Norepinephrine regulates TNF expression via the A1AR-p38 MAPK-Relish pathway in granulocytes of oyster Crassostrea gigas.

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway.

Differential bioaccumulation and tolerances of massive and branching scleractinian corals to polycyclic aromatic hydrocarbons in situ.

Scleractinian corals are capable of accumulating polycyclic aromatic hydrocarbons (PAHs) in reef environments; however, the mechanism behind their PAHs tolerance is unknown. This study investigated the occurrence and bioaccumulation of PAHs in coral reef ecosystems and examined the physiological responses induced by PAHs in coral hosts and their algal symbionts, the massive coral Galaxea fascicularis and branching coral Pocillopora damicornis. G. fascicularis had a higher PAHs accumulation capacity than P. damicornis. Both the coral hosts and algal symbionts preferentially accumulated acenaphthene, dibenzo(a,h)anthracene, and benzo(a)pyrene. The accumulated PAHs by G. fascicularis and P. damicornis hosts was accompanied by a reduction in detoxification ability. The accumulated PAHs could induce oxidative stress in P. damicorni hosts, thus G. fascicularis demonstrated a greater tolerance to PAHs compared to P. damicornis. Meanwhile, their algal symbionts had fewer physiological responses to accumulated PAHs than the coral hosts. Negative effects were not observed with benzo(a)pyrene. Taken together, these results suggest massive and branching scleractinian corals have different PAHs bioaccumulation and tolerance mechanisms, and indicate that long-term PAHs pollution could cause significant alterations of community structures in coral reef ecosystems.

Identification of Novel, Selective Ataxia-Telangiectasia Mutated Kinase Inhibitors with the Ability to Penetrate the Blood-Brain Barrier: The Discovery of AZD1390.

The inhibition of ataxia-telangiectasia mutated (ATM) has been shown to chemo- and radio-sensitize human glioma cells in vitro and therefore might provide an exciting new paradigm in the treatment of glioblastoma multiforme (GBM). The effective treatment of GBM will likely require a compound with the potential to efficiently cross the blood-brain barrier (BBB). Starting from clinical candidate AZD0156, 4, we investigated the imidazoquinolin-2-one scaffold with the goal of improving likely CNS exposure in humans. Strategies aimed at reducing hydrogen bonding, basicity, and flexibility of the molecule were explored alongside modulating lipophilicity. These studies identified compound 24 (AZD1390) as an exceptionally potent and selective inhibitor of ATM with a good preclinical pharmacokinetic profile. 24 showed an absence of human transporter efflux in MDCKII-MDR1-BCRP studies (efflux ratio <2), significant BBB penetrance in nonhuman primate PET studies (Kp,uu 0.33) and was deemed suitable for development as a clinical candidate to explore the radiosensitizing effects of ATM in intracranial malignancies.

Selective enrichment of bacteria and antibiotic resistance genes in microplastic biofilms and their potential hazards in coral reef ecosystems.

Microplastics become hotspots for bacteria to trigger a series of ecological effects, but few studies have focused on the potential impacts of microplastic biofilms in coral reef ecosystems. Here, we measured the bacterial communities and antibiotic resistance genes (ARGs) in the seawater and microplastic biofilms. Results showed that microbial biofilms were formed on the surface of microplastics. The alpha diversity of the bacterial community in the microplastic biofilms was lower than that in the seawater, and the bacterial communities were distinct between the two. Further analysis revealed that several bacteria in the microplastic biofilms carried ARGs, and the proportion of which was correlated to the concentration of antibiotics in the seawater. Specifically, Vibrio was positively correlated to sul1 in the microplastic biofilms under higher concentrations of sulfonamides. Pathway analysis reflected significant overrepresentation of human disease related pathways in the bacterial community of microplastic biofilms. These results suggest that the microplastic biofilms could selectively enrich bacteria from the reef environments, causing the development of ARGs under antibiotic driving. This may pose a serious threat to coral reef ecosystems and human health. Our study provides new insights into the ecological impacts of microplastic biofilms in coral reef ecosystems.

Chromosome-level genome assembly of the deep-sea snail Phymorhynchus buccinoides provides insights into the adaptation to the cold seep habitat.

