Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger-TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection.

In silico characterization and expression analysis of eight C-type lectins in obscure puffer Takifugu obscurus.

C-type lectins (CTLs) are a group of carbohydrate-binding proteins that play crucial roles in innate immune defense against invading pathogens. CTLs have been extensively studied in lower vertebrates, such as fish, for their roles in eliminating pathogens; however, their homologs in pufferfish are not well known. In the present study, eight CTLs from obscure puffer Takifugu obscurus (designated as ToCTL3-10 according to the order they were discovered) were obtained. All predicted ToCTL proteins contained a single carbohydrate recognition domain (CRD). ToCTL7 also contained one calcium-binding epidermal growth factor (EGF)-like domain (EGF_CA) and a transmembrane region. ToCTL9 also contained an SCP domain, an EGF domain, and an EGF-like domain. Bioinformatics analysis revealed that ToCTL3-10 mainly clustered with the corresponding CTL homologs of other pufferfish species. Tissue distribution analysis detected ToCTL3-10 in all tissues examined, including kidneys, liver, gills, spleen, intestines, and heart. Moreover, the expressions of ToCTL3-10 were significantly induced in the kidneys of obscure puffer following challenges with three Gram-negative bacterial pathogens, namely, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, and a synthetic analog of double-stranded RNA poly(I:C). The expression patterns of ToCTL3-10 in response to different immune stimulants were different. Our results indicated that the eight ToCTLs obtained herein might be involved in host defense against bacterial and poly(I:C) infections in T. obscurus.

Immunohistochemical characterization and change in location of branchial ionocytes after transfer from freshwater to seawater in the euryhaline obscure puffer, Takifugu obscurus.

The obscure puffer Takifugu obscurus is a euryhaline fish species suitable for studying the molecular mechanism of osmoregulation. The distributional changes of branchial ionocytes were detected following the transfer from freshwater (FW) to seawater (SW) based on two main ion transporters, Na+/K+-ATPase (NKA) and Na+/K+/ 2Cl- cotransporter 1 (NKCC1). The mRNA and protein expression levels of NKA and NKCC1 in the gills all increased rapidly in the first four days after transfer to SW. Double immunofluorescence staining showed that NKCC1 and NKA were colocalized in the branchial ionocytes and the immunoreaction of NKCC1 was stronger after transfer. Moreover, following transfer to SW, the number of lamellar ionocytes in the gills is reduced and the number of filament ionocytes is increased significantly. Taken together, these findings indicated that SW transfer of obscure puffer promotes the changes of distribution, function and size of branchial ionocytes.

Functional characterization of an immunity-related GTPase gene in immune defense from obscure puffer (Takifugu obscurus).

Immunity-related GTPases (IRGs) are a family of large interferon-inducible GTPases that function in effective host defense against invading pathogens. IRGs have been extensively studied in mammals for their roles in the elimination of intracellular pathogens; however, their homologs in lower vertebrates are not well known. In this study, an IRG from obscure puffer (Takifugu obscurus), ToIRG, was identified and further characterized for its functional activity. The ToIRG gene encodes a protein of 396 amino acids containing a typical N-terminal GTPase domain with three conserved motifs. Phylogenetic analysis revealed that it has a closer evolutionary relationship with mammalian GKS IRGs. Gene expression profile analysis revealed that ToIRG was ubiquitously expressed in all tested healthy tissues of obscure puffer and upregulated in response to Aeromonas hydrophila or Edwardsiella tarda challenge. The subcellular localization of ToIRG is characterized as condensed forms around the nucleus. Importantly, an antimicrobial assay in vitro suggested that ToIRG enhanced the ability of host cells to resist both intracellular (E. tarda) and extracellular pathogens (A. hydrophila). Taken together, these results provide the functional characterization of obscure puffer IRGs in immune defense, which is the first study to reveal the function of IRGs in bony fish and will provide important insights into the evolutionary divergence of IRGs.

Characterization and Functional Analysis of Two Transmembrane C-Type Lectins in Obscure Puffer (Takifugu obscurus).

C-type lectins (CTLs) have received widespread attention in animal immune responses. In the present study, two CTLs (ToCTL1 and ToCTL2) were identified from obscure puffer Takifugu obscurus. The open reading frames of ToCTL1 and ToCTL2 were 687 and 1,380 bp, respectively. The predicted ToCTL1 and ToCTL2 proteins contained a single transmembrane region and one typical carbohydrate recognition domain (CRD). Quantitative real-time polymerase chain reaction detected ToCTL1 and ToCTL2 transcripts in all examined tissues, with high levels in the intestine and kidney, and their expression levels were remarkably altered upon Vibrio harveyi and Aeromonas hydrophila infection. The recombinant proteins ToCTL1-CRD and ToCTL2-CRD agglutinated the Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner. rToCTL1-CRD and rToCTL2-CRD exhibited evident binding activities against seven kinds of bacteria and polysaccharides (lipopolysaccharide and peptidoglycan) in a Ca2+-independent manner. Moreover, rToCTL1-CRD and rToCTL2-CRD could inhibit the growth of four types of bacteria in vitro. These findings collectively demonstrated that ToCTL1 and ToCTL2 could be involved in host defense against bacterial infection in T. obscurus.

Inkjet printing assisted fabrication of polyphenol-based coating membranes for oil/water separation.

