UNC93B1 facilitates the localization and signaling of TLR5M in Epinephelus coioides.

Toll-like receptor 5 (TLR5), serving as a sensor of bacterial flagellin, mediates the innate immune response to actively engage in the host's immune processes against pathogen invasion. However, the mechanism underlying TLR5-mediated immune response in fish remains unclear. Despite the presumed cell surface expression of TLR5 member form (TLR5M), its trafficking dynamics remain elusive. Here, we have identified Epinephelus coioides TLR5M as a crucial mediator of Vibrio flagellin-induced cytokine expression in grouper cells. EcTLR5M facilitated the activation of NF-κB signaling pathway in response to flagellin stimulation and exerted a modest influence on the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling. The trafficking chaperone Unc-93 homolog B1 (EcUNC93B1) participated in EcTLR5M-mediated NF-κB signaling activation and downstream cytokine expression. In addition, EcUNC93B1 combined with EcTLR5M to mediate its exit from the endoplasmic reticulum, and also affected its post-translational maturation. Collectively, these findings first discovered that EcTLR5M mediated the flagellin-induced cytokine expression primarily by regulating the NF-κB signaling pathway, and EcUNC93B1 mediated EcTLR5M function through regulating its trafficking and post-translational maturation. This research expanded the understanding of fish innate immunity and provided a novel concept for the advancement of anti-vibrio immunity technology.

TLR5S negatively regulates the TLR5M-mediated NF-κB signaling pathway in Epinephelus coioides.

Nuclear factor kappa-B (NF-κB) pathway is a key mediator of inflammation response that plays a role in host defense for pathogen elimination, but excessive activation may lead to tissue damage or pathogen transmission. The negative regulation of NF-κB in lower vertebrates is largely unknown, hindering further understanding of immune signaling evolution. Here, we provided evidence that Epinephelus coioides soluble toll-like receptor 5 (TLR5S), a member of the TLR5 subfamily, has been newly identified as a negative regulator of NF-κB signaling. EcTLR5S was a cytoplasmic protein consisting of 17 leucine-rich repeat domains, which specifically responded to Vibrio flagellin and suppressed flagellin-induced NF-κB signaling activation and cytokine expression. The amino-terminal LRR 1-5 region was necessary for its negative regulatory function. Dual-luciferase reporter assay showed that EcTLR5S significantly inhibited the NF-κB-luc activity induced by inhibitor of NF-κB kinase α (IKKα) and IKKß. Subsequently, the functional relationship between EcTLR5M and EcTLR5S was analyzed, revealing that the negative regulatory function of EcTLR5S targeted the activation of the NF-κB pathway mediated by EcTLR5M. The above results reveal that EcTLR5S negatively regulates the flagellin-induced EcTLR5M-NF-κB pathway activation, which may prevent over-activation of immune signaling and restore homeostasis.

A novel scavenger receptor (EcSRECII) as a lipopolysaccharide recognition molecule involved in regulating NF-κB activation through extracellular EGF-like cysteine-rich repeat domains with lysosomes in Epinephelus coioides.

Scavenger receptors (SRs), as multifunctional pattern recognition receptors, play an important role in innate immunity in mammals, however, their function in fish is limited. Herein, scavenger receptor F2 in Epinephelus coioides (EcSRECII) induced an innate immune response to LPS in GS cells. EcSRECII markedly enhanced LPS-induced NF-κB and IFN-ß signaling pathways, whereas knockdown of EcSRECII significantly inhibited LPS-induced NF-κB and IFN-ß promoter activation. Interestingly, only retain of epidermal growth factor (EGF)/EGF-like domain in EcSRECII resulted in a punctate cytoplasmic distribution, while the C-terminal domain exhibited a distinct cytoskeletal cytoplasmic distribution. Moreover, devoid of this EGF/EGF-like domain fragment more sharply impaired its ability to activate EcSRECII-induced NF-κB activation than deletion of the C-terminal domain region, but both domains significantly induced IFN-ß promoter activation. Full-length EcSRECII and the deletion mutant of C-terminal domain could partly colocalize with lysosomes by LPS derived from V. parahaemolyticus (V.p. LPS) in GS cells, but there was no similar distribution in the deletion mutant of EGF/EGF-like domain. This finding firstly suggested that the N-terminal EGF/EGF-like domain was necessary for the NF-κB signaling pathway to trigger resistance to vibrio infection and its functional exertion may be associated with lysosomes, thus providing insights into the regulation of resistance to vibrio infection in teleosts.

