Boosting Electrochemical CO2 Reduction on Copper-Based Metal-Organic Frameworks via Valence and Coordination Environment Modulation.

Cu-based metal-organic frameworks (MOFs) have attracted much attention for electrocatalytic CO2 reduction to high value-added chemicals, but they still suffer from low selectivity and instability. Here, an associative design strategy for the valence and coordination environment of the metal node in Cu-based MOFs is employed to regulate the CO2 electroreduction to ethylene. A novel "reduction-cleavage-recrystallization" method is developed to modulate the Cu(II)-Trimesic acid (BTC) framework to form a Cu(I)-BTC structure enriched with free carboxyl groups in the secondary coordination environment (SCE). In contrast to Cu(II)-BTC, the Cu(I)-BTC shows higher catalytic activity and better ethylene selectivity (≈2.2-fold) for CO2 electroreduction, which is further enhanced by increasing the content of free carboxyl groups, resulting in ethylene Faraday efficiency of up to 57% and the durability of the catalyst could last for 38 h without performance decline. It indicates that the synergistic effect between Cu(I)-O coordinated structure and free carboxyl groups considerably enhances the dimerization of *CO intermediates and hinders the hydrogenation of *CO intermediates in these competitive pathways. This work unravels the strong dependence of CO2 electroreduction on the Cu valence state and coordination environment in MOFs and provides a platform for designing highly selective electrocatalytic CO2 reduction catalysts.

Regulation of apoptosis by Pacific oyster Crassostrea gigas reveals acclimation strategy to CO2 driven acidification.

Ocean acidification (OA) has posed formidable threats to marine calcifiers. In response to elevated CO2 levels, marine calcifiers have developed multiple strategies to survive, such as taking advantage of apoptosis, but its regulation mechanism remains largely unknown. Here, we used the Pacific oyster Crassostrea gigas as model to understand the apoptotic responses and regulation mechanism at short- (7 d) to long-term (56 d) CO2 exposure (pH = 7.50). The apoptosis of hemocytes was significantly induced after short-term treatment (7-21 d) but was suppressed under long-term CO2 exposure (42-56 d). Similarly, caspase-3 and caspase-9 were also increased post short-term exposure and fell back to normal levels after long-term exposure. These data together indicated diverse regulation mechanisms of apoptosis through different exposure periods. Through analysis of the B-cell lymphoma 2 (Bcl-2) family mitochondrial apoptosis regulators, we showed that only CgBcl-XL's expression kept at high levels after 42- and 56-day CO2 exposure. CgBcl-XL shared sequence, and structural similarity with its mammalian counterpart, and knockdown of CgBcl-XL in hemocytes via RNA interference promoted apoptosis. The protein level of CgBcl-XL was significantly increased after long-term CO2 exposure (28-56 d), and its distribution in hemocytes became more concentrated and dense. Therefore, CgBcl-XL serves as an essential anti-apoptotic protein for tipping the balance of cell apoptosis, which may play a key role in survival under long-term CO2 exposure. These results reveal a potential adaptation strategy of oysters towards OA and the variable environment changes through the modulation of apoptosis.

Transcriptomic profile of oyster Crassostrea gigas hemocyte after short-term cadmium exposure and bacteria stimulation.

