these findings suggested that TssJ from V. harveyi had a potential application value in vaccine industry.