Mechanisms of sex differentiation and sex reversal in hermaphrodite fish as revealed by the Epinephelus coioides genome.

Most grouper species are functional protogynous hermaphrodites, but the genetic basis and the molecular mechanisms underlying the regulation of this unique reproductive strategy remain enigmatic. In this study, we report a high-quality chromosome-level genome assembly of the representative orange-spotted grouper (Epinephelus coioides). No duplication or deletion of sex differentiation-related genes was found in the genome, suggesting that sex development in this grouper may be related to changes in regulatory sequences or environmental factors. Transcriptomic analyses showed that aromatase and retinoic acid are probably critical to promoting ovarian fate determination, and follicle-stimulating hormone triggers the female-to-male sex change. Socially controlled sex-change studies revealed that, in sex-changing fish, the brain's response to the social environment may be mediated by activation of the phototransduction cascade and the melatonin synthesis pathway. In summary, our genomic and experimental results provide novel insights into the molecular mechanisms of sex differentiation and sex change in the protogynous groupers.

Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis.

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1ß (T. nigroviridis IL-1ß). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1ß after activation.

Integrative analyses of mRNA and microRNA expression profiles reveal the innate immune mechanism for the resistance to Vibrio parahaemolyticus infection in Epinephelus coioides.

Vibrio parahaemolyticus, as one of the main pathogens of marine vibriosis, has brought huge losses to aquaculture. However, the interaction mechanism between V. parahaemolyticus and Epinephelus coioides remains unclear. Moreover, there is a lack of comprehensive multi-omics analysis of the immune response of grouper spleen to V. parahaemolyticus. Herein, E. coioides was artificially injected with V. parahaemolyticus, and it was found that the mortality was 16.7% in the early stage of infection, and accompanied by obvious histopathological lesions in the spleen. Furthermore, 1586 differentially expressed genes were screened by mRNA-seq. KEGG analysis showed that genes were significantly enriched in immune-related pathways, Acute-phase immune response, Apoptosis, Complement system and Cytokine-cytokine receptor interaction. As for miRNA-seq analysis, a total of 55 significantly different miRNAs were identified. Further functional annotation analysis indicated that the target genes of differentially expressed miRNAs were enriched in three important pathways (Phosphatidylinositol signaling system, Lysosome and Focal adhesions). Through mRNA-miRNA integrated analysis, 1427 significant miRNA-mRNA pairs were obtained and "p53 signaling pathway", "Intestinal immune network for IgA production" were considered as two crucial pathways. Finally, miR-144-y, miR-497-x, novel-m0459-5p, miR-7133-y, miR-378-y, novel-m0440-5p and novel-m0084-3p may be as key miRNAs to regulate immune signaling pathways via the miRNA-mRNA interaction network. The above results suggest that the mRNA-miRNA integrated analysis not only sheds new light on the molecular mechanisms underlying the interaction between host and V. parahaemolyticus but also provides valuable and new insights into resistance to vibrio infection.

The flagellin of Vibrio parahaemolyticus induces the inflammatory response of Tetraodon nigroviridis through TLR5M.

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1ß in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1ß and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1ß and TnIFN-γ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis.

Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.

The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.

The ERß-CXCL19/CXCR4-NFκB pathway is critical in mediating the E2-induced inflammation response in the orange-spotted grouper (Epinephelus coioides).