Oyster Crassostrea gigas, is considered as a useful environmental indicator since it is widely distributed along the intertidal zone whereby it tends to accumulate cadmium and is always exposed to various pathogen agents. However, its molecular responses to both cadmium and pathogen stimulation remain unclear. In the present study, transcriptome data of hemocytes from oysters were analyzed to reveal specific molecular responses of oyster to cadmium or cadmium/bacteria stimulation. A total of 21591, 22872 and 20107 genes were detected in the BLANK, Cd24h and Cd/Bac24h group, respectively. Among them, there were 685 differentially expressed genes collected in the comparison of Cd24h versus BLANK. GO analysis of these genes found that sixteen terms into the Molecular Function category displayed transporter activities, and were all over-enrichment by cadmium exposure, whereas twelve terms into Biological Process category involved mainly in metabolic process of the various cellular components and two terms into Cellular Component category were all under-enrichment. The 330 immune responsive genes were shared by two gene lists of CdBac24h versus BLANK and CdBac24h versus Cd24h, and seven out of thirty terms in GO analysis were related to the immune process. Further annotation of these genes from the KEGG database revealed fourteen pathways, including two nervous system related pathways, arachidonic acid pathway, four immune pathways, MAPK cascade and other four cell signaling pathways, and two energy related pathways. Twenty-two differentially expressed genes were identified to responsive to both cadmium exposure and bacteria stimulation, but in different expression patterns, suggesting that bilateral responsive genes, such as alkaline phosphatase and sodium and chloride-dependent glycine transporter gene, could be candidate biomarkers for early warning of cadmium pollution. The present results collectively indicated that a profound neuro-endocrine-immune regulatory network was activated in response to cadmium and bacteria stimulation in oyster C. gigas, and the expression pattern of some cadmium responsive genes may be either reversed or strengthened by bacteria stimulation. The results provide knowledge on the transcriptomic response profile of oyster after short-term cadmium exposure and bacteria stimulation, which would be useful for future studies on stress response mechanism of mollusc, and some cadmium-bacteria responsive genes may be explored as potential biomarkers for monitoring marine pollution.

The activated ß-integrin (CgßV) enhances RGD-binding and phagocytic capabilities of hemocytes in Crassostrea gigas.

Integrins are an important family of cell receptors that can bind foreign particles and promote cell phagocytosis after they are activated. In the present study, a novel ß integrin was identified from pacific oyster Crassostrea gigas with an extracellular domain, a single transmembrane segment, and a short cytoplasmic domain. It was phylogenetically clustered with phagocytosis-related insecta ßV, and designated as CgßV. CgßV shared a conserved NPX[Y/F] motif related to integrin activation with other phagocytosis-related ß integrins. The mRNA transcripts of CgßV were widely detected in oyster tissues including hemocytes, gonad, adductor muscle, mantle, gill, and hepatopancreas, and the expression level in hemocytes was significantly up-regulated at 12 h after lipopolysaccharide (LPS) stimulation (p < 0.05), which was 2.29-fold higher than that in the control group. CgßV proteins were mainly observed on the hemocytes surface. The oyster hemocytes were found to bind fluorescein isothiocyanate (FITC)-labeled Arg-Gly-Asp-containing peptides (RGDCPs), and the binding capability was significantly up-regulated with the peak percentage of 37.6% at 12 h post LPS stimulation, which was higher than that in the control group (8.8%, p < 0.05), suggesting the activation of RGD-binding integrins on oyster hemocytes surface. The label-free RGDCPs and anti-CgßV antibody inhibited the binding capability of hemocytes towards FITC-labeled RGDCPs, which were significant lower in RGD blocking group (7.4%, p < 0.05) and anti-CgßV blocking group (22.1%, p < 0.05) than that in the control group (37.6%), indicating that CgßV could be a RGD-binding integrin. Phagocytosis assay demonstrated that LPS could enhance the phagocytosis of hemocytes towards Escherichia coli and fluorescent beads with the phagocytic rate (PR) of 18.3% and 17.4%, and phagocytic index (PI) of 5.29 and 37.71, respectively, which were significant higher than that in the control group (11.0% and 3.65 for E. coli, 9.8% and 29.26 for fluorescent beads, respectively, p < 0.05). In addition, both the label-free RGDCPs and anti-CgßV antibody significantly hindered the phagocytosis of hemocytes towards E. coli and fluorescent beads. After the E. coli and fluorescent beads were opsonized by oyster serum, the label-free RGDCPs still inhibited the phagocytosis of hemocytes towards them, while the anti-CgßV antibody could only inhibit the phagocytosis of hemocytes towards E. coli, suggesting that only the activated CgßV was involved in the enhancing phagocytosis for bacteria in oyster. Moreover, the key components of conserved integrin-mediated phagocytosis pathway including GTPases, talin proteins, Ca2+ and cAMP were all induced by LPS in hemocytes of oyster. All these results suggested that the activated CgßV enhanced RGD-binding and phagocytic capabilities of hemocytes, shedding lights on the mechanisms of integrin-mediated phagocytosis in mollusks.

