A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor.

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

The Modification of H3K4me3 Enhanced the Expression of CgTLR3 in Hemocytes to Increase CgIL17-1 Production in the Immune Priming of Crassostrea gigas.

Increasing evidence confirms that histone modification plays a critical role in preserving long-term immunological memory. Immune priming is a novel form of immunological memory recently verified in invertebrates. Toll-like receptor (TLR) signaling and cytokines have been reported to be involved in the immune priming of the Pacific oyster Crassostrea gigas. In the present study, the expression of Toll-like receptor 3 (CgTLR3), myeloid differentiation factor 88-2 (CgMyd88-2) and interleukin 17-1 (CgIL17-1) was found to be elevated in the hemocytes of C. gigas at 6 h after the secondary stimulation with Vibrio splendidus, which was significantly higher than that at 6 h after the primary stimulation (p < 0.05). A significant increase in histone H3 lysine 4 trimethylation (H3K4me3) enrichment was detected in the promoter region of the CgTLR3 gene at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05). After the treatment with a histone methyltransferase inhibitor (5'-methylthioadenosine, MTA), the level of H3K4me3 at the promoter of the CgTLR3 gene decreased significantly at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was significantly repressed at 6 h after the secondary stimulation with V. splendidus (p < 0.05). Conversely, the treatment with monomethyl fumarate (MEF, an inhibitor of histone demethylases) resulted in a significant increase in H3K4me3 enrichment levels at the CgTLR3 promoter at 7 d after the primary stimulation (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was observed to increase significantly at 6 h after the secondary stimulation (p < 0.05). These results suggested that H3K4me3 regulated MyD88-dependent TLR signaling in the hemocytes of C. gigas, which defined the role of histone modifications in invertebrate immune priming.

A C-type lectin from Crassostrea gigas with novel EFG/FVN motif involved in recognition of various PAMPs and induction of interleukin expression.

C-type lectins (CTLs) are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which participate in the nonself-recognition and triggering the transduction pathways in the innate immunity. In the present study, a novel CTL (designated as CgCLEC-TM2) with a carbohydrate-recognition domain (CRD) and a transmembrane domain (TM) was identified from the Pacific oyster Crassostrea gigas. Two novel EFG and FVN motifs were found in Ca2+-binding site 2 of CgCLEC-TM2. The mRNA transcripts of CgCLEC-TM2 were detected in all tested tissues with the highest expression level in haemocytes, which was 94.41-fold (p < 0.01) of that in adductor muscle. The relative expression level of CgCLEC-TM2 in haemocytes significantly up-regulated at 6 h and 24 h after the stimulation of Vibrio splendidus, which was 4.94- and 12.77-fold of that in control group (p < 0.01), respectively. The recombinant CRD of CgCLEC-TM2 (rCRD) was able to bind lipopolysaccharide (LPS), mannose (MAN), peptidoglycan (PGN), and poly (I: C) in a Ca2+-dependent manner. The rCRD exhibited binding activity to V. anguillarum, Bacillus subtilis, V. splendidus, Escherichia coli, Pichia pastoris, Staphylococcus aureus and Micrococcus luteus in a Ca2+-dependent manner. The rCRD also exhibited agglutination activity to E. coli, V. splendidus, S. aureus, M. luteus and P. pastoris in a Ca2+-dependent manner. The phagocytosis rate of haemocytes towards V. splendidus significantly down-regulated from 27.2% to 20.9% after treatment of anti-CgCLEC-TM2-CRD antibody, while the growth of V. splendidus and E. coli was inhibited compared with the TBS and rTrx groups. After the expression of CgCLEC-TM2 was inhibited by RNAi, the expression level of phospho-extracellular regulated protein kinases (p-CgERK) in haemocytes, and the mRNA expressions of interleukin17s (CgIL17-1 and CgIL17-4) decreased significantly after V. splendidus stimulation, compared with that in EGFP-RNAi oysters, respectively. These results suggested that CgCLEC-TM2 with novel motifs served as a pattern recognition receptor (PRR) involved in the recognition of microorganisms, and induction of CgIL17s expression in the immune response of oysters.

