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Chronic lead (Pb) exposure causes neurodysfunction and contributes to the development of neurodegenerative disease. However, the mechanism of Pb-induced neurological dysfunction have yet to be fully elucidated. This study determined the role pyroptosis plays in Pb-induced neurodysfunction in neurons. We used both in vitro and in vivo approaches to explore whether Pb exposure induces caspase-1-mediated pyroptosis in neurons and its relationship to Pb-induced neurological disorders. Our findings showed that caspase-1-mediated pyroptosis in Pb-exposed neurons activated glycogen synthase kinase 3 protease activity by disrupting Ca2+/calmodulin-dependent protein kinase II/cAMP-response element binding protein pathway, leading to neurological disorders. Moreover, the caspase-1 inhibition VX-765 or the non-steroidal anti-inflammatory drug sodium para-aminosalicylic acid (PAS-Na) attenuated the Pb-induced neurological disorders by alleviating caspase-1 mediated neuronal pyroptosis. Our novel studies suggest that caspase-1-mediated pyroptosis in neurons represents a potential mechanism for Pb-induced neurodysfunction, identifying a putative target for attenuating the neurodegenerative effects induced by this metal. [Display omitted] • Pb induces pyroptosis in neurons. • Caspase-1 mediates neuronal pyroptosis. [ABSTRACT FROM AUTHOR]

Preeclampsia is a devastating pregnancy-related syndrome characterized by the onset of hypertension, proteinuria and edema. Insufficient invasion of trophoblasts is well-known to be correlated with preeclampsia development. The present study was performed to investigate the functional role microRNA (miRNA)-204 in trophoblastic invasion in vitro . We here found that the invasive capabilities of BeWo and JEG3 trophoblast-like cells were suppressed by miR-204 mimics, whereas enhanced by its inhibitor. Matrix metalloproteinase-9 (MMP9) was first confirmed to play a role in regulating trophoblast invasion through loss- or gain-of-function experiment. Notably, we demonstrated MMP9 as a direct target of miR-204 in BeWo cells by using the dual-luciferase assay. Moreover, forced overexpression of MMP9 was noted to partly attenuate the inhibitory effects of miR-204 on BeWo cell invasion. Taken together, our study indicates that miR-204 may contribute to the development of preeclampsia by inhibiting trophoblastic invasion, and that MMP9 is involved in miR-204-mediated trophoblast cell invasion. Our study suggests miR-204 as a novel therapeutic target for preeclampsia. [ABSTRACT FROM AUTHOR]

Abstract: Fibrinogen-related proteins (FREPs) are a kind of pattern recognition receptors (PRRs) containing fibrinogen-like (FBG) domains, and they play curial roles in the innate immune response. In the present study, a new FREP protein was identified from bay scallop Argopecten irradians (designated as AiFREP-2). The full-length cDNA of AiFREP-2 was of 1299 bp with an open reading frame of 762 bp encoding a polypeptide of 253 amino acids, including a signal sequence and an FBG domain. The FBG domain in AiFREP-2 was highly similar to those of ficolins, tenascins and other FREPs. The mRNA expression of AiFREP-2 could be detected in all the examined tissues with the highest level in gill. The mRNA expression of AiFREP-2 in hemocytes was significantly up-regulated post the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan (GLU) (P < 0.01). The recombinant AiFREP-2 (rAiFREP-2) could bind not only different PAMP ligands including LPS, PGN and GLU, but also various microbes including Gram-negative bacteria (Vibrio anguillarum), Gram-positive bacteria (Staphylococcus aureus) and fungus (Pichia pastoris and Yarrowia lipolytica). Additionally, rAiFREP-2 exhibited obvious agglutination activity towards Gram-negative bacteria V. anguillarum and Gram positive bacteria S. aureus. The results indicated that AiFREP-2 was involved in the immune response against Gram-negative bacteria, Gram-positive bacteria and fungus as a PRR in bay scallop, and the information was helpful to understand the innate immune defense mechanisms of mollusks. [Copyright &y& Elsevier]