While polyphenol-based coating has been regarded as a promising alternative to functionalize membrane surface, it usually suffers from problems of low-efficient procedure and low utilization rate of the polyphenolic compounds, hindering its large-scale implementations. To solve these problems, this study provided a first report on inkjet printing of polyphenols (catechol (CA) or tannic acid (TA)) and sodium periodate (SP) on a polyvinylidene fluoride (PVDF) membrane to improve membrane performance. A series of analyses showed the efficient formation of homogenous films on the PVDF membrane surface and the improvement of hydrophilicity by the inkjet printing technique. The PVDF membranes decorated with the optimized polyphenolic coating exhibited a promising oil/water separation efficiency (higher than 99%) with a high average water permeation flux of 5.2 times higher than that of the pristine membrane. Meanwhile, the modified membranes illustrated a good stability under acidic conditions (pH = 2-7). The novel method proposed in this study is facile, cost-saving and environment-friendly. The advantages of the proposed method and the modified membranes demonstrated the great significance of the proposed method in practical applications.

Inkjet printing assisted electroless Ni plating to fabricate nickel coated polypropylene membrane with improved performance.

While electroless nickel plating is considered as a promising candidate for fabrication of metallized polymer composite membranes with high performance, it suffers from problems of complex and high-cost pretreatment procedure, hindering its large-scale implementations. It is hypothesized that, inkjet printing integrated with electroless plating (ELP) can serve as a facile and economical membrane fabrication method to overcome above problems. The new method proposed in this study was processed by inkjet printing silver ions and pyrrole inks as catalytic layer followed by electroless Ni deposition on polypropylene (PP) membrane surface. Successful modification was verified by characterizing the surface morphology and elemental compositions of the membranes. In comparison to the pristine PP membrane, the PPy-Ag/Ni modified membrane demonstrated lower surface resistance (2.3 Ω), better hydrophilicity (44.9°) and higher pure water flux (1135.1 L m-2 h-1). When applying an external electric field (10.0 V cm-1), the average flux of the PPy-Ag/Ni membrane for yeast filtration increased from 107.8 to 137.7 L m-2 h-1, which was about 2.0 times higher than that of the pristine PP membrane. Meanwhile, the PPy-Ag/Ni membrane possessed a maximum flux recover rate when applied with an external electrical field. This work provided a facile and efficient approach for fabrication of composite conductive membranes.

The distinct evolutionary properties of the tripartite motif-containing protein 39 in the Chinese softshell turtle based on its structural and functional characterization.

The tripartite motif (TRIM)-containing proteins are a diverse family of proteins that are involved in the regulation of innate immune responses. TRIM39 is a member of the TRIM family and contains E3 ubiquitin ligase activity. In this study, a TRIM39 homolog from the Chinese softshell turtle (Pelodiscus sinensis), PsTRIM39, was identified, and its functional characterization was investigated. PsTRIM39 is a protein of 470 amino acids containing a conserved RING-finger domain, B-BOX domain, PRY domain and SPRY domain in the TRIM family. Sequence structure and phylogenetic analysis indicated PsTRIM39 has the closest relationship with that of birds. Transcriptional profiling analysis revealed that PsTRIM39 mRNA was upregulated after challenge with Aeromonas hydrophila or the soft-shelled turtle virus, iridovirus. The subcellular localization of PsTRIM39 was in the cytoplasm, which is similar to that of fish. Furthermore, PsTRIM39 colocalized with lysosomes in Fathead minnow (FHM) cells, indicating that it may play a role in immune-related function. An NFκB functional assay showed that overexpression of PsTRIM39 enhanced NFκB activity in FHM cells, which is different from that of mammalian TRIM39. Taken together, these results provide, for the first time, the structural and functional characterization of a TRIM family member in the innate immune responses of reptiles and suggest that PsTRIM39 has distinct evolutionary properties representing the transitional stage from lower vertebrates to higher vertebrates in evolution.

Comparative study of two immunity-related GTPase genes in Chinese soft-shell turtle reveals their molecular characteristics and functional activity in immune defense.

The immunity-related GTPases (IRGs) are a family of proteins that play critical roles in innate resistance to intracellular pathogens. The number and diversity of IRG genes differ greatly in different species. Although IRG proteins have been well studies in mammals, they remain poorly characterized in lower vertebrates. In this study, we cloned two IRG genes, PsIRG5 and PsIRG8, from the Chinese soft-shelled turtle and compared their characterization and functional activity with mammalian IRGs. The PsIRG5 is a gene of 1896 bp that encodes a protein of 413 amino acid and PsIRG8 is 1543 bp in length encoding another 413 aa protein. Sequence alignment between all turtle IRG-like genes and mammalian IRGs showed that both PsIRG5 and PsIRG8 were conserved with mammalian GKS IRGs, while PsIRG5 appeared a closer evolutionary relationship with mammalian GMS IRGs. The expression and subcellular characterization revealed that PsIRG5 was dramatically upregulated under Aeromonas hydrophila challenge and exhibited co-localization with lysosomes in cells; whereas PsIRG8 was downregulated and has no distinct localization. Functional activity assay demonstrated that PsIRG5 plays a role in autophagy induction and IFN-γ contributes to enhance the induction, since it has IFN-inducible elements in its promoter region. These data above unravel the molecular characterization and functional activity of IRGs in lower vertebrate for the first time and will provide insights into the comparative immunity and evolutionary relationships of IRGs between mammals and reptiles.