Increased oxygen stimulation promotes chemoresistance and phenotype shifting through PLCB1 in gliomas.

Gliomas, the most common CNS (central nerve system) tumors, face poor survival due to severe chemoresistance exacerbated by hypoxia. However, studies on whether altered hypoxic conditions benefit for chemo-sensitivity and how gliomas react to increased oxygen stimulation are limited. In this study, we demonstrated that increased oxygen stimulation promotes glioma growth and chemoresistance. Mechanically, increased oxygen stimulation upregulates miR-1290 levels. miR-1290, in turn, downregulates PLCB1, while PLCB1 facilitates the proteasomal degradation of ß-catenin and active-ß-catenin by increasing the proportion of ubiquitinated ß-catenin in a destruction complex-independent mechanism. This process inhibits PLCB1 expression, leads to the accumulation of active-ß-catenin, boosting Wnt signaling through an independent mechanism and ultimately promoting chemoresistance in glioma cells. Pharmacological inhibition of Wnt by WNT974 could partially inhibit glioma volume growth and prolong the shortened survival caused by increased oxygen stimulation in a glioma-bearing mouse model. Moreover, PLCB1, a key molecule regulated by increased oxygen stimulation, shows promising predictive power in survival analysis and has great potential to be a biomarker for grading and prognosis in glioma patients. These results provide preliminary insights into clinical scenarios associated with altered hypoxic conditions in gliomas, and introduce a novel perspective on the role of the hypoxic microenvironment in glioma progression. Furthermore, the outcomes reveal the potential risks of utilizing hyperbaric oxygen treatment (HBOT) in glioma patients, particularly when considering HBOT as a standalone option to ameliorate neuro-dysfunctions or when combining HBOT with a single chemotherapy agent without radiotherapy.

An LQT2-related mutation in the voltage-sensing domain is involved in switching the gating polarity of hERG.

BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

Involvement of metabotropic glutamate receptors in regulation of immune response in the Pacific oyster Crassostrea gigas.

Metabotropic glutamate receptors (mGluRs) play a pivotal role in the neuroendocrine-immune regulation. In this study, eight mGluRs were identified in the Pacific Oyster Crassostrea gigas, which were classified into three subfamilies based on genetic similarity. All CgmGluRs harbor variable numbers of PBP1 domains at the N-terminus. The sequence and structural features of CgmGluRs are highly similar to mGluRs in other species. A uniformly upregulated expression of CgmGluRs was observed during D-shaped larval stage compared to early D-shaped larval stage. The transcripts of CgmGluRs were detectable in various tissues of oyster. Different CgmGluR exhibited diverse expression patterns response against different PAMP stimulations, among which CgmGluR5 was significantly downregulated under these stimulations, reflecting its sensitivity and broad-spectrum responsiveness to microbes. Following LPS stimulation, the mRNA expression of CgmGluR5 and CgCALM1 in haemocytes was suppressed within 6 h and returned to normal levels by 12 h. Inhibition of CgmGluR5 activity resulted in a significant reduction in CgCALM1 expression after 12 h. Further KEGG enrichment analysis suggested that CgmGluR5 might modulate calcium ion homeostasis and metabolic pathways by regulating CgCALM1. This research delivers the systematic analysis of mGluR in the Pacific Oyster, offering insights into evolutionary characteristics and immunoregulatory function of mGluR in mollusks.

The involvement of AMP-activated protein kinase α in regulating glycolysis in Yesso scallop Patinopecten yessoensis under high temperature stress.

AMP-activated protein kinase α subunit (AMPKα), the central regulatory molecule of energy metabolism, plays an important role in maintaining energy homeostasis and helping cells to resist the influence of various adverse factors. In the present study, an AMPKα was identified from Yesso scallop Patinopecten yessoensis (PyAMPKα). The open reading frame (ORF) of PyAMPKα was of 1599 bp encoding a putative polypeptide of 533 amino acid residues with a typical KD domain, a α-AID domain and a α-CTD domain. The deduced amino acid sequence of PyAMPKα shared 59.89-74.78% identities with AMPKαs from other species. The mRNA transcripts of PyAMPKα were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in adductor muscle. PyAMPKα was mainly located in cytoplasm of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyAMPKα, the phosphorylation level of PyAMPKα at Thr170 and the lactic acid (LD) content in adductor muscle all increased significantly, while the glycogen content decreased significantly. The activity of pyruvate kinase (PyPK) and the relative mRNA expression level of phosphofructokinase (PyPFK) were significantly up-regulated at 3 h after high temperature stress treatment (25 °C). Furthermore, the PyAMPKα activator AICAR could effectively upregulate the phosphorylation level of PyAMPKα, and increase activities of PyPFK and pyruvate kinase (PyPK). Meanwhile the glycogen content also declined under AICAR treatment. These results collectively suggested that PyAMPKα was involved in the high temperature stress response of scallops by enhancing glycolysis pathway of glycogen. These results would be helpful for understanding the functions of PyAMPKα in maintaining energy homeostasis under high temperature stress in scallops.