The main physiological function of 17ß-estradiol (E2) in vertebrates is to regulate sexual development and reproduction. In fish, especially hermaphroditic fish, estrogen is often used to aid reproduction, but it also can trigger an inflammatory response. However, the molecular mechanism for this E2-induced inflammatory reaction is not clear. In this study, we found that the ERß-CXCL19/CXCR4-NFκB cascade regulated the E2-induced inflammatory response in the orange-spotted grouper (Epinephelus coioides). Strikingly, E2 treatment resulted in significantly high expression of inflammatory cytokines and induced phosphorylation and degradation of IκBα and translocation of NFκB subunit p65 to the nucleus in grouper spleen cells. However, the E2-induced inflammatory response could be prevented by the broad estrogen receptor (ER) ligand ICI 182,780. Moreover, the luciferase assay showed that E2 induced the inflammatory response by activating the promotor of chemokine CXCL19 through ERß1 and ERß2. Knockdown of CXCL19 blocked the E2-induced inflammatory response and NFκB nucleus translocation. Additionally, knockdown of chemokines CXCR4a and CXCR4b together, but not alone, blocked the E2-induced inflammatory response. The immunofluorescence assay and co-immunoprecipitation analysis showed that CXCL19 mediated the E2-induced inflammatory response by activating CXCR4a or CXCR4b. Taken together, these results showed that the ERß-CXCL19/CXCR4-NFκB pathway mediated the E2-induced inflammatory response in grouper. These findings are valuable for future comparative immunological studies and provide a theoretical basis for mitigating the adverse reactions that occur when using E2 to help fish reproduce.

Recognition of DAP and activation of NF-κB by cytosolic sensor NOD1 in Oreochromis niloticus.

As a lower vertebrate, the immune defense mechanism of fish mainly depends on the innate immune system. Nucleotide-binding oligomerization domain-like receptors (NLRs) are an important class of pattern recognition receptors in the innate immune system. In this study, NOD1 gene was cloned and characterized in Nile tilapia (Oreochromis niloticus). The ORF of Nile tilapia NOD1 gene was 2826 bp long and encoded 941 amino acid residues with a structure of CARD-NACHT-LRRs that was similar to the other counterparts in mammals and fishes. Phylogenetic and synteny analysis showed that NOD1 was conserved among different fishes and existed at least in the early stage of fish evolution. Expression pattern revealed that NOD1 mRNA was constitutively expressed in the tested tissues, while had high expression level in main immune organs and mucosal immune tissues (liver, head kidney, spleen, blood, gill, and intestine). Following Streptococcus agalactiae challenge, Nile tilapia NOD1 mRNA expression levels were altered in immune organs (liver, head kidney, spleen, blood), and the expression pattern was similar in liver, spleen and blood. Furthermore, the ligand recognition and signaling pathway of Nile tilapia NOD1 were also analyzed, it showed that NOD1 could recognize Tri-DAP intracellularly and activated NF-κB signaling pathway. In summary, our results indicated that the Nile tilapia NOD1 may play an important role in innate immune system and provided a basis for the functional study of NOD1 in teleost.

23S rRNA from Vibrio parahaemolyticus regulates the innate immune response via recognition by TLR13 in orange-spotted grouper (Epinephelus coioides).

Toll-like receptors (TLRs) are major pattern recognition receptors (PRRs) that recognize multiple pathogen-associated molecular patterns (PAMPs) through the leucine-rich repeat (LRR) domain and mount effective immune responses. Vibrio parahaemolyticus is the main pathogen that causes vibriosis in aquatic animals, yet the mechanisms of its recognition by innate immune system in teleost fish remain unknown. Here, the results reveal that TLR13 in orange-spotted grouper (Epinephelus coioides) (EcTLR13) recognizes a conserved 23S ribosomal RNA (23S rRNA) sequence in V. parahaemolyticus, and the 13-nucleotide motif near the 23S rRNA ribozyme activation site (VP13) acts as a PAMP. After challenge with RNA and 23S rRNA from V. parahaemolyticus and with the synthetic oligoribonucleotide VP13, the expression of EcTLR13 in grouper spleen cells (GS cells) was significantly increased. EcTLR13-knockdowned GS cells were stimulated with the same stimulants as listed above, the expression of IL-6, IL-12, IL-1ß and TNFα was significantly reduced. RNA-protein immunoprecipitation revealed that VP13 could directly bind to EcTLR13. The dual-luciferase reporter assay also showed that EcTLR13 enhanced the fluorescence activity of IFNß rather than that of NF-κB when the cells were challenged with RNA from V. parahaemolyticus or with synthetic VP13. Our study established the mechanism of fish TLR13-mediated recognition of microbial products during V. parahaemolyticus infection.

Comparative Metabolomics and Proteomics Reveal Vibrio parahaemolyticus Targets Hypoxia-Related Signaling Pathways of Takifugu obscurus.