The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas.

The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as CgSmac) was identified from oyster Crassostrea gigas. The open reading frame of CgSmac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. CgSmac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that CgSmac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSmac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of CgSmac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of CgSmac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of CgSmac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in dsCgSmac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of CgSmac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in dsCgSmac + LPS group than those of dsEGFP + LPS group. Collectively, CgSmac could activate mitochondrial apoptosis pathway by promoting caspase-3 activity in oyster hemocytes against exogenous LPS invasion. These results provided new insights on oyster apoptosis and the immune defense mechanisms in invertebrates.

CgNrdp1, a conserved negative regulating factor of MyD88-dependent Toll like receptor signaling in oyster Crassostrea gigas.

Toll like receptor (TLR) signaling cascades are under precise regulations to ensure the proper immune responses during various pathogen invasions. The neuregulin receptor degradation protein-1 (Nrdp1) has been demonstrated to be a novel negative regulator of TLR signaling by targeting MyD88 to induce degradation in mammals. In the present study, an Nrdp1 homologue, CgNrdp1, was identified from the genome of Pacific oyster Crassostrea gigas. It contained an open reading frame encoding a polypeptide of 315 amino acids which shared high identities with other homologues from different species. There was a conserved RING domain in CgNrdp1, indicating the functional E3 ubiquitin ligase activity. The bacterially expressed recombinant CgNrdp1 and CgMyD88 showed much stronger affinity compared to control groups in the ELISA assay, showing the interacting ability between CgNrdp1 and CgMyD88. When CgMyD88 or HsMyD88 was co-transfected with CgNrdp1 into HEK293T cells, the luciferase activities of NF-κB were significantly decreased compared to those in MyD88 single-transfection groups, indicating the conserved negative regulating function of CgNrdp1 on the MyD88 induced TLR signaling. These results indicated that CgNrdp1 was a negative regulator of TLR signaling in oyster and the Nrdp1-MyD88 axis was functional and highly conserved from mollusks to mammals in the negative regulation of TLR signaling.

The modulation role of serotonin in Pacific oyster Crassostrea gigas in response to air exposure.

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a critical neurotransmitter in the neuroendocrine-immune regulatory network and involved in regulation of the stress response in vertebrates and invertebrates. In the present study, serotonin was found to be widely distributed in the tissues of Pacific oyster Crassostrea gigas, including haemolymph, gonad, visceral ganglion, mantle, gill, labial palps and hepatopancreas, and its concentration increased significantly in haemolymph and mantle after the oysters were exposed to air for 1 d. The apoptosis rate of haemocytes was significantly declined after the oysters received an injection of extra serotonin, while the activity of superoxide dismutase (SOD) in haemolymph increased significantly. After the stimulation of serotonin during air exposure, the apoptosis rate of oyster haemocytes and the concentration of H2O2 in haemolymph were significantly decreased, while the SOD activity was significantly elevated. Furthermore, the survival rate of oysters from 4th to 6th d after injection of serotonin was higher than that of FSSW group and air exposure group. The results clearly indicated that serotonin could modulate apoptotic effect and redox during air exposure to protect oysters from stress.

A norepinephrine-responsive miRNA directly promotes CgHSP90AA1 expression in oyster haemocytes during desiccation.

Oyster Crassostrea gigas is one model mollusc inhabiting in the intertidal zone and is frequently stressed by desiccation. The adaptation mechanism of oyster to environmental stress involves multiple levels, and miRNA is one of the most important regulators in post-transcriptional level. In the present study, an oyster norepinephrine-responsive miRNA cgi-miR-365 was proved to contribute to the host adaptation against desiccation by directly promoting the expression of CgHSP90AA1. Briefly, a significant increase of cgi-miR-365 was observed from the first day after aerial exposure and the up-regulation was vigorously repressed when oysters were injected with adrenoceptors antagonists. A total of 15 genes involved in biological regulation, metabolic process and response to stimulus were predicted to be modulated by cgi-miR-365. Among these genes, CgHSP90AA1 was up-regulated significantly during desiccation and could be down-regulated after simultaneous injection of adrenoceptors antagonists. The interaction between cgi-miR-365 and CgHSP90AA1 was subsequently verified in vitro, and a significant promotion of CgHSP90AA1 transcripts was observed after overexpressing cgi-miR-365 in either in vitro luciferase reporter assay or primarily cultured haemocytes. Meanwhile, CgHSP90AA1 transcripts decreased in vivo when cgi-miR-365 was repressed by its inhibitor during desiccation. Collectively, it was suggested that cgi-miR-365 could be induced by norepinephrine during desiccation and promote CgHSP90AA1 expression directly after binding to its 3'-UTR, which would provide new evidence in miRNA-mediated adaptation mechanism in oysters against intertidal stress.