Effect of high temperature stress on glycogen metabolism in gills of Yesso scallop Patinopecten yessoensis.

Glycogen is the main energy storage material in mollusc, and the regulation of its metabolism is essential for the response against high temperature stress. In the present study, the alternation of lactic acid (LD) content, glycogen reserves, mRNA expression level of genes encoding glycogen metabolism enzymes and activities of glycogen metabolism enzymes in gills of Yesso scallop Patinopecten yessoensis after an acute high temperature treatment at 25 °C were examined to understand the effect of high temperature on glycogen metabolism. The activity of T-ATPase in gills of scallops presented a gradual increase trend especially at 6 h after the acute high temperature treatment (p < 0.05). The glycogen reserves did not change significantly even there was a downward trend at 24 h after the acute high temperature treatment (p > 0.05). The mRNA transcripts of glycogen synthase (PyGCS) in gills of scallops decreased significantly at 1, 3, 6 and 12 h (p < 0.05), and recovered to normal level at 24 h (p > 0.05) after the acute high temperature treatment, while that of glycogen phosphorylase a (PyGPa) and phosphoenol pyruvate carboxy kinase (PyPEPCK) were both significantly down-regulated from 1 h to 24 h (p < 0.05) after the acute high temperature treatment. The activity of PyGPa at 1, 12 and 24 h and the content of LD at 3 and 24 h in gills of scallops after the acute high temperature treatment both increased significantly (p < 0.05). Furthermore, the mRNA transcripts of hexokinase (PyHK) and pyruvate kinase (PyPK) in gills of scallops increased significantly (p < 0.05) after the acute high temperature treatment, and the response of PyHK was stronger. However, there was no significant difference on the activity of PyPK in gills of scallops between the experimental samples and the blank samples (p > 0.05). In addition, the mRNA transcripts of citrate synthase (PyCS) in gills of scallops were significantly down-regulated at 6 h and 12 h (p < 0.05), and finally returned to normal level at 24 h (p > 0.05) after the acute high temperature treatment. These results collectively indicated acute high temperature stress leaded the alternation of glycogen metabolism in the gills of Yesso scallop, glycogenesis, gluconeogenesis and TCA cycle were inhibited, and the glycolysis pathway of glycogen was enhanced to produce more energy for coping with environmental pressure.

The intestinal bacterial community over seasons and its relationship with physiological status of Yesso scallop Patinopecten yessoensis.

Emerging evidence indicates that the intestinal bacterial communities associated with eukaryotes play critical roles in the physiological activities and health of their hosts. Yesso scallop Patinopecten yessoensis, one of the cold-water aquaculture species in the North Yellow Sea of China, has suffered from massive mortality in recent years. In the present study, P. yessoensis were collected from Zhangzi Island, Dalian from March 2021 to January 2022 to investigate the intestinal bacterial community and physiological indices. 16S rRNA gene sequencing data revealed that the diversity of intestinal bacteria changed significantly over seasons, with the highest Chao1 (237.42) and Shannon (6.13) indices detected in January and the lowest Chao1 (115.44) and Shannon (2.73) indices detected in July. Tenericutes, Proteobacteria and Firmicutes were dominant phyla in the intestinal bacteria of P. yessoensis, among which Firmicutes and Proteobacteria significantly enriched in August and January, respectively. Mycoplasma was the most abundant genus during the sampling period, which exhibited the highest abundance in October (75.26%) and lowest abundance in August (13.15%). The functional profiles of intestinal bacteria also exhibited seasonal variation, with the pathways related to pentose phosphate and deoxyribonucleotides biosynthesis enriched in August while the glycogen biosynthesis pathway enriched in October. Redundancy analysis showed that seawater pH, dissolved inorganic nitrogen and silicate were major environmental factors driving the temporal succession of scallop intestinal bacteria. Correlation clustering analysis suggested that the relative abundances of Endozoicomonas and Vibrio in the intestine were positively correlated with superoxide dismutase activity in hepatopancreas while negatively correlated with malondialdehyde content in hepatopancreas and glycogen content in adductor muscle. All the results revealed that the intestine harbored a lower bacterial diversity and a higher abundance of Vibrio in August, compared to January, which were closely related to the oxidative stress status of scallop in summer. These findings will advance our understanding of the relationship between seasonal alteration in the intestinal bacteria and the physiological status of scallops.

Functional characterization and in vitro pharmacological rescue of KCNQ2 pore mutations associated with epileptic encephalopathy.

Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.

Applications of Induced Pluripotent Stem Cell-Derived Glia in Brain Disease Research and Treatment.

Glia are integral components of neural networks and are crucial in both physiological functions and pathological processes of the brain. Many brain diseases involve glial abnormalities, including inflammatory changes, mitochondrial damage, calcium signaling disturbance, hemichannel opening, and loss of glutamate transporters. Induced pluripotent stem cell (iPSC)-derived glia provide opportunities to study the contributions of glia in human brain diseases. These cells have been used for human disease modeling as well as generating new therapies. This chapter introduces glial involvement in brain diseases, then summarizes different methods of generating iPSC-derived glia disease models of these cells. Finally, strategies for treating disease using iPSC-derived glia are discussed. The goal of this chapter is to provide an overview and shed light on the applications of iPSC-derived glia in brain disease research and treatment.

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.

CgCaspase-3 activates the translocation of CgGSDME in haemocytes of Pacific oyster Crassostrea gigas.

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas.

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

The involvement of CgCaspase-8-2 in regulating the expressions of cytokines, antibacterial peptide and autophagy-related genes in oysters.

Caspase-8 has been reported to be involved not only in apoptosis, but also in many other important immune response processes, such as inflammation and autophagy. In the present study, the open reading frame of CgCaspase-8-2 was cloned from the Pacific oyster Crassostrea gigas, which was of 2160 bp encoding 737 amino acids. There were two death effector domains (DEDs) and a cysteine aspartase cysteine structural (CASc) domain in the deduced amino acid sequences of CgCaspase-8-2. The mRNA expressions of CgCaspase-8-2 in haemocytes and gills all increased significantly after Vibrio splendidus stimulation at 3 h, 6 h, and 24 h. The cleaved CgCaspase-8-2 protein was observed in haemocytes at 3 h after V. splendidus stimulation and the expression of CgCaspase-8-2 protein was relatively higher in granulocytes, compared with that in agranulocytes. In CgCaspase-8-2-RNAi oysters, the mRNA expressions of CgIL17s (CgIL17-1, -2, -3, -4, -6), CgTNF, CgIFNLP and CgBigDef1 all decreased significantly at 12 h after V. splendidus stimulation. Meanwhile, the mRNA expressions of CgATG5 and CgBeclin1 decreased significantly at 12 h after V. splendidus stimulation, while CgBcl2 increased significantly. These results indicated that CgCaspase-8-2 was involved in not only the regulation of cytokine and antibacterial peptide production, but also autophagy-related gene expressions.

A negative feedback loop of H19/miR-675/VDR mediates therapeutic effect of cucurmin in the treatment of glioma.

Curcumin (CUR) shows a remarkable antitumor activity against a wide range of cancers such as glioma, but its underlying mechanism remains elusive. In this study, we aimed to explore the potential role of H19/miR-675/vitamin D receptor (VDR) in the effect of CUR against glioma. Real-time polymerase chain reaction and western-blot analysis were used to study the effect of CUR or 1,25-dihydroxyvitamin D (1,25(OH)2 D3 ) on the expression of H19, miR-675, and VDR. In addition, the effect of H19 on VDR expression was also studied. Furthermore, the expression of H19, miR-675, and VDR between CUR-loaded nanoparticles (NPs) and NP groups was compared, and the interaction among H19, miR-675, and VDR was analyzed by in-silicon and luciferase assays. In a dose-dependent manner, CUR and 1,25(OH)2 D3 both downregulated the expression of H19 and miR-675 but increased the expression of VDR. In addition, H19 evidently reduced the mRNA and protein levels of VDR. Furthermore, VDR was confirmed as a target gene of miR-675, which significantly reduced the expression of VDR. Finally, the administration of CUR evidently decreased tumor volume. CUR-loaded NP group exhibited lower levels of H19 and miR-675, while the NP group showed higher levels of VDR mRNA and protein. In summary, it is the first time that the involvement of a negative feedback loop of H19/miR-675/VDR has been demonstrated in the development of glioma. Therefore, H19 might serve as a new biomarker for the diagnosis and treatment of glioma.

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.

AP-1 regulates the expression of IL17-4 and IL17-5 in the pacific oyster Crassostrea gigas.