BACKGROUND: The deep-sea snail Phymorhynchus buccinoides belongs to the genus Phymorhynchus (Neogastropoda: Raphitomidae), and it is a dominant specie in the cold seep habitat. As the environment of the cold seep is characterized by darkness, hypoxia and high concentrations of toxic substances such as hydrogen sulfide (H2S), exploration of the diverse fauna living around cold seeps will help to uncover the adaptive mechanisms to this unique habitat. In the present study, a chromosome-level genome of P. buccinoides was constructed and a series of genomic and transcriptomic analyses were conducted to explore its molecular adaptation mechanisms to the cold seep environments. RESULTS: The assembled genome size of the P. buccinoides was approximately 2.1 Gb, which is larger than most of the reported snail genomes, possibly due to the high proportion of repetitive elements. About 92.0% of the assembled base pairs of contigs were anchored to 34 pseudo-chromosomes with a scaffold N50 size of 60.0 Mb. Compared with relative specie in the shallow water, the glutamate regulative and related genes were expanded in P. buccinoides, which contributes to the acclimation to hypoxia and coldness. Besides, the relatively high mRNA expression levels of the olfactory/chemosensory genes in osphradium indicate that P. buccinoides might have evolved a highly developed and sensitive olfactory organ for its orientation and predation. Moreover, the genome and transcriptome analyses demonstrate that P. buccinoides has evolved a sulfite-tolerance mechanism by performing H2S detoxification. Many genes involved in H2S detoxification were highly expressed in ctenidium and hepatopancreas, suggesting that these tissues might be critical for H2S detoxification and sulfite tolerance. CONCLUSIONS: In summary, our report of this chromosome-level deep-sea snail genome provides a comprehensive genomic basis for the understanding of the adaptation strategy of P. buccinoides to the extreme environment at the deep-sea cold seeps.

A bone morphogenetic protein regulates the shell formation of Crassostrea gigas under ocean acidification.

Bone morphogenetic proteins (BMPs) are key factors controlling osteoblast differentiation, which have been proved to be involved in the hard tissue formation of marine mollusks. In the present study, a member of BMPs gene (CgBMP7) was identified from Pacific oyster Crassostrea gigas (C. gigas) with the aim to understand its possible role in the regulation of shell formation under ocean acidification (OA) conditions. The open reading frame (ORF) of CgBMP7 was of 1254 bp encoding a polypeptide of 417 amino acids. The deduced amino acid sequence of CgBMP7 was comprised of one signal peptide, one prodomain and one TGF-ß domain, which shared 21.69%-61.10% identities with those from other species. The mRNA transcript of CgBMP7 was ubiquitously expressed in all the tested tissues of adult oysters with a higher expression level in mantle, notably highest in the middle fold (MF) of the three folds of mantle. The expression level of bone morphogenetic protein type I receptor (CgBMPR1B) mRNA was also highest in the MF and up-regulated dramatically post recombinant BMP7 protein (rCgBMP7) stimulation. After the blockage of BMPR1B with inhibitor LDN193189 (LDN), the mRNA expression level and phosphorylation level of CgSmad1/5/8 in mantle were decreased, and the mRNA expression levels of CgCaM and Cgengrailed-1 were down-regulated significantly. During the oysters were exposed to acidified seawater for weeks, the expression levels of CgBMP7, CgBMPR1B and CgSmad1/5/8 in the MF decreased significantly (p < 0.01) at the 4th week, and CgCaM and Cgengrailed-1 also exhibited the same variable expression patterns as CgBMP7. In addition, the growth of shell in the treatment group (pH 7.8) was slower than that in the control group (pH 8.1). These results collectively indicated that BMP7 was able to trigger the BMPR-Smad signaling pathway and involved in controlling the formation of oyster calcified shell under OA conditions.

Acute hypoxia induces reduction of algal symbiont density and suppression of energy metabolism in the scleractinian coral Pocillopora damicornis.