The transcriptional response of the Pacific oyster Crassostrea gigas against acute heat stress.

The Pacific oyster, Crassostrea gigas, has evolved sophisticated mechanisms to adapt the changing ambient conditions, and protect themselves from stress-induced injuries. In the present study, the expression profiles of mRNA transcripts in the haemocytes of oysters under heat stress were examined to reveal the possible mechanism of heat stress response. There were 23,315, 23,904, 23,123 and 23,672 transcripts identified in the haemocytes of oysters cultured at 25 °C for 0, 6, 12, and 24 h (designed as B, H6, H12, H24), respectively. And 22,330 differentially expressed transcripts (DTs) were yielded in the pairwise comparisons between the above four samples, which corresponded to 8074 genes. There were 9, 12 and 22 Gene Ontology (GO) terms identified in the DT pairwise comparison groups of H6_B, H12_H6 and H24_H12, respectively, and the richest GO terms in biological process category were cellular catabolic process, translational initiation and apoptotic process, respectively. There were 108, 102 and 102 KEGG pathways successfully retrieved from DTs comparison groups DTH6_B, DTH12_H6 and DTH24_H12, respectively, among which 93 pathways were shared by all three comparison groups, and most of them were related to metabolism of protein, carbohydrate and fat. The expression patterns of 12 representative heat stress response-relevant genes detected by quantitative real-time PCR (qRT-PCR) were similar to those obtained from transcriptome analysis. By flow cytometric analysis, the apoptosis rate of haemocytes increased significantly after oysters were treated at 25 °C for 24 h and recovered at 4 °C for 12 h (p < 0.05) and 36 h (p < 0.01), and it also increased significantly when the heat treatment lasted to 60 h (p < 0.01). The present results indicated that, when oysters encountered short term heat stress, the expression of genes related to energy metabolism, as well as unfolded protein response (UPR) and anti-apoptotic system, were firstly regulated to maintain basic life activities, and then a large number of genes involved in stabilizing protein conformation and facilitating further protein refolding were activated to repair the stress injury. However, the stress injury gradually became irreparable with the stress persisting, and apoptosis was activated when the heat treatment prolonged to 24 h. The information was useful to better understand the molecular mechanism of heat stress response and develop strategies for the improvement of oyster survival rate during summer high-temperature period.

High-Throughput Identification of Antimicrobial Peptides from Amphibious Mudskippers.

Widespread existence of antimicrobial peptides (AMPs) has been reported in various animals with comprehensive biological activities, which is consistent with the important roles of AMPs as the first line of host defense system. However, no big-data-based analysis on AMPs from any fish species is available. In this study, we identified 507 AMP transcripts on the basis of our previously reported genomes and transcriptomes of two representative amphibious mudskippers, Boleophthalmus pectinirostris (BP) and Periophthalmus magnuspinnatus (PM). The former is predominantly aquatic with less time out of water, while the latter is primarily terrestrial with extended periods of time on land. Within these identified AMPs, 449 sequences are novel; 15 were reported in BP previously; 48 are identically overlapped between BP and PM; 94 were validated by mass spectrometry. Moreover, most AMPs presented differential tissue transcription patterns in the two mudskippers. Interestingly, we discovered two AMPs, hemoglobin ß1 and amylin, with high inhibitions on Micrococcus luteus. In conclusion, our high-throughput screening strategy based on genomic and transcriptomic data opens an efficient pathway to discover new antimicrobial peptides for ongoing development of marine drugs.

