Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components.

Toll like receptor (TLR) 5 and 9 are important members of the TLR family that play key roles in innate immunity in all vertebrates. In this study, paTLR5 and paTLR9 were identified in silver pomfret (Pampus argenteus), a marine teleost of great economic value. Open reading frames (ORFs) of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively. Sequence analysis revealed several conserved characteristic features, including signal peptides, leucine-rich repeat (LRR) motifs, and a Toll/interleukin-I receptor (TIR) domain. Sequence, phylogenetic and synteny analysis revealed high sequence identity with counterparts in other teleosts, confirming their correct nomenclature and conservation during evolution. Quantitative real-time PCR revealed that the that both TLRs were ubiquitously expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin. In vitro immunostimulation experiments revealed that Aeromonas hydrophila lipopolysaccharide (LPS) and Vibrio anguillarum flagellin induced higher levels of paTLR9 and paTLR5 mRNA expression in isolated fish intestinal epithelial cells (FIECs) than Lactobacillus plantarum lipoteichoic acid (LTA), but all increased the secretion of IL-6 and TNF-α and induced cell apoptosis and necrosis. Together, these results indicate that paTLR5 and paTLR9 may function in the response to bacterial pathogens. Our findings enhance our understanding of the function of TLRs in the innate immune system of silver pomfret and other teleosts.

Molecular characterization and expression analysis of toll-like receptor 2 in response to bacteria in silvery pomfret intestinal epithelial cells.

Toll-like receptor 2 (TLR2) has been shown to play a crucial role in the host defense of pathogenic microbes in innate immunity. In this study, the full-length cDNA of TLR2 in silvery pomfret (Pampus argenteus) was cloned by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of TLR2 was 2932 bp, containing an open reading frame (ORF) of 2469 bp encoding 822 amino acids. A multiple alignment analysis of the silvery pomfret TLR2 protein-coding sequence with other known TLR2 sequences from Oplegnathus fasciatus, Epinephelus coioides, Larimichthys crocea, Miichthys miiuy, Oreochromis niloticus, Paralichthys olivaceus, Trematomus bernacchii, Sparus aurata, and Chionodraco hamatus exhibited a high degree of homology of 78.83%, 75.91%, 74.21%, 74.94%, 71.95%, 72.57%, 73.68%, 75%, and 72.52 respectively, between these fish. Analysis of the TLR2 domain structures indicated that TLR2 from the silvery pomfret has the typical structural features of proteins that belong to the TLR family, including one transmembrane domain, eleven leucine-rich repeats (LRRs), and one Toll/IL-1 receptor homology domain (TIR). In vitro immunostimulation experiments revealed that Lactobacillus plantarum and Clostridium butyricum induce high levels of TLR2 mRNA and protein expression, but they induce only moderate levels of IL-8 and TNF-α production compared to Vibrio anguillarum. This suggests that TLR2 might play a vital role in the L. plantarum and C. butyricum-mediated immune response. In contrast, V. anguillarum significantly increased the secretion of IL-8 and TNF-α and induced cell apoptosis and necrosis. Due to the lower expression of TLR2 and higher levels of IL-8 and TNF-α induced by V. anguillarum, we hypothesize that a V. anguillarum infection is independent of the TLR2-induced production of pro-inflammatory cytokines. These results indicate that TLR2 may be involved in molecular interactions between the host and commensal bacteria, that exist in the silvery pomfret intestinal tract.

Ability of Lactobacillus plantarum lipoteichoic acid to inhibit Vibrio anguillarum-induced inflammation and apoptosis in silvery pomfret (Pampus argenteus) intestinal epithelial cells.