Loss of oxygen in the ocean is accelerating and threatening the coral reef ecosystem. In this study, the impacts of hypoxia on the scleractinian coral Pocillopora damicornis were explored. The algal symbiont density, chlorophyll a + c2 content, energy consumption of corals, as well as energy available and consumption of their symbionts, decreased significantly post hypoxia stress. Meanwhile, the malondialdehyde contents in corals and symbionts, together with the caspase-3 activation level in corals, increased significantly in response to hypoxia stress. Furthermore, it was revealed that activities such as coral cell division and calcification were inhibited under hypoxia. These results collectively suggest that acute hypoxia stress reduces symbiont density and chlorophyll a + c2 content in the coral P. damicornis by elevating intracellular oxidative pressure and apoptotic level, which further suppresses energy metabolism in the symbiotic association and negatively affects a series of activities such as coral cell division and calcification.

The scleractinian coral Pocillopora damicornis relies on neuroendocrine regulation to cope with polycyclic aromatic hydrocarbons under heat stress.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic environmental pollutants and are threatening scleractinian corals. In this study, PAHs treatment did not induce significant physiological responses of the coral Pocillopora damicornis and its algal symbionts, but biological processes including response to toxin, drug metabolic, and oxidation reduction were triggered at the mRNA level. These results implied that PAHs could be a group of slow-acting environmental toxicants, whose effects were moderate but persistent. Besides, it was interesting to find that PAHs activated the neuroendocrine system in the coral by triggering the expression of monoaminergic and acetylcholinergic system related genes, indicating that PAHs might function as environmental hormones. Moreover, the combined treatments of PAHs and heat caused a much obvious effect on the coral and its algal symbionts by elevating antioxidant activity and suppressing photosynthesis in the symbionts. Results from the transcriptome data further indicated that corals might perform stress responses upon PAHs and heat challenges through the TNF and apoptosis pathways, which perhaps was modulated by the neuroendocrine system of corals. Collectively, our survey demonstrates that the PAHs can function as environmental hormones and activate the neuroendocrine regulation in scleractinian corals, which may contribute to the stress responses of symbiotic association by modulating photosynthesis, antioxidation, and apoptosis.

RGD-Labeled Hemocytes With High Migration Activity Display a Potential Immunomodulatory Role in the Pacific Oyster Crassostrea gigas.

Immunocyte migration to infection sites is important for host cellular defense, but the main types of migrating hemocytes and their mechanisms against pathogen invasions are unclear in invertebrates. In the present study, a population of hemocytes in the Pacific oyster Crassostrea gigas labeled with a fluorescein isothiocyanate (FITC)-conjugated Arg-Gly-Asp (RGD)-containing peptide was sorted. RGD+ hemocytes were characterized by a smaller cell size and cytoplasmic-nucleo ratio, fewer cytoplasmic granules, and higher levels of myeloperoxidase, reactive oxygen species, and intracellular free calcium concentration. RGD+ hemocytes exhibited a high level of migration activity, which was further induced after V. splendidus infection. Transcriptome analysis revealed that RGD+ hemocytes highly expressed a series of migration-related genes, which together with migration-promoting genes were significantly upregulated after V. splendidus infection. The neuroendocrine system was also proven to regulate the migration activity of RGD+ hemocytes, especially with the excitatory neuroendocrine factor dopamine, which promoted migration activity as confirmed by receptor blocking assays. Meanwhile, RGD+ hemocytes could highly express immunomodulatory factor interleukin (IL)-17s and their receptor genes, which was positively related to the production of antimicrobial peptides in whole hemocytes after V. splendidus infection. Collectively, this study identified a specific hemocyte population, i.e., RGD+ hemocytes, that shows high migration activity in response to pathogen infection and exerts a potential immunomodulatory role by highly expressing IL-17s that might enhance the hemocytes' antimicrobial peptide production in oysters.

Cortisol modulates glucose metabolism and oxidative response after acute high temperature stress in Pacific oyster Crassostrea gigas.