The inhibitory role of γ-aminobutyric acid (GABA) on immunomodulation of Pacific oyster Crassostrea gigas.

γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter to suppress the immune-mediated pro-inflammatory reactions, and it has been used in the treatment of many inflammation-related diseases in vertebrates, while its immunomodulatory role in invertebrates has never been reported. In the present study, GABA was found to exist in the hemolymph of Pacific oyster Crassostrea gigas, and its concentration decreased slightly from 8.00 ± 0.37 µmol L(-1) at normal condition to 7.73 ± 0.15 µmol L(-1) at 6 h after LPS stimulation, and then increased to 9.34 ± 0.15 µmol L(-1), 8.86 ± 0.68 µmol L(-1) at 12 h and 48 h, respectively. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the protein expression of NOS increased significantly, and these increased trends were remarkably inhibited by GABA stimulation. At the same time, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. The results collectively demonstrated that GABA was an indispensable inhibitory agent for both humoral and cellular immune response, which mainly functioned at the late phase of immune response to avoid the excess immune reactions and maintain the immune homeostasis.

Cgi-miR-92d indirectly regulates TNF expression by targeting CDS region of lipopolysaccharide-induced TNF-α factor 3 (CgLITAF3) in oyster Crassostrea gigas.

Tumor necrosis factor alpha (TNF-α) mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria, during which microRNAs have been found crucial by controlling multiple TNF-α-related genes. In the present study, cgi-miR-92d was annotated as a member of miR-17-92 family and could target the CDS region of lipopolysaccharide (LPS)-induced TNF-α factor (CgLITAF3) in oyster Crassostrea gigas. It was observed that cgi-miR-92d could be vigorously modulated by Vibrio splendidus or LPS stimulation while CgLITAF3 altered oppositely. Two putative binding sites of cgi-miR-92d were then found at CDS region of CgLITAF3. The interaction between cgi-miR-92d and CgLITAF3 was subsequently verified both in vitro and in vivo. As a result, a significant decrease of cellular luminescence was observed in CgLITAF3 luciferase reporter assay when cgi-miR-92d was overexpressed. The luminescent decrease was then recuperated when cgi-miR-92d inhibitor was co-transfected with miRNA mimics. Besides, CgLITAF3 transcripts were significantly down-regulated when cgi-miR-92d was overexpressed in vivo during V. splendidus challenge. Gain-of-function assay of CgLITAF3 was then conducted in HEK293T cells to verify its function. Consequently, a significant increase of TNF-α was observed during the assay. At the meantime, CgTNF was also down-regulated in gain-of-function assay of cgi-miR-92 in vivo, which was a member of TNF superfamily in oysters which could be robustly induced after pathogen stimulation. Together, these results verify the interaction between CgLITAF3 and cgi-miR-92d, which might dedicate crucially in the repaid activation of CgTNF expression during inflammatory response of oysters.

Polymorphism in a serine protease inhibitor gene and its association with the resistance of bay scallop (Argopecten irradians) to Listonella anguillarum challenge.

Serine protease inhibitors (SPIs) play a crucial role in regulation of both host and bacterial serine protease. They are classified into several protein families, where Kazal-type inhibitors are one of families with multi-domain. In the present study, the polymorphism of AiSPI from Bay scallop Argopecten irradians was found to be associated with disease resistance of bay scallop against Listonella anguillarum. Nine single nucleotide polymorphisms (SNPs) were identified in the exon region of AiSPI, where five SNPs were non-synonymous mutation. Three of these mutations were located in "kazal-like 3"domain, two SNP loci positioned at +536, +1312 were selected for further association studies. For the locus +536, the genotype frequency of A/G in the resistant stock (12.8%) was significantly lower (p < 0.05) than that in the susceptible stock (35.1%), while, the genotype A/A in the resistant stock (87.2%) was significantly higher in comparison with susceptible stock (64.9%) (p < 0.05). The G allele frequencies were 6.4% and 17.6% in resistant stock and susceptible stock, respectively, and χ2-test revealed a significant difference in the frequency distribution between the two stocks (p < 0.05). But there was no significant association between the mutation C-T at locus +1312 with either resistant or susceptible group (p > 0.05). The genotype frequencies of T/T, T/C, C/C at locus +1312 were 94.6%, 2.7% and 2.7% respectively in the susceptible stock, while 100%, 0% and 0% respectively in the resistant stock. The amino acid change for the mutation at locus +536 A-G was from asparagine to serine, and the predicted homology model of this amino acid variation could affect its function as well as the structural integrity of the domain. In vitro elastase inhibition assay of the protein variants at locus +536 was conducted to explicate the effect of SNP. The increasing concentration of protein (0 mmol/L- 2.93 mmol/L) was incubated with 80 nmol/L elastase where the residual enzyme activity values for rAiSPI (N) with A variant and rAiSPI (S) with G variant were started to reduce from 0.40 to 0.215 and 0.435 to 0.356, respectively. The elastase inhibition ability of rAiSPI (N) variant was significantly higher than that of rAiSPI (S) (p < 0.01). The results suggested that the mutation at locus +536A/A significantly associated with disease resistance of bay scallop would shed light for selective breeding program.