Abstract: Rab GTPase is essential for the control of intracellular membrane trafficking in all eukaryotic cells and further affects the ability of phagocytic cells to scavenge pathogen. In the present study, the cDNAs for two crab Rab proteins (EsRab-1 and EsRab-3) were identified from the Chinese mitten crab Eriocheir sinensis by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNAs of EsRab-1 and EsRab-3 were of 892 bp and 965 bp with ORFs of 615 bp and 630 bp, respectively. The cDNAs encoded two peptides of 204 and 209 amino acid residues with the conserved GTP/Mg2+ binding sites, Switch I region and Switch II region in RAB domains. The mRNA transcripts of EsRab-1 and EsRab-3 were both highest expressed in hepatopancreas, and marginally expressed in other tissues including hemocytes, muscle, gonad, gill and heart. After the crabs were challenged by bacteria Vibrio anguillarum, the expression levels of both EsRab-1 and EsRab-3 in hemocytes were significantly up-regulated, and reached the highest level at 1.5 h post-stimulation, which was 7-fold (P < 0.05) and 6-fold (P < 0.01) of blank group for EsRab-1 and EsRab-3, respectively. No significant change of mRNA expression was detected for either EsRab-1 or EsRab-3 in crabs stimulated by Pichia pastoris. These results clearly suggested the involvement of Rab proteins in crab anti-bacterial immunity. [Copyright &y& Elsevier]

Abstract: C-type lectins are a large family of Ca2+-dependent carbohydrate binding proteins which play crucial roles to recognize and eliminate pathogens in innate immunity. In the present study, a novel C-type lectin was identified from Eriocheir sinensis (designated as EsCTL). The full-length cDNA of EsCTL was of 789 bp with an open reading frame of 468 bp encoding a polypeptide of 156 amino acids with a signal sequence and single carbohydrate-recognition domain (CRD). The potential tertiary structure of the CRD adopted a typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. An EPQ motif to determine carbohydrate binding specificity was identified in the CRD of EsCTL. The mRNA transcripts of EsCTL were mainly detected in hepatopancreas and its relative expression level in hemocytes was significantly up-regulated after the challenges of Vibrio anguillarum (P < 0.05) and Pichia pastoris (P < 0.05). The recombinant EsCTL protein (rEsCTL) could bind different PAMPs, including LPS, PGN, β-glucan, and polyI:C; and also bind various microorganisms including three Gram-positive bacteria, three Gram-negative bacteria and two yeasts. Moreover, rEsCTL could significantly enhance the in vitro encapsulation of crab hemocytes. All these results suggested that EsCTL functioned as an important PRR involved in immune defense against invading pathogen in crab. [Copyright &y& Elsevier]

Abstract: The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F′ as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system. [Copyright &y& Elsevier]

Abstract: Poly(ADP-ribose) polymerase (PARP) inhibitors have neuroprotective effects after retinal ischemia and reperfusion (I/R) injury, but mechanisms of this action are not clear. A second generation PARP inhibitor, GPI 15427, was administrated to mice to investigate the possible mechanisms underlying its neuroprotective effects after retinal I/R injury. Ischemia was induced by increasing intraocular pressure to 80–90mm Hg for 60min followed by reperfusion, and mice were treated with GPI 15427 (40mg/kg−1 day−1, orally) 2days before or 1day after injury. Histopathology caused by the retinal I/R injury was estimated by TUNEL assay and histological analyses. Relative gene expressions were evaluated by RT-PCR, Western blotting and immunohistological studies. GPI 15427 inhibited the retinal I/R-induced PARP activation and glial cell activation. GPI 15427 also significantly inhibited the I/R-induced neurodegeneration, as well as increase in TUNEL-positive cells. I/R-induced PERK-eIF2α-CHOP activation and Bip over-expression were inhibited by GPI 15427, while it did not suppress I/R-induced CHOP over-expression and degeneration of retinal capillaries. Our results suggest that GPI 15427 inhibited retinal I/R-induced neurodegeneration and glial cell activation, and this was associated with an effect of the drug to suppress PERK-eIF2α-CHOP activation and Bip over-expression. These results provide evidence that GPI 15427 inhibits retinal I/R injury at least in part via inhibition of endoplasmic reticulum stress. [Copyright &y& Elsevier]

