A high-protein retained PES hemodialysis membrane with tannic acid as a multifunctional modifier.

A high protein retention polyethersulfone (PES) membrane was prepared by nonsolvent-induced phase separation and surface coating, which exhibited enhanced hemocompatibility and antioxidant stress performance. The cross-linked network was constructed by tannic acid (TA) and alpha-lipoic acid (α-LA) on the surface of the membrane, which controlled the pores to a reasonable size. The enrichment of heparin-like groups on the membrane surface, implemented by "hydrophobic interaction" and "click reaction", confers anticoagulant properties; the presence of a large number of phenolic hydroxyl groups from TA and the introduction of α-LA allows the modified membranes to intervene in oxidative stress. The hemocompatibility characterizations included plasma recalcification time (PRT), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and hemolysis rate (HR). Additionally, the DPPH ABTS radical scavenging capacity was tested to evaluate the antioxidant performance. The results show that the modified membrane presents an outstanding protein retention rate (99.3%) along with permeability. In addition, the PRT is prolonged to 341.7 s, and the DPPH• scavenging ability reaches 0.74 µmol•cm-2. The membranes can be easily prepared and present excellent comprehensive performance. This work provides a simple and facile strategy for the fabrication of hemodialysis membranes with controllable pore sizes.

Two c-type lectins from Venerupis philippinarum: Possible roles in immune recognition and opsonization.

In the study, two c-type lectins were identified and characterized from the manila clam Venerupis philippinarum (designed as VpClec-1 and VpClec-2, respectively). Multiple alignments and phylogenetic analysis strongly suggested that they were new members of the c-type lectin superfamily. In normal tissue of clams, both VpClec-1 and VpClec-2 transcripts were highly expressed in the tissue of hepatopancreas. After Vibrio anguillarum challenge, the temporal expression of both VpClec-1 and VpClec-2 transcripts was up-regulated in the hemocytes of manila clams. The recombinant protein VpClec-1 (rVpClec-1) showed obvious binding activities to lipopolysaccharide (LPS), peptidoglycan (PGN), glucan and zymosan in vitro, while the recombinant protein VpClec-2 (rVpClec-2) could only bind LPS, glucan and zymosan. Coinciding with the PAMPs binding assay, both rVpClec-1 and rVpClec-2 displayed broad agglutination and antibacterial activities towards Vibrio harveyi, Vibrio splendidus, Vibrio anguillarum, Enterobacter cloacae and Aeromonas hydrophila. Moreover, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rVpClec-1 and rVpClec-2. Notably, the rVpClec-1 but not rVpClec-2 elicited a chemotactic response from hemocytes. All the results showed that VpClec-1 and VpClec-2 functioned as pattern recognition receptors (PRRs) with distinct recognition spectrum, and involved in the innate immune responses of manila clams.

A sialic acid-binding lectin with bactericidal and opsonic activities from Ruditapes philippinarum.

In the present study, a sialic acid-binding lectin was cloned and characterized from Manila clam Ruditapes philippinarum (designed as RpSabl). The open reading frame of RpSabl encoded a polypeptide of 162 amino acids with a calculated molecular mass of 17.7 kDa. Analysis of the conserved domain suggested that RpSabl was a new member of the sialic acid-binding lectins family. In non-stimulated clams, RpSabl transcripts were constitutively expressed in all five tested tissues, especially in hepatopancreas. After Vibrio anguillarum challenge, the expression of RpSabl mRNA in hepatopancreas was significantly up-regulated at 3 h (3.8-fold, P < 0.05), 6 h (4.9-fold, P < 0.05), 12 h (12.3-fold, P < 0.01) and 24 h (9.7-fold, P < 0.01), while RpSabl transcripts in hemocytes was only significantly up-regulated at 6 h (8.5-Fold, P < 0.01). RNAi-mediated knockdown of RpSabl transcripts affected the survival rates of Manila clam against V. anguillarum, perhaps mainly due to the inhibited expression of antibacterial effectors (e.g. lysozyme and defensin). Moreover, recombinant protein of RpSabl (rRpSabl) possessed binding activities towards lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan in vitro. Coinciding with the Pathogen-associated molecular patterns (PAMPs) binding assay, rRpSabl displayed broad bacterial-agglutination properties towards Vibrio harveyi, Vibrio splendidus, V. anguillarum, Enterobacter cloacae and Aeromonas hydrophila. Meanwhile, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rRpSabl incubation. All these results showed that RpSabl could function as a versatile molecule involved in the innate immune responses of R. philippinarum.