The activator protein-1 (AP-1) plays an important role in inducing the immune effector production in response to cellular stress and bacterial infection. In the present study, an AP-1 was identified from Pacific oyster Crassostrea gigas (designed as CgAP-1) and its function was investigated in response against lipopolysaccharide (LPS) stimulation. CgAP-1 was consisted of 290 amino acids including a Jun domain and a basic region leucine zipper (bZIP) domain. CgAP-1 shared 98.6% similarities with ChAP-1 from oyster C. hongkongensis, and assigned into the branch of invertebrates in the phylogenetic tree. The mRNA transcripts of CgAP-1 gene were detected in all tested tissues with highest expression level in hemocytes, especially in granulocytes. The mRNA expression level of CgAP-1 gene in hemocytes was significantly up-regulated (8.53-fold of that in PBS group, p < 0.01) at 6 h after LPS stimulation. CgAP-1 protein could be translocated into the nucleus of oyster hemocytes after LPS stimulation. The mRNA transcripts of interleukin17s (CgIL17-4 and CgIL17-5) in the hemocytes of CgAP-1-RNAi oysters decreased significantly at 24 h after LPS stimulation, which were 0.37-fold (p < 0.05) and 0.17-fold (p < 0.01) compared with that in EGFP-RNAi oysters, respectively. The results suggested that CgAP-1 played an important role in the immune response of oyster by regulating the expression of CgIL17s.

A conserved interferon regulation factor 1 (IRF-1) from Pacific oyster Crassostrea gigas functioned as an activator of IFN pathway.

Interferon regulatory factors (IRFs), a family of transcription factors with a novel helix-turn-helix DNA-binding motif, play important roles in regulating the expression of interferons (IFNs) and IFN-stimulated genes. In the present study, an interferon regulation factor 1 was identified from oyster Crassostrea gigas (designated CgIRF-1), and its immune function was characterized to understand the regulatory mechanism of interferon system against viral infection in invertebrates. The open reading frame (ORF) of CgIRF-1 was 990 bp, encoding a polypeptide of 329 amino acids with a typical IRF domain (also known as DNA-binding domain). The mRNA transcripts of CgIRF-1 were detected in all the tested tissues with the highest expression level in hemocyte. CgIRF-1 protein was distributed in both nucleus and cytoplasm of the oyster hemocyte. The mRNA expression of CgIRF-1 in hemocytes was significantly up-regulated at 48 h after poly (I:C) stimulation (p < 0.05). The recombinant CgIRF-1 (rCgIRF-1) could interact with classically IFN-stimulated response elements (ISRE) in vitro. The relative luciferase activity of interferon-like protein promotor reporter gene (pGL-CgIFNLP promotor) was significantly (p < 0.05) enhanced in HEK293T cell after transfection of CgIRF-1. These results indicated that CgIRF-1 could bind ISRE and regulate the expression of CgIFNLP as a transcriptional regulatory factor, and participated in the antiviral immune response of oysters.

Inositol-requiring enzyme 1 involved in regulating hemocyte apoptosis upon heat stress in Patinopecten yessoensis.

The inositol-requiring enzyme 1 (IRE1), one of the primary endoplasmic reticulum (ER) transmembrane receptor proteins, is involved in regulating unfolded protein response (UPR) signaling pathway and plays an import role in maintaining cell homeostasis. In the present study, an IRE1 homologue was identified from Patinopecten yessoensis (designated as PyIRE1). The cDNA of PyIRE1 was of 3314 bp with a 2646 bp open reading frame (ORF) of IRE1 encoding a polypeptide of 881 amino acids. There was a signal peptide, four pyrrolo-quinoline quinine (PPQ) domains, a transmembrane helix region, a Serine/Threonine protein kinases domain (S_TKc) and a protein kinases or N-glycanases containing protein domain (PUG) in the deduced amino acid sequence of PyIRE1. The PyIRE1 mRNA was constitutively expressed in all the tested tissues, with the highest expression level in gills. PyIRE1 protein was mainly located in the ER of P. yessoensis hemocytes. The expression profiles of PyIRE1, glucose-regulated protein 94 (designated as PyGRP94) and glucose-regulated protein 78 (designated as PyGRP78) were determined by SYBR Green qRT-PCR after heat shock treatment. The mRNA expression levels of all these three genes were significantly up-regulated and reached their peak values at 2 h (3.97-fold, p < 0.05), 8 h (19.67-fold, p < 0.05) and 4 h (27.37-fold, p < 0.05) in hemocytes, 2 h (3.55-fold, p < 0.05), 12 h (8.58-fold, p < 0.05) and 8 h (35.31-fold, p < 0.05) in gills after heat shock treatment, respectively. After the injection with PyIRE1 dsRNA, the mRNA expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2) family member PyBax and the activity of caspase-3 significantly decreased in comparison with the control group (p < 0.05) after heat shock treatment. These results collectively suggested that PyIRE1, as an ER stress sensor, was potentially involved in the response upon heat stress by regulating the expression of PyBax and apoptosis of hemocytes in P. yessoensis.

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.

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.

A new fibrinogen-related protein from Argopecten irradians (AiFREP-2) with broad recognition spectrum and bacteria agglutination activity.

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 ß-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.