[Display omitted] Precise modulation of immuno-inflammatory response is crucial to control periodontal diseases and related systemic comorbidities. The present nanosystem with the controlled-release and cell-penetrating manner enhances the inflammation modulation effects of baicalein in human gingival epithelial cells (hGECs) for better oral healthcare. We constructed a red-emissive mesoporous silica nanoparticle-based nanosystem with cell-penetrating poly(disulfide) (CPD) capping, through a facile in-situ polymerization approach. It was featured with a glutathione-responsive manner and instant cellular internalization capacity for precisely delivering baicalein intracellularly. Laboratory experiments assessed whether and how the nanosystem per se with the delivered baicalein could modulate immuno-inflammatory responses in hGECs. The in-situ polymerized CPD layer capped the nanoparticles and yet controlled the release of baicalein in a glutathione-responsive manner. The CPD coating could facilitate cellular internalization of the nanosystem via endocytosis and thiol-mediated approaches. Notably, the intracellularly released baicalein effectively downregulated the expression of pro-inflammatory cytokines through inhibiting the NF-κB signaling pathway. The nanosystem per se could modulate immuno-inflammatory responses by passivating the cellular response to interlukin-1β. This study highlights that the as-synthesized nanosystem may serve as a novel multi-functional vehicle to modulate innate host response via targeting the NF-κB pathway for precision healthcare. [ABSTRACT FROM AUTHOR]

Cisplatin, carboplatin, and oxaliplatin are the only three platinum-based antineoplastic drugs that have been accepted worldwide for treating various cancers. Up to 83.6% of patients treated with platinum-based antineoplastic drugs will develop chemotherapy-induced peripheral neuropathy (CIPN), manifesting as sensory paresthesias, dysesthesias, and hypoesthesias that can cause significant adverse impact to daily activities. To investigate how these three platinum-based drugs affect mitochondrial function and myelination state of Schwann cells and the signalling pathway involved. 2 μM Cisplatin, 20 μM carboplatin, and 1 μM oxaliplatin were used to inhibit the growth of CAL-27 by 20% respectively. These drugs were then used to induce chemotherapy-induced peripheral neuropathy in Rat Schwann Cells (RSC96). The changes in cell metabolism and myelin formation in RSC96 were investigated. Cisplatin and carboplatin, but not oxaliplatin increased intracellular and mitochondrial reactive oxygen species in RSC96. Only Cisplatin and carboplatin decreased mitochondrial membrane potential (ΔΨm) and ATP production in RSC96. Both Cisplatin and carboplatin led to demyelination of RSC96, characterized by increased expression of p75NTR and decreased expression of myelin protein zero (MPZ). Cisplatin and carboplatin, but not oxaliplatin, caused mitochondrial dysfunction and induced demyelination in RSC96 while showing similar toxicity to head and neck cancer cells. Oxaliplatin may be a potential chemotherapy drug to prevent CIPN in patients with head and neck cancer. • Cisplatin and carboplatin increase reactive oxygen species in Schwann cells. • Cisplatin and carboplatin induce mitochondrial dysfunction and impair ATP production. • Cisplatin and carboplatin induce Schwann cell dedifferentiation. • Oxaliplatin is a potential chemotherapy drug to prevent CIPN. [ABSTRACT FROM AUTHOR]

