Differential mantle transcriptomics and characterization of growth-related genes in the diploid and triploid pearl oyster Pinctada fucata.

To explore the molecular mechanism of triploidy effect in the pearl oyster Pinctada fucata, two RNA-seq libraries were constructed from the mantle tissue of diploids and triploids by Roche-454 massive parallel pyrosequencing. The identification of differential expressed genes (DEGs) between diploid and triploid may reveal the molecular mechanism of triploidy effect. In this study, 230 down-regulated and 259 up-regulated DEGs were obtained by comparison between diploid and triploid libraries. The gene ontology and KEGG pathway analysis revealed more functional activation in triploids and it may due to the duplicated gene expression in transcriptional level during whole genome duplication (WGD). To confirm the sequencing data, a set of 11 up-regulated genes related to growth and development control and regulation were analyzed by RT-qPCR in independent experiment. According to the validation and annotation of these genes, it is hypothesized that the set of up-regulated expressed genes had the correlated expression pattern involved in shell building or other interactive probable functions during triploidization. The up- regulation of growth-related genes may support the classic hypotheses of 'energy redistribution' from early research. The results provide valuable resources to understand the molecular mechanism of triploidy effect in both shell building and producing high-quality seawater pearls.

DNA methylation is associated with expression level changes of galectin gene in mantle wound healing process of pearl oyster, Pinctada fucata.

Galectin is one important member of pattern recognition proteins that plays a pivotal role in regulating innate immune response of invertebrates. In this study, we cloned the promoter sequence of a tandem-repeat galectin from the pearl oyster Pinctada fucata (P. fucata). The quantitative real-time PCR analysis revealed that galectin mRNA expression in mantle tissues were firstly up-regulated from time points of 2 h-24 h, and then down-regulated from time points of 24 h-168 h after mantle injury. The genome methylation level of mantle tissue was inversely related to galectin mRNA expression (Pearson correlation: -0.554, P: 0.154). The "CpG4-6" methylation level in promoter region of galectin was significant positive correlated with the mRNA expression (Pearson correlation: 0.313, P: 0.049). The results indicated that galectin gene may be involved in immune response in mantle wound healing process of P. fucata, and DNA methylation may be a regulation factor of gene expression.

Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction.

OBJECTIVE: To investigate the use of citrulline as an indicator for diagnosing septic acute intestinal dysfunction (SAID) in a rat model. METHODS: SD rats were divided into three groups: a normal group (A), a model group (B), and a glutamine group (C). Group B was divided into a 36-h group (B1) and a 72-h feeding group (B2). The concentrations of serum citrulline, intestinal fatty acid-binding protein (I-FABP) and intestinal glutamine and histopathological changes were measured. RESULTS: The lengths of the villus and thicknesses of the mucosal layer in groups B1, B2 and C were significantly different from those in group A. Citrulline concentrations in groups B1, B2 and C were lower than in group A; the serum concentrations in group C were significantly greater than in groups B1 and B2. The I-FABP levels of groups B1, B2 and C were higher than group A; I-FABP levels of groups B1 and B2 were higher than group C. Intestinal glutamine levels of groups B1 and B2 were lower than groups A and C. The serum citrulline of group C was negatively correlated with I-FABP and Chiu's score. CONCLUSIONS: Serum citrulline could be used as the diagnostic indicator of SAID.

Human CRP defends against the toxicity of circulating histones.

C-reactive protein (CRP) is an acute-phase protein that plays an important defensive role in innate immunity against bacterial infection, but it is also upregulated in many noninfectious diseases. The generic function of this highly conserved molecule in diseases that range from infection, inflammation, trauma, and malignancy is not well understood. In this article, we demonstrate that CRP defends the human body against the toxicity of histones released into the circulation after extensive cell death. In vitro, CRP significantly alleviates histone-induced endothelial cell damage, permeability increase, and platelet aggregation. In vivo, CRP rescues mice challenged with lethal doses of histones by inhibiting endothelial damage, vascular permeability, and coagulation activation, as reflected by significant reductions in lung edema, hemorrhage, and thrombosis. In patients, elevation of CRP significantly increases the capacity to neutralize extracellular histones in the circulation. We have also confirmed that CRP interacts with individual histones in vitro and forms CRP-histone complexes in serum from patients with both elevated CRP and histones. CRP is able to compete with phospholipid-containing liposomes for the binding to histones. This explains how CRP prevents histones from integrating into cell membranes, which would otherwise induce calcium influx as the major mechanism of cytotoxicity caused by extracellular histones. Because histone elevation occurs in the acute phase of numerous critical illnesses associated with extensive cell death, CRP detoxification of circulating histones would be a generic host defense mechanism in humans.