Coronavirus disease 2019 (COVID-19) raises the issue of how hypoxia destroys normal physiological function and host immunity against pathogens. However, there are few or no comprehensive omics studies on this effect. From an evolutionary perspective, animals living in complex and changeable marine environments might develop signaling pathways to address bacterial threats under hypoxia. In this study, the ancient genomic model animal Takifugu obscurus and widespread Vibrio parahaemolyticus were utilized to study the effect. T. obscurus was challenged by V. parahaemolyticus or (and) exposed to hypoxia. The effects of hypoxia and infection were identified, and a theoretical model of the host critical signaling pathway in response to hypoxia and infection was defined by methods of comparative metabolomics and proteomics on the entire liver. The changing trends of some differential metabolites and proteins under hypoxia, infection or double stressors were consistent. The model includes transforming growth factor-ß1 (TGF-ß1), hypoxia-inducible factor-1α (HIF-1α), and epidermal growth factor (EGF) signaling pathways, and the consistent changing trends indicated that the host liver tended toward cell proliferation. Hypoxia and infection caused tissue damage and fibrosis in the portal area of the liver, which may be related to TGF-ß1 signal transduction. We propose that LRG (leucine-rich alpha-2-glycoprotein) is widely involved in the transition of the TGF-ß1/Smad signaling pathway in response to hypoxia and pathogenic infection in vertebrates as a conserved molecule.

Dual-Function Analysis of Astaxanthin on Golden Pompano (Trachinotus ovatus) and Its Role in the Regulation of Gastrointestinal Immunity and Retinal Mitochondrial Dysfunction Under Hypoxia Conditions.

The present study investigated the potential mechanisms of astaxanthin in the regulation of gastrointestinal immunity and retinal mitochondrial function of golden pompano (Trachinotus ovatus). Triplicate groups of juvenile T. ovatus (mean initial weight: 6.03 ± 0.01 g) were fed one of six diets (D1, D2, D3, D4, D5, and D6) for 8 weeks, with each diet containing various concentrations of astaxanthin (0, 0.0005, 0.001, 0.005, 0.01, or 0.1%, respectively). Growth performance of fish fed the D2-D5 diets was higher than that of fish fed the D1 diet; however, growth performance and survival of fish deteriorated sharply in fish fed the D6 diet. Gut villus in fish fed the D2-D5 diets were significantly longer and wider than that of fish fed the D6 diet. Feeding with D2-D5 diets led to increased abundance of Bacillus, Pseudomonas, Oceanobacillus, Lactococcus, Halomonas, Lactobacillus, and Psychrobacter while abundance of Vibrio and Bacterium decreased. Additionally, feeding with the D6 diet resulted in a sharp decline in Pseudomonas and Lactobacillus abundance and a sharp increase in Vibrio abundance. A low dissolved oxygen environment (DO, 1.08 mg/L) was conducted for 10 h after the rearing trial. No fish mortality was observed for any of the diet treatments. Lysozyme (LZY) activity in fish fed the D6 diet decreased sharply and was significantly lower than that in other groups. ROS production also decreased sharply in fish fed the D6 diet. Moreover, the conjunctiva and sclera in the fish fed the D6 diet were indistinguishable. Suitable dietary astaxanthin supplementation levels (0.005-0.1%) exerting a neuroprotective effect from low dissolved oxygen environments is due to up-regulated expression of anti-apoptotic factors, such as phosphorylated Bcl-2-associated death promoter (pBAD), phosphorylated glycogen synthase kinase-3ß (pGSK-3ß), Bcl-2 extra large (Bcl-xL), and down-regulated expression of Bcl-2-associated X protein (Bax) pro-apoptotic factor in retinas. Furthermore, suitable dietary astaxanthin levels (0.0005-0.01%) suppressed up-regulation of critical mitochondrial components, such as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), mitochondrial transcription factor A (TFAM), and mitochondrial DNA (mtDNA), while excessive astaxanthin supplementation produces the opposite effect. In brief, high-dose astaxanthin arouses and aggravates low dissolved oxygen-induced inflammation, oxidative stress, intestinal disorder, retinal apoptosis, and retinal mitochondrial dysfunction in T. ovatus. Second-degree polynomial regression of WG indicated that the optimum dietary astaxanthin for juvenile T. ovatus is 0.049%.