Lipoteichoic acid (LTA) is a major constituent of the cell wall of Gram-positive bacteria. The structure and immunomodulation of LTA vary greatly between different species. LTA from Lactobacillus plantarum has been shown to exert anti-pathogenic effects. Vibrio anguillarum is a major causative agent of vibriosis, one of the most prevalent fish diseases. The purpose of this study was to examine the effects of L. plantarum LTA on V. anguillarum growth, adhesion, and induced inflammation and apoptosis in intestinal epithelial cells of silvery pomfret (Pampus argenteus). Our results showed that L. plantarum LTA was unable to inhibit V. anguillarum growth; however, it significantly inhibited adhesion of V. anguillarum. It also showed significant inhibitory effects on EHEC-induced inflammation and apoptosis by modulating the expression of NF-κB (nuclear factor kappa B), IκB (inhibitor of NF-κB), Bcl2 (B-cell leukemia/lymphoma-2), BAX (Bcl-2-associated X protein), IL-8 (interleukin 8) and TNF-α (tumor necrosis factor-α), and via inhibition of caspase-9 and caspase-3 activation. These data extend our understanding of the beneficial effects of L. plantarum LTA, which is related to the inhibition of V. anguillarum, and suggest that L. plantarum LTA has potential as a new therapeutic agent against V. anguillarum-caused vibriosis in fish.

Exploring the Effects of Nano-CaCO3 on the Core-Shell Structure and Properties of HDPE/POE/Nano-CaCO3 Ternary Nanocomposites.

To address the dilemma of the stiffness and toughness properties of high-density polyethylene (HDPE) composites, titanate coupling agent-treated CaCO3 nanoparticles (nano-CaCO3) and ethylene-octene copolymer (POE) were utilized to blend with HDPE to prepare ternary nanocomposites via a two-sequence-step process. Meanwhile, a one-step process was also studied as a control. The obtained ternary nanocomposites were characterized by scanning electron microscopy (SEM), Advanced Rheometrics Expansion System (ARES), Dynamic Mechanical Analysis (DMA), wide-angle X-ray diffraction analysis (WXRD), and mechanical test. The SEM results showed one or two CaCO3 nanoparticles were well-encapsulated by POE and were uniformly dispersed into the HDPE matrix to form a core-shell structure of 100-200 nm in size by the two-step process, while CaCO3 nanoparticles were aggregated in the HDPE matrix by the one-step method. The result of the XRD showed that the nano-CaCO3 particle played a role in promoting crystallization in HDPE nanocomposites. Mechanical tests showed that the synergistic effect of both the POE elastomer and CaCO3 nanoparticles should account for the balanced performance of the ternary composites. In comparison with neat HDPE, the notched impact toughness of the ternary nanocomposites of HDPE/POE/nano-CaCO3 was significantly increased. In addition, the core-shell structure absorbed the fracture impact energy and prevent further propagation of micro-cracks, thus obtaining a higher notched Izod impact strength.

Comparison of physical properties and fishing performance between biodegradable PLA and conventional PA trammel nets in grey mullet (Mugil cephalus) and red-lip mullet (Liza haematocheila) fishery.

Marine plastic pollution and continuous capture of marine animals, so-called "ghost fishing", by abandoned, lost, or otherwise discarded fishing gear (ALDFG) are global concerns. This study investigated whether biodegradable polylactic acid (PLA) monofilaments can be used to replace conventionally used non-biodegradable polyamide (PA) in trammel net fishery for limiting ALDFG associated effects. It evaluated the physical properties of PLA and PA monofilaments and compared fishing performance of PLA and PA trammel nets in a commercial mullet fishery in the Yellow Sea, China. Although PA monofilament exhibited superior physical properties, no significant differences in catch efficiency between PA and PLA trammel nets were observed. Fish of both species were mainly captured by pocketing which can further explain observed similar catch efficiency. These initial results suggest a potential for applying biodegradable materials in trammel net fisheries. Therefore, further long-term testing is encouraged to investigate whether this promising performance is persistent over long-term.

Fabrication of micro-nano structure nanofibers by solvent etching.