Enzalutamide is an effective drug for the treatment of castration-resistant prostate cancer (CRPC), but acquired enzalutamide resistance is usually unavoidable within the short term in many patients. Lycopene, a safe and effective phytochemical, has been documented to have anticancer activity in a variety of tumors, especially for prostate cancer (PCa). The aim of this study was to provide data support for the combination of lycopene and enzalutamide in the treatment of CRPC. To this end, tissues from patients with primary prostate cancer (PPC) and CRPC were examined by immunohistochemical analysis and found that p -AKT and p -EZH2 were overexpressed in CRPC. Furthermore, Kaplan-Meier survival analysis showed that the high expression of p -AKT and p -EZH2 may be related to the poor prognosis of patients. In addition, the expression of p -AKT, p -EZH2 and androgen receptor (AR) were significantly down-regulated in 22RV1 and C4–2B cells and the proliferation and invasion of CRPC cells were inhibited after treatment with lycopene, while SC79 (an AKT agonist) markedly rescue this effect. Of note, a combination of lycopene and enzalutamide significantly inhibited the proliferation and invasion of CRPC cells in vitro, as well as tumor growth and bone metastasis in vivo. These results suggest that the enhanced antitumor effects of enzalutamide by lycopene may be related to the reduction of AR protein levels through lycopene-mediated inhibition of AKT/EZH2 pathway, which may provide a new approach to improve the efficacy of enzalutamide in CRPC. • Lycopene suppressed CRPC cells proliferation and invasion. • p -AKT and p -EZH2 are overexpressed and associated with prognosis significance in CRPC. • The inhibitory effect of lycopene on the proliferation and invasion of CRPC cells through AKT/EZH2/AR pathway. • Lycopene strengthens the effect of Enzalutamide on CRPC Cell Lines in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Low-density lipoprotein receptor-related protein (LRP) is a group of important endocytic receptors contributing to binding ligands and maintaining internal environment. In the present study, an LRP-like molecule was identified from Zhikong scallop Chlamys farreri (CfLPR), and its mRNA expression profiles, tissue location, and immunology activities were analyzed to explore its possible function in the innate immune system. The ORF of CfLRP was of 1971 bp encoding a polypeptide of 656 amino acids with ten low-density lipoprotein-receptor YWTD (LY) domains and one scavenger receptor cysteine-rich (SRCR) domain. It shared similar structure with out-membrane domains of LRP family members in mammalian. The mRNA transcripts of CfLRP were dominantly expressed in hepatopancreas and mantle (P < 0.01), and its mRNA level in hemocytes was up-regulated (P < 0.01) significantly after the stimulations of lipopolysaccharides (LPS), peptidoglycan (PGN) and β-glucan. Western blotting assay using polyclonal antibody specific for CfLRP revealed that CfLRP was localized in the plasma. The recombinant protein of CfLRP (rCfLRP) could bind acetylated low density lipoprotein (Ac-LDL), metalloprotease SPF1 of Vibrio splendidus and mannan, but could not bind other typical PAMPs such as LPS, PGN, β-glucan and zymosan. Meanwhile, rCfLRP also exhibited strong bacteriostatic activity to Gram-negative bacteria Vibrio anguillarum and V. splendidus. These results indicated that CfLRP could serve as a receptor to recognize and eliminate the invading pathogens, which provided a new implication in the function of LRP-like molecules in invertebrate immunity. [Copyright &y& Elsevier]

Abstract: The adrenergic receptors are a class of G protein-coupled receptors, through which norepinephrine and epinephrine trigger the second messenger to modulate the immune response in immunocytes of vertebrate. In the present study, a gene coding the homologue of α-adrenergic receptor was identified from scallop Chlamys farreri (designated CfαAR). Its deduced protein comprised 318 amino acids, and contained a conserved 7tm_1 domain. After CfαAR protein was expressed in the HEK293 cells, the stimulation of octopamine, tyramine, epinephrine and isoprenaline (β-adrenergic receptor agonist) did not change significantly the intracellular cAMP concentration, whereas the stimulation of norepinephrine and phenylephrine (α-adrenergic receptor agonist) lowered significantly the cAMP level to 0.52 and 0.84 pmol μl−1 (P < 0.05), respectively. The CfαAR transcripts were ubiquitously detected in the tested tissues including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest expression in the gill. The expression level of CfαAR mRNA decreased significantly (0.21-fold, P < 0.05) at 3 h after the challenge of bacteria Vibrio anguillarum. Then, it began to increase (4.74-fold, P < 0.05) at 12 h, and reached the highest level (4.92-fold, P < 0.05) at 24 h after bacteria challenge. The addition of α-adrenergic receptor agonist to the primary scallop haemocytes repressed significantly the increase of phagocytic and antibacterial activity induced by LPS stimulation, while the induction was reverted by the addition of α-adrenergic receptor antagonist. These results collectively suggested that α-adrenergic receptor could be regulated dynamically in the transcriptional level during the immune response, and it could modulate the haemocyte phagocytic and antibacterial function through the second messenger cAMP, which might be requisite for pathogen elimination and the homeostasis maintenance in scallop. [Copyright &y& Elsevier]