MicroRNA-29b modulates the innate immune response by suppressing IFNγs production in orange-spotted grouper (Epinephelus coioides).

Interferon-γ (IFNγ), a type II interferon, is essential to host resistance against various infections. Unlike other vertebrates, fish have two types of IFNγs, IFNγ1 (also named IFNγ-rel) and IFNγ2. MicroRNAs (miRNAs) regulate multiple biological processes by suppressing mRNA translation or inducing mRNA degradation. Among them, miR-29 can directly target IFNγ and affact innate and adaptive immune responses in mice. There are five members of the miR-29 family in orange-spotted grouper (Epinephelus coioides), which share the same miRNA seed region. However, whether miR-29 directly targets E. coioides IFNγs and regulate IFNγ production is still unknown. In the present study, the negative correlation between miR-29b and both IFNγs in immune tissues of healthy E. coioides and grouper spleen cells (GS cells) stimulated with LPS or poly I:C was demonstrated. Moreover, dual-luciferase reporter assays and western blotting were performed to demonstrate that miR-29b suppressed E. coioides IFNγ production. Studies of NO production in GS cells after miR-29b transfection revealed that miR-29b overexpression affected NO production through the downregulation of IFNγ expression. Taken together, our results suggest that miR-29b may directly target E. coioides IFNγs and modulate IFNγ-mediated innate immune responses by suppressing E. coioides IFNγs production.

Molecular characterization and functional analysis of IKKα in orange-spotted grouper (Epinephelus coioides).

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKß-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.

Type F scavenger receptor expressed by endothelial cells (SREC)-II from Epinephelus coioides is a potential pathogen recognition receptor in the immune response to Vibrio parahaemolyticus infection.

Scavenger receptors play a central role in defending against infectious diseases in mammals. However, the function of SRECII remains unknown in teleost fish. In this study, type F scavenger receptor expressed by endothelial cells-II (SRECII) cDNA sequence was first identified from Epinephelus coioides, named EcSRECII, which contained an N-terminal signal peptide, eight EGF/EGF-like cysteine-rich motifs and a C-terminal low-complexity region. The gene location maps revealed that EcSRECII has the conservation of synteny among selected species. Subcellular localization showed that EcSRECII was mainly located in the cytoplasm in HEK293T cells and GS cells. In healthy E. coioides, EcSRECII mRNA was highly expressed in spleen, skin, gill, thymus and head kidney. The relative EcSRECII mRNA expression after Vibrio parahaemolyticus infection was significantly up-regulated at 12 h in spleen, head kidney and thymus, but downregulated at 1 d in skin and reduced at 3 d and 1 w in spleen. Furthermore, overexpression of EcSRECII activated NF-κB and IFN-ß signaling pathway in vitro. Taken together, these results indicated that EcSRECII could be as the potential pathogen recognition receptor for involving in bacterial infection by regulating innate immunity responses in E. coioides.

Physicochemical stability of extemporaneously prepared clonidine solutions for use in neonatal abstinence syndrome.

WHAT IS KNOWN AND OBJECTIVE: Extemporaneously prepared clonidine admixture is increasingly used for the management of neonatal abstinence syndrome. However, its stability beyond 15 minutes at room temperature is currently unknown. Therefore, healthcare professionals must prepare clonidine admixtures multiple times a day while the treatment is indicated, resulting in subsequent limitations and problems. The aim of this study was to investigate the physicochemical stability of clonidine in commonly used pharmaceutical diluents at clinically relevant concentrations and temperatures. METHODS: Glass bottles (n = 18) and plastic syringes (n = 18) containing 0.5 and 5 µg/mL of clonidine in either 5% glucose, 10% glucose or 0.9% normal saline were prepared and stored at 4°C for 7 days and at 35°C for 24 hours, respectively. Aliquots were withdrawn at predefined time points and analysed for the concentration of clonidine, changes in pH and colour, and particle content. RESULTS AND DISCUSSION: No evidence of particle formation, or colour or pH change was observed throughout the study period. Clonidine retained more than 98% of its initial concentration when stored in the tested diluents at 4°C for 7 days and at 35°C for 24 hours. WHAT IS NEW AND CONCLUSION: Our findings will allow healthcare professionals to prepare weekly dose of clonidine in glass bottles for storage in a refrigerator. The daily required dose of clonidine can be drawn aseptically from the glass bottle each day and stored in a plastic syringe at room temperature. Clonidine present in a plastic syringe can be administered via the nasogastric route 4-6 times a day. This practice would not only save nursing time and avoid delays in the timely administration of clonidine, but also reduce the risk of potential medication errors as well as preparation-associated costs.