The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

The involvement of ecdysone and ecdysone receptor in regulating the expression of antimicrobial peptides in Chinese mitten crab, Eriocheir sinensis.

The ecdysone, 20-hydroxyecdysone (20E) and ecdysone receptor (EcR), are regarded as the key regulators of development, metamorphosis, and growth in arthropods. In the present study, the role of 20E and EsEcR in regulating the expression of antimicrobial peptides (AMPs) was investigated in Chinese mitten crab, Eriocheir sinensis. The concentration of 20E in plasma was significantly (p < 0.05) up-regulated from 3 h to 12 h after lipopolysaccharide (LPS) stimulation. The mRNA expression level of EsEcR-4 in hemocytes was significantly (p < 0.01) up-regulated from 6 h to 24 h after LPS stimulation, while no significant changes of EsEcR-2 and EsEcR-3 transcripts were observed. After 20E injection, EsEcR-4 expression level was significantly increased from 12 h to 48 h with the highest level at 24 h (4.34-fold compared to the control group, p < 0.01), and the mRNA expression levels of AMPs (EsALF-2, EsLYZ and EsCrus) in hemocytes were significantly increased from 6 h to 24 h with the peak level of 2.93-fold (p < 0.01), 2.33-fold (p < 0.01) and 2.75-fold (p < 0.01) at 12 h, respectively. After EsEcR-4 expression was interfered with specific dsRNA, a significant reduction of EsALF-2 (0.56-fold compared to the control group, p < 0.01), EsLYZ (0.27-fold, p < 0.01) and EsCrus (0.41-fold, p < 0.01) mRNA expression level was observed in dsEsEcR-4+LPS group at 12 h post LPS stimulation. Moreover, the mRNA expression levels of EsDorsal and EsJNK in hemocytes were significantly (p < 0.05) increased from 6 h to 24 h post 20E injection, and the phosphorylation of Dorsal and JNK in the hemocytes were significantly (p < 0.01) up-regulated at 3 h post 20E injection, while that in dsEsEcR-4+LPS group were significantly decreased after LPS stimulation compared to dsEsEGFP+LPS group. Taken together, these results suggested that 20E and EsEcR-4 play important roles in regulating the expression level of AMPs in the immune responses of E. sinensis by regulating the mRNA expression level and phosphorylation of Dorsal and JNK.

The involvement of a regucalcin in suppressing hemocyte apoptosis in Pacific oyster Crassostrea gigas.

Regucalcin (RGN), also known as senescence marker protein-30 (SMP30), plays a vital role in the regulation of Ca2+ homeostasis. In the present study, a regucalcin (designated as CgRGN) was identified from Pacific oyster Crassostrea gigas. The complete cDNA sequence of CgRGN was of 1059 bp, containing an open reading frame of 933 bp which encoded a protein of 310 amino acids. The deduced amino acid sequence of CgRGN shared similarity with other RGNs from the genome of C. gigas as well as other species. The mRNA transcripts of CgRGN were universally detected in all tested tissues, with higher level in hepatopancreas, labial palp, and gills. The relative expression level of CgRGN in hemocytes was significantly up-regulated (p < 0.05) at 3, 12, 72, and 96 h after the stimulation of lipopolysaccharide (LPS). After CgRGN expression was interfered by specific CgRGN-dsRNA, the hemocytes apoptosis rate increased dramatically at 12 h post LPS stimulation (1.56 fold, p < 0.01), compared to the control group. The caspase-3 activity in hemocytes and NO concentration in hemolymph increased significantly (p < 0.05) in dsCgRGN injection oysters. These results collectively indicated that CgRGN could suppress LPS-induced apoptosis and be involved in the immune response of oysters.

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.

The efficacy and safety of first-line therapies for preventing chronic post-surgical pain: a network meta-analysis.