An oyster species-specific miRNA scaffold42648_5080 modulates haemocyte migration by targeting integrin pathway.

miRNAs are important gene regulators at post-transcriptional level and can modulate diverse biological processes, including immune response. Dozens of species-specific miRNAs have been identified in oyster Crassostrea gigas while their functions remain largely unknown. In the present study, an oyster species-specific miRNA scaffold42648_5080 was found responsive to LPS stimulation and might target a total of 31 oyster genes possibly involved in cell communication, cellular localization and cellular response to stimulus. Besides, in gain-of-function assay of scaffold42648_5080 in vivo, the phagocytosis (30.90% in miRNA group verse 23.20% in miRNA control group), apoptosis (3.10% in miRNA group verse 5.30% in miRNA control group) and migration rate (13.88% in miRNA group verse 21.03% in miRNA control group) of oyster haemocytes were found significantly altered after the injection of scaffold42648_5080 mimics. Among the target genes, integrin-linked kinase (CgILK) was considered crucial in cell migration and its interaction with scaffold42648_5080 was then verified both in vitro and in vivo. Consequently, a significant decrease of relative luciferase ratio was observed in CgILK 3'-UTR luciferase reporter assay after transfection of scaffold42648_5080 mimics (0.70-fold of that in blank group, p < 0.01). Meanwhile, when scaffold42648_5080 was overexpressed in vivo (5.41-fold of miRNA control group, p < 0.01), the expression of CgILK declined significantly to 0.25-fold of miRNA control group (p < 0.01). Comparatively, a significant decrease of the haemocyte migration rate (19.76% verse 34.82% in siEGFP control group, p < 0.01) was observed after knock-down of CgILK in vivo. The present study, as far as we know, for the first time revealed the immunomodulation role of an oyster species-specific miRNA, which might provide new insights into miRNA-mediated adaptation mechanism of oysters.

CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca2+ increased significantly (p < 0.05). But, this increasing of Ca2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response.

[The Application of X-Ray Photoelectron Spectroscopy on Refining Catalyst].

Abstract XPS analysis provides qualitative, quantitative and chemical state information for surface elements of solid materials. Therefore, XPS is widely applied in the characterization of refining catalyst. In the present paper, the applications of XPS in the field of typical refining catalysts, including hydrogenation catalyst, S Zorb sorbent and rare-earth modified Y zeolite, are illustrated and exemplified. For sulfided Co (Ni)-Mo (W)/Al2O3(-SiO2) hydrodesulfurization catalysts, the anhydrous oxygen-free transfer process from the reactor to XPS chamber was illustrated. The identification and peak fitting of S(2p) , Mo(3d), W (4f), Co(2p) and Ni(2p) XPS spectra were summarized. The typical chemical states of the active elements were described. Based on these results, the sulfidation extents of the active metals and the cause for the sulfidation inadequency of the catalysts were deduced. As for the application of XPS in S Zorb sorbent, the existence form of zinc was obtained from ZnLMM Auger spectra, and the fracture mechanism and deactivation reason of the sorbent were derived. The distribution of sulfur along the vertical direction was investigated using XPS and argon ion sputtering XPS. Besides, in situ XPS was applied to study the conversion of sulfur- and nickel-containing species for spent sorbent under hydrogen condition. Finally, for cerium modified Y zeolite, the location of cerium ion inside and outside Y zeolite cage was investigated. The results indicate that the liquid phase method is more suitable for the migration of cerium ion toward zeolite as compared with the solid phase method.