Abstract: Serine protease inhibitors (serpins) represent an expanding superfamily of endogenous inhibitors that regulate proteolytic events and involve in a variety of physiological processes. A serine protease inhibitor, namely Esserpin, was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) analysis. The full-length cDNA of Esserpin was of 2367 bp, including an open reading frame (ORF) of 1371 bp encoding a polypeptide of 456 amino acids with estimated molecular mass of 49.95 kDa and theoretical isoelectric point of 6.03. A putative signal peptide of 23 amino acids and a classical serpin domain were identified in Esserpin. The deduced amino acid sequence of Esserpin shared homology with serpins from Fenneropenaeus chinensis and Pacifastacus leniusculus. The mRNA transcripts of Esserpin could be detected in all the examined tissues including heart, gill, hemocytes, muscle, gonad and hepatopancreas, and the highest expression level was present in gonad. After the crabs were challenged by Vibrio anguillarum and Pichia pastoris, the expression levels of Esserpin transcripts in hemocytes were significantly up-regulated, and peaked at 24 h (5.18-fold of blank group, P < 0.05) and 3 h (2.87-fold of blank group, P < 0.05), respectively. The functional activity of Esserpin was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombinant Esserpin (rEsserpin) could inhibit trypsin activities in a dose-dependent manner, and it could lead to 100% inhibition of trypsin activities under the concentration of 873.76 nM, while there was no evident inhibition of chymotrypsin observed with rEsserpin. Moreover, rEsserpin inhibited the growth of E. coli at the final concentration of 1747.52 nM, and it also significantly depressed (P < 0.05) the phenoloxidase activity in the plasma at the final concentration of 873.76 nM. These results indicated that Esserpin was a homologue of serpin in crab and it could be induced after immune stimulation and mediate immune response possibly via the inhibition of bacterial growth and the regulation of prophenoloxidase-activating system. [Copyright &y& Elsevier]

Abstract: Opioid growth factor receptor (OGFR) is a receptor for [Met5]-enkephalin and plays important roles in the regulation of cell growth and embryonic development. In the present study, a cDNA of 2381 bp for the scallop Chlamys farreri OGFR (designated as CfOGFR) was identified by rapid amplification of cDNA ends (RACE) approach and expression sequence tag (EST) analysis. The complete cDNA sequence of CfOGFR contained an open reading frame (ORF) of 1200 bp, which encoded a protein of 399 amino acids. The amino acid sequence of CfOGFR shared 33–64% similarity with other OGFRs. There was a low complexity domain and a conserved OGFR_N domain at the N-terminal of CfOGFR. The mRNA transcripts of CfOGFR were constitutively expressed in the tested tissues with the highest expression level in hepatopancreas. During the early embryonic development, the mRNA transcripts of CfOGFR could be detected in different development stages, where the expression level presented a downward trend as a whole. The stimulations of LPS, Glu and poly (I:C) significantly induced the expression of CfOGFR mRNA in hemocytes (P < 0.05), while PGN stimulation exerted no influence. Co-IP and western blot results revealed that the CfOGFR in hemocytes displayed high affinity and specificity to [Met5]-enkephalin. Exogenous [Met5]-enkephalin was observed to inhibit the proliferation of HEK293T cells transfected with pcDNA3.1(+)-CfOGFR in a time and dosage dependent manner. These results collectively indicated that CfOGFR, as a homolog of OGFRs in C. farreri, played an important role in cells proliferation, and might be involved in the immune response of scallops. [Copyright &y& Elsevier]