Cortisol is the main stress hormone that plays crucial roles in energy metabolism and immune response in vertebrates. In the present study, the homologues of 11ß-hydroxysteroid dehydrogenase type 1 (designated Cg11ß-HSD1) and 5α-reductase 1 (designated Cg5αR1), the key enzymes related to cortisol metabolism, were identified from Pacific oyster Crassostrea gigas. The Cg11ß-HSD1 harbored a conserved SDR domain, and Cg5αR1 contained a Steroid_dh domain and three transmembrane domains. The mRNA transcripts of Cg11ß-HSD1 and Cg5αR1 were constitutively expressed in all the examined tissues of oysters, with the highest expression level in haemocytes and labial palp, respectively. After acute high temperature stress (28 °C), the mRNA expression level of Cg11ß-HSD1 in hepatopancreas significantly up-regulated at 6 h and 12 h, and that of Cg5αR1 significantly up-regulated at 6 h, compared with the Blank group (11 °C). The concentration of cortisol and glucose, as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in hepatopancreas all significantly up-regulated after acute high temperature stress, while the glycogen concentration in adductor muscle decreased significantly at 6 h and 12 h. After the blockage of Cg11ß-HSD1 with metyrapone, the cortisol concentration and the activities of SOD and CAT significantly decreased after acute high temperature stress, the glucose concentration in hepatopancreas significantly increased at 24 h, and the glycogen concentration in adductor muscle significantly increased at 6 h. These results collectively suggested that cortisol played a crucial role in regulating glucose metabolism and oxidative response in oysters upon acute high temperature stress.

Dynamics of phytoplankton community in scallop farming waters of the Bohai Sea and North Yellow Sea in China.

BACKGROUND: As the major suppliers of food for higher consumers, phytoplankton are closely related to the yield, nutritional ingredients and even toxin contents of mariculture animals, potentially influencing the human health when they are consumed. With the increase of shellfish culture density, phytoplankton in the coastal waters have been excessively consumed in recent years, and the nutrients they depend on are becoming more and more limited, which severely restrict the shellfish mariculture and threaten the coastal ecosystems. RESULTS: In the present study, nutrient concentrations, phytoplankton assemblages and scallop growth status were investigated in the main bay scallop farming waters of the Bohai Sea (Qinhuangdao site) and North Yellow Sea (Zhuanghe site) in 2018. Both phosphate and silicate limitations were observed at the two sites, with the major determinant of phytoplankton assemblages being silicate in Qinhuangdao and phosphate in Zhuanghe, respectively. The phytoplankton assemblages at the two sites displayed different community structures and succession patterns. The phytoplankton community was dominated by dinoflagellates and diatoms in Qinhuangdao, while dinoflagellates were the most abundant group in Zhuanghe, which accounted for 41.9% of the total phytoplankton abundance. The dominant genera of diatoms in Qinhuangdao were Skeletonema, Thalassiosira and Leptocylindrus, while those in Zhuanghe were Thalassiosira and Cyclotella. Greater biomass and more appropriate structure of phytoplankton contributed to higher growth rate and glycogen content of cultured bay scallops. CONCLUSIONS: Our study characterized the relationship between nutrient concentration, phytoplankton community and scallop mariculture in the main bay scallop farming waters in northern China. The results suggest that, as nutrient limitation intensified, dinoflagellates are becoming the dominant phytoplankton species in the scallop farming waters of the Bohai Sea and the North Yellow Sea, which is harmful to the coastal mariculture.

Discovery of a Potent and Selective ATAD2 Bromodomain Inhibitor with Antiproliferative Activity in Breast Cancer Models.

ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.

Unraveling the metabolic effects of benzophenone-3 on the endosymbiotic dinoflagellate Cladocopium goreaui.

As a well-known pseudo-persistent environmental pollutant, oxybenzone (BP-3) and its related organic ultraviolet (UV) filters have been verified to directly contribute to the increasing mortality rate of coral reefs. Previous studies have revealed the potential role of symbiotic Symbiodiniaceae in protecting corals from the toxic effects of UV filters. However, the detailed protection mechanism(s) have not been explained. Here, the impacts of BP-3 on the symbiotic Symbiodiniaceae Cladocopium goreaui were explored. C. goreaui cells exhibited distinct cell growth at different BP-3 doses, with increasing growth at the lower concentration (2 mg L-1) and rapid death at a higher concentration (20 mg L-1). Furthermore, C. goreaui cells showed a significant BP-3 uptake at the lower BP-3 concentration. BP-3 absorbing cells exhibited elevated photosynthetic efficiency, and decreased cellular carbon and nitrogen contents. Besides, the derivatives of BP-3 and aromatic amino acid metabolism highly responded to BP-3 absorption and biodegradation. Our physiological and metabolic results reveal that the symbiotic Symbiodiniaceae could resist the toxicity of a range of BP-3 through promoting cell division, photosynthesis, and reprogramming amino acid metabolism. This study provides novel insights into the influences of organic UV filters to coral reef ecosystems, which urgently needs increasing attention and management.