Hemocytic immune responses triggered by CpG ODNs in shrimp Litopenaeus vannamei.

CpG oligodeoxynucleotides (CpG ODNs), also called bacterial DNA or synthetic oligodeoxynucleotides, can induce apparent immunity protection against various pathogens, and they are widely used as functional immunostimulant or vaccine adjuvant in mammals. In the present study, CpG-rich plasmid pUC57-CpG was constructed and employed to stimulate the shrimp Litopenaeus vannamei, and the total hemocyte count, percentage of apoptotic hemocytes, regeneration of circulating hemocytes, the ability of phagocytosis and generation of reactive oxygen species (ROS) were measured to reveal the possible protection mechanism of CpG ODNs. After the injection of pUC57-CpG, the total hemocyte count significantly decreased (p < 0.01) to 2.56 × 10(7) cell/mL at the first day post stimulation, while the apoptosis increased (p < 0.01), which was 1.72-fold of that in control group. At the same time, the regeneration of circulating hemocytes fluctuated in a similar trend, and a significant increase was observed at the first day post stimulation. The phagocytotic activity including the percentage of phagocytosis and phagocytotic index, experienced an upward tend during the whole experimental period and the ROS level increased by 22% (p < 0.05) compared to that in the control group at first day post stimulation. These results together suggested that pUC57-CpG could promote the apoptosis and regeneration of circulating hemocytes, and enhance the phagocytosis and ROS production, which might contribute to the boosted immunity against the infection of pathogens.

The expression of immune-related genes during the ontogenesis of scallop Chlamys farreri and their response to bacterial challenge.

In the present study, the expression of some immune-related genes was examined as indicator to understand the development of immune defense system during the ontogenesis of scallop Chlamys farreri. The mRNA transcripts of pattern-recognition receptors (PRRs) including CfPGRP-S1, CfLGBP, CfLec-1 and CfLec-3 were observed at a low level or even undetected at early developmental stages from eggs to blastula, and then began to increase overwhelmingly in trochophore. For the genes of immune effector including CfLYZ, CfLBP/BPI, CfSOD and CfCAT, their mRNA transcripts were higher expressed in embryos, and increased significantly in D-hinged or early veliger larvae. The whole-mount immunofluorescence assay revealed two immunoreactive spots of CfPGRP-S1 were first observed in the mid-ventral region of prototroch in trochophore, and the immunopositive fluorescence of CfLGBP, CfLec-1 and CfLec-3 appeared at the same spots in early D-hinged larvae. Most of the PRRs were located in velum, mouth, esophagus and stomach region in early and mid-veliger larvae, and especially the strong immunopositive fluorescence of CfLec-3 was observed in velum. The immunoreactive areas of CfLYZ, CfLBP/BPI, CfSOD and CfCAT were observed in trochophore and early D-hinged larvae. After D-hinged larvae, they distributed in different tissues from the edge of velum, mouth, esophagus to the region around digestive gland. After bacterial challenge, the mRNA expression of CfLGBP, CfLec-1 and CfLec-3 did not change significantly in trochophore, while a down-regulation of CfPGRP-S1 was observed at 6 h (P < 0.05). The expression of CfPGRP-S1 and CfLGBP decreased or increased inversely in D-hinged and late veliger larvae respectively, whereas CfLec-1 and CfLec-3 increased significantly during 6-24 h after bacterial challenge in the two stages (P < 0.05). In contrast, the expressions of immune effectors in trochophore and late veliger larvae were significant up-regulated at 6 h, 12 h or 24 h after bacterial challenge (P < 0.05). However, in late D-hinged larvae, CfLYZ and CfSOD expressions were significantly down-regulated at 6 h, while CfLBP/BPI expression was up-regulated at 6 h and 24 h post challenge (P < 0.05). These results indicated that the immune defense system of scallop might appear firstly in the mid-ventral region of prototroch in trochophore, and developed maturely after late D-hinged larvae. The developing immune system in the D-hinged and late veliger larvae could respond to the immune stimulation in different manner.