Integrative Biomarker Assessment of the Influence of Saxitoxin on Marine Bivalves: A Comparative Study of the Two Bivalve Species Oysters, Crassostrea gigas, and Scallops, Chlamys farreri.

Harmful algae blooms have expanded greatly in recent decades, and their secreted toxins pose a severe threat to human health and marine ecosystems. Saxitoxin (STX) is a main paralytic shellfish poison naturally produced by marine microalgae of the genus Alexandrium. Despite numerous studies have assessed the impacts of STX on marine bivalves, comparative in vivo study on the toxicity of STX on bivalves with distinct accumulation ability (such as oysters and scallops) has been seldom investigated. The aim of this study was to identify whether distinct sensitivity exists between oysters, Crassostrea gigas, and scallops, Chlamys farreri under the same amount of STX exposure using multiple biomarker responses. The responses of different biochemical markers including oxidative stress markers (catalase, superoxide dismutase, glutathione S-transferase, and lipid peroxidation) and immunotoxicity biomarkers (hemocyte phagocytosis rate, reactive oxidative species production, and DNA damages) were evaluated in bivalves after 12, 48, and 96 h of exposure to STX. The integrated biomarker responses value combined with two-way ANOVA analysis suggested that STX posed slightly severer stress on scallops than oysters for the extended period of time. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess the toxicity associated with STX exposure in marine bivalves.

Molecular characterization, expression, and antibacterial activity of a c-type lysozyme isolated from the manila clam, Ruditapes philippinarum.

Lysozyme is an antibacterial enzyme that is widely distributed in nature and plays an important role in the prevention of bacterial infections. In this study, a c-type lysozyme (designated as "RpCLYZ") was cloned and characterized from the manila clam, Ruditapes philippinarum. The full-length cDNA was 619 bp with an open reading frame (ORF) of 447 bp, and encoded a polypeptide of 148 amino acids with a calculated molecular mass of 17.0 kDa and an isoelectric point of 4.83. RpCLYZ was found to share high sequence similarity with c-type lysozymes from other invertebrates. The mRNA transcript of RpCLYZ was universally expressed in a wide range of tissues, especially in gills and mantle. Challenge with Vibrio anguillarum, significantly induced mRNA expression of RpCLYZ, which reached a maximum level 48 h after bacterial challenge. Recombinant RpCLYZ (rRpCLYZ) exhibited antibacterial activities against both Gram-positive and Gram-negative bacteria. Additionally, the optimal pH and temperature for rRpCLYZ activity were determined to be 4.5 and 20 °C, respectively. These results suggest that RpCLYZ participates in innate immune responses against bacterial invasion.

Two macrophage migration inhibitory factors (MIFs) from the clam Ruditapes philippinarum: Molecular characterization, localization and enzymatic activities.

Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient cytokine-like factor and plays a critical role in both innate and adaptive immunity. In the present study, two MIFs (designed as RpMIF-1 and RpMIF-2, respectively) were identified and characterized from the clam Ruditapes philippinarum by rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of RpMIF-1 and RpMFI-2 consisted of 531 and 722 nucleotides, encoding a polypeptide of 113 and 114 amino acid residues, respectively. Multiple alignments and phylogenetic analysis revealed that both RpMIF-1 and RpMIF-2 belonged to the MIF family. The conserved catalytic-site Pro2 for tautomerase activity was identified in the deduced amino acid sequences of RpMIFs. Both RpMIF-1 and RpMIF-2 transcripts were constitutively expressed in examined tissues of R. philippinarum with dominant expression in hepatopancreas, gills and hemocytes. Immunolocalization analysis showed that RpMIF-1 and RpMIF-2 proteins were expressed in examined tissues with the exception of adductor muscle and foot. After Vibrio anguillarum and Micrococcus luteus challenge, the mRNA expression of RpMIFs was significantly modulated in hemocytes, gills and hepatopancreas. Recombinant RpMIF-1 and RpMIF-2 proteins possessed significant tautomerase activity and oxidoreductase activity, indicating that these two proteins was perhaps involved in inflammatory responses. In summary, our results suggested that RpMIF-1 and RpMIF-2 played an important role in the innate immunity of R. philippinarum.