A myxovirus resistance like protein involved in CgIFNLP mediated immune response of oyster Crassostrea gigas.

The myxovirus resistance (Mx) proteins belong to interferon (IFN)-induced dynamin GTPase and play a pivotal role in the inhibition of replication of numerous viruses. In the present study, an Mx homologue (designated as CgMx1) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgMx1 cDNA was of 1689 bp encoding a peptide of 562 amino acid residues. There was an N-terminal dynamin GTPase domain in the predicted peptide, which consisted of a tripartite GTP-binding motif (GDXXSGKS, DLPG and T/NKXD). The deduced amino acid sequence of CgMx1 shared 30-39% similarity with other Mx family members. And CgMx1 was clustered with Mx from H. discus, and then assigned into the invertebrate branch of the phylogenetic tree. The mRNA transcripts of CgMx1 were constitutively distributed in all the tested tissues, with the highest level in haemocytes (1342.45-fold of labial palps, p < 0.05). The mRNA expression of CgMx1 in haemocytes was significantly up-regulated to the highest level at 6 h (13.14-fold, p < 0.001) after poly (I:C) treatment and at 24 h (66.28-fold, p < 0.001) after recombinant IFN-like protein (rCgIFNLP) stimulation, respectively. CgMx1 protein was found to distribute in both the cytoplasm and nucleus of haemocytes. In the oysters with CgIFNLP and signal transducer and activator of transcription (CgSTAT) silenced by RNAi, the mRNA expression of CgMx1 decreased significantly in the haemocytes at 12 h after poly (I:C) stimulation, which was 0.02-fold and 0.04-fold of that in EGFP-RNAi oysters (p < 0.001), respectively. Meanwhile, EMSA assay revealed that CgSTAT was able to transactivate CgMx1 promoter through directly binding to its interferon-stimulated response element (ISRE) and gamma interferon activation site (GAS). The above results indicated that CgMx1 participated in the immune response of C. gigas through the signal pathway mediated by CgIFNLP and CgSTAT.

A novel CgIFNLP receptor involved in regulating ISG expression in oyster Crassostrea gigas.

Interferons (IFNs) are the key coordinators of antiviral immunity by binding to their receptors to orchestrate a complex transcriptional network in vertebrates. Recently, the existence of molluscan IFN-like system has been certified by the identification of important components in IFN system, such as IFN-like protein (CgIFNLP) from oyster Crassostrea gigas. In the present study, a novel CgIFNLP receptor (designed CgIFNLPR-1) was identified from C. gigas. The open reading frame (ORF) of CgIFNLPR-1 cDNA was of 1962 bp encoding a peptide of 653 amino acid residues with five fibronectin type III (FNIII) domains and one transmembrane helix region. The mRNA transcripts of CgIFNLPR-1 were constitutively distributed in all the tested tissues, with the highest level in gonad. After Poly (I:C) stimulation, the mRNA expression of CgIFNLPR-1 in haemocytes was significantly up-regulated to the highest level at 48 h (4.54-fold of that in control group, p < 0.05). CgIFNLPR-1 protein was mainly distributed in the cytoplasm and membrane of oyster haemocytes. CgIFNLP and CgIFNLPR-1 were able to interact with each other in vitro. After the CgIFNLPR-1 was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (ISGs), including CgMx, CgViperin and CgIFNIP-44, were significantly inhibited after Poly (I:C) stimulation, which was 0.17, 0.31 and 0.53-fold of that in EGFP group, respectively (p < 0.01). These findings suggested that CgIFNLPR-1 was a novel CgIFNLP receptor in the oyster to recognize CgIFNLP and regulate the expressions of CgISGs.

The cGAS/STING-TBK1-IRF Regulatory Axis Orchestrates a Primitive Interferon-Like Antiviral Mechanism in Oyster.