MicroRNA-182-3p negatively regulates cytokines expression by targeting TLR5M in orange-spotted grouper, Epinephelus coioides.

Toll-like receptors (TLRs) as essential pattern recognition receptors in innate immunity, can recognize pathogens and trigger immune response to eliminate invading pathogens. MicroRNAs regulates multiple biological processes by suppressing mRNA translation or resulting in mRNA degradation. MiR-182 has previously been implicated in DNA repair, disease and cancer aspects. The potential role of miR-182-3p in TLR signaling pathway against pathogens is unclear. In this study, we found that the expression of miR-182-3p was up-regulated after Vibrio parahaemolyticus flagellin stimulation in grouper spleen (GS) cells, and negatively correlated with the expression of orange-spotted grouper (Epinephelus coioides) TLR5M (EcTLR5M). Then we found that miR-182-3p could directly target EcTLR5M by using bioinformatic analysis and dual-luciferase reporter assay. Dual-luciferase reporter assay also showed that miR-182-3p down-regulated the wild-type EcTLR5M 3'UTR in luciferase activity rather than the mutant group in HEK 293T cells. We further verified the effect of miR-182-3p on the activation of Nuclear factor-κB (NF-κB) signaling pathway, and found that miR-182-3p inhibitors significantly augmented flagellin-induced NF-κB phosphorylation. Additionally, we also demonstrated that the increased expression of miR-182-3p significantly suppressed the flagellin-induced EcTLR5M, pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA expression. And the endogenous miR-182-3p knockdown experiments reversely verified the regulatory effect of miR-182-3p. These results suggested that miR-182-3p post-transcriptionally controls EcTLR5M expression and thereby suppresses the expression of pro-inflammatory cytokines. This study is the first to demonstrate that miR-182-3p suppresses pro-inflammatory cytokines expression by regulating the TLR signaling pathway.

Development of attenuated erythromycin-resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture.

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re-attenuated S. agalactiae (named TFJ-ery) was developed from a natural low-virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ-ery were determined. The results indicated that TFJ-ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ-ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ-ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ-ery at a dose of 5.0 × 107 CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post-vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post-vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ-ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.

Vibrio parahaemolyticus flagellin induces cytokines expression via toll-like receptor 5 pathway in orange-spotted grouper, Epinephelus coioides.

Vibrio parahaemolyticus is the major pathogen of vibriosis in aquatic animals and causes inflammation that may be related to tissue damage. Here, we have established a V. parahaemolyticus flagellin stimulation model using grouper spleen (GS) cell line. Purified V. parahaemolyticus flagellin was used to stimulate GS cells. Our results showed that the mRNA levels of orange-spotted grouper (Epinephelus coioides) toll-like receptor 5M (EcTLR5M), EcTLR5S and downstream cytokines, such as interferon-γ2 (IFN-γ2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were all significantly increased after stimulation with V. parahaemolyticus flagellin in GS cells. Gene silencing of the EcTLR5M and EcTLR5S in GS cells by using small interfering RNA resulted in suppression of the V. parahaemolyticus flagellin-induced cytokines expression. We further demonstrated that activation of both mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were required for cytokines expression. We observed that the phosphorylation of NF-κB inhibitor-α (IκBα), extracellular signal-regulated kinase (ERK) and p38 were induced following treatment with flagellin. Additionally, most of p65, a NF-κB subunit, was found to translocate to the nucleus after 60 min stimulation. Overall, our results suggest that V. parahaemolyticus flagellin influences cytokines expression, such as IFN-γ2, IL-6 and TNF-α, via EcTLR5s recognition and MAPKs/NF-κB signaling pathway activation in GS cells.