BACKGROUND: Due to conflicting evidence regarding first-line therapies for chronic post-surgical pain (CPSP), here we comparatively evaluated the efficacy and safety of first-line therapies for the prevention of CPSP. MATERIALS AND METHODS: MEDLINE, EMBASE, and Cochrane CENTRAL databases were searched for randomized, controlled trials (RCTs) of systemic drugs measuring pain three months or more post-surgery. Pairwise meta-analyses (a frequentist technique directly comparing each intervention against placebo) and network meta-analyses (a Bayesian technique simultaneously comparing several interventions via an evidence network) compared the mean differences for primary efficacy (reduction in all pain), secondary efficacy (reduction in moderate or severe pain), and primary safety (drop-out rate from treatment-related adverse effects). Ranking probabilities from the network meta-analysis were transformed using surface under the cumulative ranking analysis (SUCRA). Sensitivity analyses evaluated the impact of age, gender, surgery type, and outlier studies. RESULTS: Twenty-four RCTs were included. Mexiletine and ketamine ranked highest in primary efficacy, while ketamine and nefopam ranked highest in secondary efficacy. Simultaneous SUCRA-based rankings of the interventions according to both efficacy and safety revealed that nefopam and mexiletine ranked highest in preventing CPSP. Through the sensitivity analyses, gabapentin and ketamine remained the most-highly-ranked in terms of efficacy, while nefopam and ketamine remained the most-highly-ranked in terms of safety. CONCLUSIONS: Nefopam and mexiletine may be considered as first-line therapies for the prevention of CPSP. On account of the paucity of evidence available on nefopam and mexiletine, gabapentin and ketamine may also be considered. Venlafaxine is not recommended for the prevention of CPSP.

Quality of life in adolescent patients with idiopathic scoliosis after brace treatment: A meta-analysis.

BACKGROUND: Whether brace-treated adolescents with idiopathic scoliosis (AIS) have improved quality of life (QoL) is still unknown. Thus, we conducted a meta-analysis to compare the QoL of brace-treated AIS patients with untreated AIS patients. The pain, self-image/appearance, mental health, function/activity, satisfaction with management, total score without satisfaction, and total score of patients were used to measure the QoL after the intervention. METHODS: Multiple electronic databases including PubMed, Web of Science, and Embase were searched for all years up to June 30, 2016. Articles in English that used the Scoliosis Research Society-22 (SRS-22) or a modified version of the SRS-22 questionnaire to evaluate the QoL differences between brace-treated AIS patients and untreated AIS patients were included in the meta-analysis. The Newcastle-Ottawa Scale was used in the quality of literature evaluation. The pooled standardized mean difference (SMD) with its corresponding 95% confidence interval (CI) for each parameter was computed. Egger test and Begg test were used to test for publication bias. RESULTS: The SRS-22 or a modified SRS-22 questionnaire was used to evaluate the QoL after surgery. There was no significant difference in pain (SMD = 0.123, 95% CI: -0.101 to 0.347, P = .282), self-image/appearance (SMD = 0.108, 95% CI: -0.116 to 0.332, P = .334), mental health (SMD = 0.031, 95% CI: -0.130 to 0.201, P = .365), function/activity (SMD = 0.202, 95% CI: -0.022 to 0.425, P = .077), and total score without satisfaction (SMD = 0.123, 95% CI: -0.232 to 0.478, P = .497) between the untreated (observation) and brace-treated AIS patients, whereas a significant difference was observed in satisfaction with management (SMD = 0.393, 95% CI: 0.127-0.659, P = .004) and total score (SMD = 0.312, 95% CI: 0.054-0.571, P = .018) between the 2 groups. CONCLUSION: Our meta-analysis indicated that brace-treated AIS patients had a higher QoL. However, further analysis could not be performed because of insufficient data, such that we were unable to make subgroup analysis of QoL for different types of AIS and the therapeutic methods chosen by brace-treated AIS patients.