Interferon (IFN) system is considered as the first defense line against viral infection, and it has been extensively studied in vertebrates from fish to mammals. In invertebrates, Vagos from arthropod and IFN-like protein (CgIFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the CgIFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After CgIFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (CgISGs) was significantly inhibited. Both cyclic GMP-AMP synthase (CgcGAS) and stimulator of interferon gene (CgSTING) identified from oyster were able to recognize the double-stranded nucleic acid [Poly (I:C) and dsDNA] and expressed at high level after Poly (I:C) stimulation. The expression of CgIFNLP and interferon regulatory factors (CgIRF1/8) and the nuclear translocation of CgIRF8 were all suppressed in CgcGAS-RNAi or CgSTING-RNAi oysters after Poly (I:C) stimulation. The expression level of CgSTING and TANK binding kinase1 (CgTBK1) did not decrease in CgcGAS-RNAi oysters. After CgSTING was knocked down, the high expression of CgTBK1 induced by Poly (I:C) was prevented significantly. These results indicated that there was a primitive IFN-like antiviral mechanism dependent on the cGAS/STING-TBK1-IRFs regulatory axis in mollusks, which was different from the classic cGAS-STING-TBK1 signal pathway in mammals.

A tripartite motif protein (CgTRIM1) involved in CgIFNLP mediated antiviral immunity in the Pacific oyster Crassostrea gigas.

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases involved in many biological processes, such as inflammation and antiviral immunity. In the present study, a novel TRIM protein homolog named CgTRIM1 was identified from Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgTRIM1 was of 1914 bp encoding a putative polypeptide of 637 amino acid residues. There were three classical domains in the predicted CgTRIM1 protein, including one RING domain, two b-box domains and one coiled-coil domain in N-terminal. For the lack of C-terminal domains, the CgTRIM1 was classified as the member of C-V TRIM subfamily. The mRNA transcripts of CgTRIM1 were detected in all the tested tissues and haemocytes, with the highest expression level in gill. The mRNA and protein levels of CgTRIM1 in gill were significantly up-regulated at 6 h after poly (I:C) stimulation. Moreover, the nuclear translocation of CgTRIM1 was observed in haemocytes of oysters after poly (I:C) stimulation. After IFN-like protein (CgIFNLP) was knocked down by RNA interference (RNAi), the expression of CgTRIM1 in gill was markedly inhibited in both mRNA (0.14-fold, p < 0.001) and protein levels after poly (I:C) stimulation. Furthermore, after knocking down of CgTRIM1, the mRNA expression levels of IFN-stimulated genes, including myxovirus resistance of oyster (CgMx) and Interferon-induced protein 44 (CgIFI44) were significantly down-regulated post poly (I:C) stimulation, while no significant change of the CgIFNLP expression was observed. These results indicated that CgTRIM1 participated in the antiviral response of C. gigas by regulating the mRNA expressions of IFN-stimulated genes.

A hexokinase from the oyster Crassostrea gigas is involved in immune recognition as a pattern recognition receptor.

Hexokinase (HK) is generally recognized as a crucial enzyme participating in glycolysis. In the present study, a HK (named CgHK) was identified as a potential pattern recognition receptor (PRR) from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgHK was of 1395 bp, encoding a peptide of 464 amino acids with one Hexokinase_1 domain and one Hexokinase_2 domain. The predicted amino acid sequence of CgHK shared 17%-29% similarities with that of other known HKs. The mRNA transcripts of CgHK were constitutively detected in all the examined tissues, with relative high expression level in labial palp and haemocytes. CgHK protein was mainly observed in the cytoplasm of oyster haemocytes. The mRNA expression level of CgHK in haemocytes was significantly up-regulated and peaked at 3 h after Vibrio splendidus (7.64-fold, p < 0.001) and lipopolysaccharide (LPS) (11.86-fold, p < 0.001) stimulations. The recombinant CgHK protein (rCgHK) exhibited Mg2+-dependent adenosine triphosphate (ATP) binding activity in vitro and activity to bind D-(+)-glucose (GLU) and various pathogen-associated molecular pattern (PAMPs) such as LPS and peptidoglycan (PGN) in the absence of Mg2+. It also displayed higher binding activity towards V. splendidus and relatively lower binding activity towards Staphylococcus aureus, Escherichia coli, and Micrococcus luteus. After the mRNA expression of CgHK in haemocytes was knocked down by dsRNA interference, the expression of CgIL17-5 mRNA in haemocytes was considerably down-regulated at 3 h after the stimulation with V. splendidus (0.33-fold, p < 0.001). These results collectively indicated that CgHK was able to recognize various PAMPs and pathogenic bacteria as a PRR apart from being the enzyme to exert ATP binding activity in glycolysis, and activate the anti-bacterial immune response by promoting the expression of pro-inflammatory cytokines CgIL17-5 in oyster haemocytes.