Micro-Nano structure nanofibrous affinity membranes of poly(ether sulfones) (PES) blended with a functional polymer poly(ethyleneimine) (PEI) were fabricated by electrospinning technique followed by solvent etching in crosslinking solution. The surface SEM image of the water washed PES/PEI nanofibrous membrane confirmed that PEI was concentrated on the fiber surface. The nanofibrous PES/PEI membranes were crosslinked in a mixture of acetone and water with glutaraldehyde (crosslinking agent, GA), and the micro-nano structural surface of the nanofibrous membranes was created by solvent etching due to the solvation between PEI and the solvent water in the crosslinking solution during the crosslinking process. The influence of the component of the crosslinking bath on the mophology of the resulting PES/PEI nanofibers was investigated. It was found that the relatively uniform micro-nano spherules grew on the surface of the nanofibers when the content of water in crosslinking solution was more than 20 wt%, and the diameters of the spherules were in the range of 50-250 nm. The advantage of the micro-nano structrue for the heavy metal ions removal in wastwater has been demonstrated by taking a series of static adsorption experiments. It was found that the micro-nano structrue of PES/PEI nanofibrous membranes could bring high performance of adsorption capacity for heavy metal ions, indicating that the unique morphology could bring much more large surface area per unit mass and high effectivity for heavy metal ions removal from aqueous solutions.

Structure and Properties of Polyoxymethylene/Silver/Maleic Anhydride-Grafted Polyolefin Elastomer Ternary Nanocomposites.

In the present study, silver (Ag) nanoparticles and maleic anhydride-grafted polyolefin elastomer (MAH-g-POE) were used as enhancement additives to improve the performance of the polyoxymethylene (POM) homopolymer. Specifically, the POM/Ag/MAH-g-POE ternary nanocomposites with varying Ag nanoparticles and MAH-g-POE contents were prepared by a melt mixing method. The effects of the additives on the microstructure, thermal stability, crystallization behavior, mechanical properties, and dynamic mechanical thermal properties of the ternary nanocomposites were studied. It was found that the MAH-g-POE played a role in the bridging of the Ag nanoparticles and POM matrix and improved the interfacial adhesion between the Ag nanoparticles and POM matrix, owing to the good compatibility between Ag/MAH-g-POE and the POM matrix. Moreover, it was found that the combined addition of Ag nanoparticles and MAH-g-POE significantly enhanced the thermal stability, crystallization properties, and mechanical properties of the POM/Ag/MAH-g-POE ternary nanocomposites. When the Ag/MAH-g-POE content was 1 wt.%, the tensile strength reached the maximum value of 54.78 MPa. In addition, when the Ag/MAH-g-POE content increased to 15wt.%, the elongation at break reached the maximum value of 64.02%. However, when the Ag/MAH-g-POE content further increased to 20 wt.%, the elongation at break decreased again, which could be attributed to the aggregation of excessive Ag nanoparticles forming local defects in the POM/Ag/MAH-g-POE ternary nanocomposites. Furthermore, when the Ag/MAH-g-POE content was 20 wt.%, the maximum decomposition temperature of POM/Ag/MAH-g-POE ternary nanocomposites was 398.22 °C, which was 71.39 °C higher than that of pure POM. However, compared with POM, the storage modulus of POM/Ag/MAH-g-POE ternary nanocomposites decreased with the Ag/MAH-g-POE content, because the MAH-g-POE elastomer could reduce the rigidity of POM.

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification.

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.

Molecular characterization of TLR3 and TRIL in silvery pomfret (Pampus argenteus) and their expression profiles in response to bacterial components.