Abstract: The complex interactions among host, pathogen and environment are believed to be the main causes for the mass mortality of cultured scallops during summer period. In the present study, the temporal variations of immune and energy parameters of Chlamys farreri under Vibrio anguillarum challenge, higher temperature (29°C) exposure as well as their combined treatment were investigated in order to better understand the energetic mechanisms of scallop immune defense. After the treatments, the superoxide anion level, the activities of superoxide dismutase (SOD) and acid phosphatase, as well as heat shock protein 70 expression level in the hemolymph of scallops increased substantially within 48h. And as time progressed, the malondialdehyde content in the serum of scallops in the higher temperature treated and the combined stress treated groups were significantly increased, while the SOD activity was significantly depressed (96h, P <0.05). After 3h, a significant decline (P <0.05) in glycogen reserves was observed in the examined tissues of all the scallops in the bacteria challenged, higher temperature treated and the combined stress treated groups. The cellular energy allocation (CEA) in the examined tissues dropped considerably when the treatments lasted 48h. There was a significant decline in the CEA and a significant increase in the energy consumption in the examined tissues compared with other treatments when the scallops were exposed to the combined stress for 96h (P <0.05). All the results demonstrated that the antioxidant systems and acute phase response system in scallops were not enough to wholly repair oxidative damage caused by higher temperature and the combined stress with bacteria challenge, and glycogen reserved in relative tissues were mobilized to meet the increased energy demands during the process of immune defense. Immune defense against the combined stress imposed greater costs on scallop''s energy expenditure than either stressor alone, and CEA could be a useful tool to evaluate energetic allocation. The information provided valuable insights into possible mechanisms of scallop mass mortalities during summer period. [Copyright &y& Elsevier]

Abstract: Peptidoglycan recognition protein (PGRP) is a pattern recognition receptor, playing important roles in the innate immune response against invasive pathogens. The single nucleotide polymorphism (SNP) loci in scallop PGRP gene (CfPGRP) were screened from Chlamys farreri to investigate their association with disease resistance of scallop against Listonella anguillarum. Thirteen SNP sites were identified in PGRP domain of CfPGRP, and two of them at positions 4407 and 4408 which are located in the same codon resulted in a nonsynonymous substitution. The genotype frequency of CG/CG in the resistant stock was significantly lower than that in susceptible stock (0% vs 32.4%), while that of CG/TA in the resistant stock was significantly higher than that in susceptible stock (P < 0.01). The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfPGRP-S1 (R) with CG variant in 4407–4408 site, rCfPGRP-S1 (Y) with TA variant in 4407–4408 site, were elucidated by examining their P/N value at 405 nm with ELISA assay. The in vitro binding activities of the two rCfPGRP-S1 variants to both lipopolysaccharide (LPS) and peptidoglycan (PGN) varied (P < 0.05) in a dose-dependent manner, and rCfPRPP-S1(Y) exhibited significantly higher affinity to PGN and LPS than that of rCfPGRP-S1(R) (P < 0.05). The growth inhibition assay was conducted to find the antibacterial activities of the two variants. Both rCfPGRP-S1(R) and rCfPGRP-S1 (Y) displayed obvious activity to suppress the growth of Escherichia coli, but there was no significant difference in suppression activity of two variants (P > 0.05). The results suggested that the polymorphism at locus 4407–4408 of CfPGRP-S1 considerably affected its PAMP binding activity, and the SNP locus 4407–4408 CG/TA was associated with disease resistance of scallop against L. anguillarum infection, which could be used as a candidate marker for future selection in zhikong scallop breeding program. [Copyright &y& Elsevier]

Abstract: Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3–6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg−1 respectively, but it decreased to 10.82 U mg−1 and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg−1 in the present of trypsinase and Cu2+. These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop. [Copyright &y& Elsevier]