The glutaminase (CgGLS-1) mediates anti-bacterial immunity by prompting cytokine synthesis and hemocyte apoptosis in Pacific oyster Crassostrea gigas.

Glutaminase, an amidohydrolase enzyme that hydrolyzes glutamine to glutamate, plays crucial roles in various immunomodulatory processes such as cell apoptosis, proliferation, migration, and secretion of cytokines. In the present study, a glutaminase homologue (designated as CgGLS-1) was identified from Pacific oyster Crassostrea gigas, whose open reading frame was of 1836 bp. CgGLS-1 exhibited high sequence identity with vertebrate kidney-type GLS, and closely clustered with their homologues from mollusc C. virginica. The enzyme activity of recombinant CgGLS-1 protein (rCgGLS-1) was estimated to be 1.705 U/mg. CgGLS-1 mRNA was constitutively expressed in all the tested tissues of oysters, with the highest expression level in hemocytes. CgGLS-1 mRNA expression in hemocytes was significantly up-regulated and peaked at 6 h (2.07-fold, p < 0.01) after lipopolysaccharide (LPS) stimulation. The CgGLS-1 protein was mainly distributed in the cytoplasm with a significant co-location with mitochondria in oyster hemocytes. The content of Glu in the oyster serum was significantly decreased after the inhibition of CgGLS-1 using specific inhibitor Bis-2- [5-(phenyl acetamido)-1,3,4-thiadiazol-2-yl] ethyl sulfide (BPTES), and the expression levels of CgmGluR6, CgAP-1, cytokines CgIL17-5 and CgTNF-1 were significantly decreased after BPTES and LPS stimulation. The transcripts of CgCaspase3 as well as the apoptosis index of hemocytes were also decreased. These results collectively suggest that CgGLS-1 is the enzyme to synthesize Glu in oyster, which can modulate anti-bacterial immunity by regulating the secretion of pro-inflammatory cytokines CgIL17-5 and CgTNF-1, as well as hemocyte apoptosis.

A truncated intracellular Dicer-like molecule involves in antiviral immune recognition of oyster Crassostrea gigas.

The enzyme Dicer is best known for its role as an endoribonuclease in the small RNA pathway, playing a crucial role in recognizing viral double-stranded RNA (dsRNA) and inducing down-stream cascades to mediate anti-virus immunity. In the present study, a truncated Dicer-like gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDCL) of 530 amino acids. The CgDCL contained one N-terminal DEAD domain and a C-terminal helicase domain, but lack the conserved PAZ domain, ribonuclease domain (RIBOc) and dsRNA binding domain. The mRNA transcripts of CgDCL were detected in all the examined tissues with high expression levels in lip, gills and haemocytes, which were 62.06-fold, 48.91-fold and 47.13-fold (p < 0.05) of that in mantle, respectively. In the primarily cultured oyster haemocytes, the mRNA transcripts of CgDCL were significantly induced at 12 h after poly(I:C) stimulation, which were 4.04-fold (p < 0.05) of that in control group. The expression level of CgDCL mRNA in haemocytes was up-regulated significantly after dsRNA and recombinant interferon-like protein (rCgIFNLP) injection, which was 12.87-fold (p < 0.01) and 3.22-fold (p < 0.05) of that in control group, respectively. CgDCL proteins were mainly distributed in the cytoplasm of haemocytes. The recombinant CgDCL protein displayed binding activity to dsRNA and poly(I:C), but no obvious dsRNA cleavage activity. These results collectively suggest that truncated CgDCL from C. gigas was able to be activated by poly(I:C), dsRNA and CgIFNLP, and functioned as an intracellular recognition molecule to bind nucleic acid of virus, indicating a potential mutual cooperation between RNAi and IFN-like system in anti-virus immunity of oysters.