Toll-like receptors (TLRs) play important roles in the innate system by recognizing pathogen-associated molecular patterns derived from various microbes. In this study, we reported the cloning and identification of paTLR3 and paTLR4 interactor with leucine rich repeats (TRIL) cDNA from silvery pomfret (Pampus argenteus). The full-length paTLR3 and paTRIL cDNA were 2996 and 3163 bp long, respectively. Both of the two proteins contained many LRR domains, one LRR-C terminal domain and one transmembrane region, which fits with the characteristic TLR and its analogue domain architecture. Phylogenetic analyses revealed that paTLR3 and paTRIL shared the closest relationship with Lateolabrax japonicas and Notothenia coriiceps, respectively. The expression levels of paTLR3 and paTRIL varied greatly among the examined tissues with the highest expression both in liver. Following exposure to V. anguillarum flagellin, A. hydrophila lipopolysaccharide (LPS) and L. plantarum lipoteichoic acid (LTA), paTLR3 and paTRIL were all up-regulated. V. anguillarum flagellin induced the highest expression levels of paTLR3 and paTRIL. A. hydrophila flagellin and A. hydrophila LPS induced the highest expression levels of IL-1ß and IL-8, respectively. The present results will provide the valuable information for understanding the structure, function and the immune defense process of paTLR3 and paTRIL in silvery pomfret.

Effect of Silver Nanoparticles on the Microstructure, Non-Isothermal Crystallization Behavior and Antibacterial Activity of Polyoxymethylene.

Silver (Ag) nanoparticles were synthesized by a facile route in the presence of oleic acid and n-propylamine. It was shown that the average primary size of the as-synthesized Ag nanoparticles was approximately 10 nm and the surface of as-synthesized Ag nanoparticles was capped with monolayer surfactants with the content of 19.6%. Based on as-synthesized Ag nanoparticles, polyoxymethylene (POM)/Ag nanocomposites were prepared. The influence of Ag nanoparticles on non-isothermal crystallization behavior of POM was investigated by differential scanning calorimetry (DSC). The Jeziorny, Jeziorny-modified Avrami, Ozawa, Liu and Mo, Ziabicki and Kissinger models were applied to analyze the non-isothermal melt crystallization data of POM/Ag nanocomposites. Results of half time (t1/2), crystallization rate parameter (CRP), crystallization rate function (K(T)), kinetic parameter (F(T)), the kinetic crystallizability at unit cooling rate (GZ) and the crystallization activation energy (∆E) were determined. Small amounts of Ag nanoparticles dispersed into POM matrix were shown to act as heterogeneous nuclei, which could enhance the crystallization rate of POM, increase the number of POM spherulites and reduce POM spherulites size. However, the higher loading of Ag nanoparticles were easily aggregated, which restrained POM crystallization to some degree. Furthermore, the POM/Ag nanocomposites showed robust antibacterial activity against Escherichia coli and Staphylococcus aureus.

Anionic Surfactant-Triggered Steiner Geometrical Poly(vinylidene fluoride) Nanofiber/Nanonet Air Filter for Efficient Particulate Matter Removal.

The emergence of Steiner minimal tree is of fundamental importance, and designing such geometric structure and developing its application have practical effect in material engineering and biomedicine. We used a cutting-edge nanotechnology, electrospinning/netting, to generate a Steiner geometrical poly(vinylidene fluoride) (PVDF) nanofiber/nanonet filter for removing airborne particulate matter (PM). Manipulation of surface morphologies by precise control of charged situation enabled the creation of two-dimensional nanonets with Steiner geometry. A significant crystalline phase transition of PVDF from α-phase to ß-phase was triggered by the dipole orientation and the intermolecular interactions derived from the electrostatic potential analysis. Particularly, the synergy of electrical interaction (ion-dipole and dipole-dipole) and hydrophobic interaction facilitated the formation of Steiner geometric structure during the evolution process of nanonets. The resultant PVDF nanofiber/nanonet air filter exhibited high filtration efficiency of 99.985% and low pressure drop of 66.7 Pa under the airflow velocity of 32 L/min for PM0.26 removal by the safest physical sieving mechanism. Furthermore, such filter possessed robust structure integrity for reusability, comparable optical transmittance, superior thermal stability, and prominent purification capacity for smoke PM2.5. The successful construction of such fascinating Steiner geometrical PVDF nanonets will provide new insights into the design and exploitation of novel filter media for air cleaning and haze treatment.