Abstract: Iodothyronine deiodinase is responsible for the deiodination of thyroxine T4 to T3, and involved in the complex neuroendocrine-immune regulatory network to optimize the immune response in vertebrate. In this study, the full-length cDNA of an iodothyronine deiodinase (designated as CfDx) was cloned from scallop Chlamys farreri. The complete cDNA sequence of CfDx was of 1404 bp and contained an open reading frame of 900 bp encoding a polypeptide of 299 amino acids. The deduced amino acid sequence of CfDx contained an in-frame TGA stop codon probably encoding an essential selenocysteine (SeC), and there was a conserved region of about 15 amino acids surrounding the SeC residue. The CfDx mRNA transcripts were detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, gonad, gill and mantle, with the higher expression level in hepatopancreas and kidney. After LPS stimulation, the CfDx mRNA expression level in haemocytes increased significantly at 12 h (25.35-fold, P < 0.05) and 24 h (7.62-fold, P < 0.05), and the concentration of T3 in haemolymph increased significantly at 12 h (3.62 ng dL−1, P < 0.05) even the concentration of T4 did not change significantly. After scallop received an injection of 50 μg CfDx dsRNA, the expression level of CfDx mRNA in haemocytes began to decrease significantly at 36 h and maintained the relative low level (about 0.3-fold of the PBS control group) from 36 to72 h, but the ratio of T4/T3 in haemolymph began to increase at 36 h (2.31-fold, P < 0.05) and kept increasing from 36 to 72 h comparing with that in the PBS control group. These results indicated that CfDx was a homologue of iodothyronine deiodinase in scallop C. farreri, and it might be involved in the immunomodulation via regulating the concentration of thyroid hormones T3 and T4 in the haemolymph of scallop. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and β-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and β-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and β-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum. [Copyright &y& Elsevier]

Abstract: As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159bp, containing a 5′ untranslated region (UTR) of 90bp, a 3′ UTR of 866bp with a poly (A) tail, and an open reading frame (ORF) of 1203bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3kDa, which shared 59.6–64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly (P <0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6h and 12h after the stimulation of L. anguillarum, while P. pastoris significantly (P <0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris. [Copyright &y& Elsevier]

Abstract: C-type lectins are a superfamily of proteins that can bind pathogen-associated molecular patterns (PAMPs) and microorganisms through the recognition of carbohydrates, thus they are directly involved in innate defense mechanisms as part of the acute-phase response to infection. In this study, the cDNA of a novel C-type lectin (designated as AiCTL-7) was cloned from bay scallop Argopecten irradians by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of AiCTL-7 was of 651 bp containing a 525 bp open reading frame which encoded a signal peptide of 15 residues and a conserved carbohydrate-recognition domain (CRD) of 174 residues with the EPD and WSD motifs instead of the invariant EPN and WND motifs for determining the carbohydrate-binding specificity and constructing Ca2+-binding site 2 in vertebrates. The deduced amino acid sequence of AiCTL-7 CRD shared homology not only with the CRDs of C-type lectins in mollusks, but also with the fish lectin CRDs. The mRNA transcripts of AiCTL-7 were mainly detected in the tissue of hepatopancreas and also marginally detectable in kidney, gonad, hemocytes, heart and adductor of health scallop. After challenge with fungi Pichia pastoris GS115 and Gram-negative bacteria Listonella anguillarum, the relative expression level of AiCTL-7 was up-regulated significantly in hepatopancreas and hemocytes. The CRD of AiCTL-7 was recombined and expressed in Escherichia coli, and the recombinant protein (rAiCTL-7) aggregated P. pastoris remarkably in a Ca2+-dependent manner, and this agglutination could be inhibited by d-mannose, but not by d-galactose or β-1,3-glucan. However, rAiCTL-7 displayed no obvious agglutinating activity against L. anguillarum. These results collectively indicated that AiCTL-7 was involved in the primitive acute-phase response to microbial invasion as an important pattern recognition receptor (PRR) in the innate immune system of scallops. [Copyright &y& Elsevier]