The CpG ODNs enriched diets enhance the immuno-protection efficiency and growth rate of Chinese mitten crab, Eriocheir sinensis.

CpG oligodeoxynucleotides (ODNs), the well-known vaccine adjuvant in mammals, have been proved to mount innate immune responses in crustaceans. In the present study, CpG ODNs was employed as supplements in diets to fed crab Eriocheir sinensis, and the changes of immune parameters as well as weight gain were investigated to evaluate its possible application in crab farming. After the crabs were fed with 40 mg/kg and 100 mg/kg CpG ODNs containing diets (designated as C40 and C100 group) for four weeks, the lysozyme activities were significantly enhanced (p < 0.01) in both groups, while the catalase activity was only increased (p < 0.01) in the C40 group. When those crabs were subsequently challenged with Aeromonas hydrophila, the cumulative mortalities in C40 and C100 groups were declined by 10.4% and 10.8% (p < 0.05) compared with that of control group, respectively. Interestingly, the final weights of crabs were increased after four weeks' feeding of CpG ODNs, and the percentage of weight gain in C40 group reached 124.5 ± 14.2%, which was significantly higher (p < 0.05) than that of control group (78.1 ± 19.2%) and C100 group (107.3 ± 28.2%). The uptake of CpG ODNs by haemocytes and the possible mechanism of CpG ODNs to active the immune response were investigated by using the laser scanning confocal microscope. CpG ODNs (labeled with 5'-end-FAM) could be internalized by the haemocytes after incubation of 20 min, with strong signals detected at the cell membrane and in the cytoplasm. In the cytoplasm, most of the CpG ODNs were localized in lysosome, and some of them escaped from the lysosomal compartments and aggregated around the nuclear. The results clearly demonstrated that CpG ODNs could be internalized directly by crab haemocytes and mostly located in the late endosome. The enhancements of immuno-protection efficiency and growth rate from CpG ODNs as supplements in diets might depend on the uptaking and locating processes, and they could be used as a potential immunostimulant for the crab aquaculture.

A tailless Dscam from Eriocheir sinensis diversified by alternative splicing.

Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF), plays an essential role in pathogen recognition and further involves in the innate defense of invertebrates. In the present study, the cDNA of a Dscam from Chinese mitten crab Eriocheir sinensis (designated EsDscam) was cloned and characterized. It contained a 5-terminal untranslated region (UTR) of 60 bp, a 3-UTR of 216 bp with a poly (A) tail, and an open reading frame (ORF) of 4848 bp encoding a polypeptide of 1615 amino acids with the putative molecular mass of 178.4 kDa and theoretical isoelectric point of 6.31. The EsDscam protein shared higher sequence identities and similar domain architecture with Dscams from other invertebrate, including typical 10 immunoglobulin (Ig) domains, 6 fibronectin type 3 domains (FNIII) and one cell attachment sequence (RGD) in extracellular region, while it lacked the expected transmembrane domain and cytoplasmic tail compared with other members of Dscam family. After sequencing 80 separate clones of Ig2, 3 and Ig7 regions from pooled cDNA libraries constructed from normal and bacterial-infected crabs, 44 alternative sequences were detected in the N-terminal of Ig2, 39 ones in Ig3, and 31 ones in Ig7 domain, suggesting that EsDscam could potentially encode at least 53196 unique isoforms. Furthermore, two 3'UTR isoforms and two 5'UTR isoforms of EsDscam were also identified by RACE strategy. EsDscam mRNA was most abundantly expressed in the tissues of nerve, muscle, hepatopancreas and gill, and weakly expressed in heart, gonad and hemocytes. Western blotting and immunofluorescence analysis revealed that EsDscam protein was mainly distributed in serum, and few on the membrane of crab hemocytes. These results suggested that this tailless EsDscam was one member of crustacean Dscam family, and the generation of diverse isoforms through alternative splicing allowed it to recognize various pathogens and play an active role in immune defense of crabs.