Time-series transcriptomic analysis of the kelp grouper Epinephelus moara in response to low salinity stress.

The Kelp grouper Epinephelus moara is one of the most widely consumed and economically important marine fish in China. The species can tolerate a wide range of salinity, but genomic resources are not available, and the molecular mechanisms underlying adaptation to salinity at the transcriptomic level remain largely unclear. In this study, the transcriptomic responses of the liver of E. moara under low salinity were investigated using the Illumina digital gene expression system. After de novo assembly, 499,356 transcripts were generated and contributed 445,068 unigenes. A total of 14, 19, 33 and 3101 genes were differentially expressed following exposure to low salinity stress for 2, 6, 24 and 48 h, respectively. Only two genes were differentially expressed in all groups. Four genes related to metabolism and ambient salinity adaption were randomly selected to validate the differentially expressed genes (DEGs) by real-time PCR. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyse the functional significance of DEGs, including those responding to salinity through diverse biological processes, cellular components, molecular functions, and pathways associated with metabolic and osmotic responses. This work provides new insight into the response to salinity challenges in E. moara, and the findings expand our knowledge of the molecular basis of metabolic regulation mechanisms in this species. Additionally, the transcriptional data provide a valuable resource for future molecular and genetic studies on E. moara.

Diverse and abundant antibiotic resistance genes from mariculture sites of China's coastline.

With the rapid development of mariculture in China, large amounts of antibiotics are being discharged into the aquatic environment. Little information is available regarding antibiotics and corresponding antibiotic resistance genes (ARGs) associated with maricultural environments in China. Sediments from eleven typical mariculture areas along the whole coastline of China were collected, and the sediment in Meijijiao in southern China was used as a non-mariculture control. The results revealed that antibiotics and their corresponding ARGs were widely distributed in most maricultural sediments, and present at low concentrations in samples from Meijijiao. The sulfonamide-resistance genes were prevalent, and the sul1 and sul2 in Penglai were the highest detected by using quantitative PCR. Moreover, remarkable differences in ARGs among different sites were observed. Due to the limited availability of primers to detect ARGs, illumina high-throughput sequencing was also used for profiling ARGs, and the results showed that the abundance of bacA in all samples was the highest compared to other ARGs, followed by mexF and mexB. This is the first study to comprehensively investigate the antibiotic resistance profile in typical mariculture areas along the whole coast of China. This study provides insights into the impacts of mariculture on the profiles of bacterial and ARG compositions in China.

Cloning, characterization, and function of MyD88 in silvery pomfret (Pampus argenteus) in response to bacterial challenge.

Myeloid differentiation factor 88 (MyD88) is a key and universal downstream adapter for most Toll-like receptors (TLRs) and plays an important role in both the innate and adaptive immune response. In this study, the full-length cDNA of MyD88 (PAMyD88) from silvery pomfret (Pampus argenteus) was cloned and characterized. The PAMyD88 cDNA is 1545bp in length and contains an 876bp open reading frames (ORF). Multiple sequence alignment and phylogenetic tree analyzes revealed that the amino acid sequence of PAMyd88 was homologous to a variety of previously MyD88 molecules characterized from other species. The quantitative real-time reverse transcription-PCR analysis showed that the PAMyD88 mRNA was broadly expressed in all examined tissues, with higher levels observed in the immune-relevant organs. The results showed a significant up-regulation of the TLR2 and PAMyD88 transcript levels in response to L. plantarum and C. butyricum and a substantial expression level of TLR4 and PAMyD88 induced by V. anguillarum. Additionally, a challenge with V. anguillarum resulted in significant apoptosis, whereas the L. plantarum and C. butyricum induced only low levels of apoptosis. These data provide insight into the roles of PAMyD88 in the TLR signaling pathway in response to probiotic and pathogenic bacteria in silvery pomfrets.