Molecular characterization of interferon regulatory factor 2 (IRF-2) homolog in pearl oyster Pinctada fucata.

Interferon regulatory factors (IRFs) control many facets of the innate and adaptive immune responses, regulate the development of the immune system itself and involve in reproduction and morphogenesis. In the present study, the IRF-2 homology gene, PfIRF-2 from pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfIRF-2 encodes a putative protein of 350 amino acids, and contains a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. Comparison and phylogenetic analysis revealed that PfIRF-2 shared a relatively higher identity with other mollusk but relatively lower identity with vertebrate IRF-2, and was clustered with IRF-1 subfamily composed of IRF-2 and IRF-1. Furthermore, gene expression analysis revealed that PfIRF-2 involved in the immune response to LPS and poly(I:C) stimulation. Immunofluorescence assay showed that the expressed PfIRF-2 was translocated into the nucleus and dual-luciferase reporter assays indicated that PfIRF-2 could involved and activate interferon signaling or NF-κB signal pathway in HEK293 cells. The study of PfIRF-2 may help better understand the innate immune in mollusk.

Gigabase-scale transcriptome analysis on four species of pearl oysters.

Pearl oysters have been found to secrete nacre and form pearls with good quality and significant commercial interest. However, the transcriptomic and genomic resources for pearl oysters are still limited. To improve this situation, transcriptome sequencing was conducted from four species of pearl oysters with Illumina HiSeq™ 2000. There were four gigabase-scale transcriptomes for four species of pearl oysters, ∼26.3 million reads with ∼2.37 gigabase base pairs (Gbp) in Pinctada fucata, ∼26.5 million reads with ∼2.39 Gbp in Pinctada margaritifera, ∼27.0 million reads with ∼2.43 Gbp in Pinctada maxima, and ∼25.9 million reads with ∼2.33 Gbp in Pteria penguin, respectively. After sequence assembly and blastx alignment, the numbers of annotated unigenes ≥200 bp were 33,882 in P. fucata, 30,666 in P. margaritifera, 26,420 in P. maxima, and 29,928 in P. penguin. Based on these annotated unigenes among four species of pearl oysters, CDSs were extracted and predicted and furthermore, analyses of GO and KEGG assignments were performed. In addition, 60 putative genes of growth factors and their receptors from four species of pearl oysters were predicted. This study established an excellent resource for gene discovery and expression in pearl oysters, but also offered a significant platform for functional genomics and comparative genomic studies for mollusks.

Molecular cloning, characterization and expression analysis of tumor necrosis factor receptor-associated factor 3 (TRAF3) from pearl oyster Pinctada fucata.

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.

Molecular cloning and characterization of class I NF-κB transcription factor from pearl oyster (Pinctada fucata).

NF-κB transcription factors play central roles in many important physiological and pathological processes including innate immune responses. Here we report the cloning of an NF-κB transcription factor, PfRelish from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfRelish full-length cDNA is 3916 bp with an open reading frame of 3558 bp encoding a putative protein of 1186 amino acids. The deduced PfRelish contains a N-terminal RHD, a nucleus localization signal, an IκB-like domain with six ankyrin repeats and a death domain at the C-terminus, which is similar to class I NF-κB transcription factors. Comparison and phylogenetic analysis revealed that class I NF-κBs in mollusks including PfRelish might have most distant relationship to the arthropod Relish. Further expression analysis showed that PfRelish was apparently upregulated after Vibrio alginolyticus injection, which suggested that PfRelish was involved in the immune response to V. alginolyticus.

Effects of angiopoietin-1 on attachment and metastasis of human gastric cancer cell line BGC-823.

AIM: To evaluate the effects of angiopoietin-1 (Ang-1) on adhesion of gastric cancer cell line BGC-823 and expression of integrin beta1, CD44V6, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP-2). METHODS: BGC-823 cells were transfected transiently with adenovirus-Ang-1 (Ad-Ang-1). Cells transfected transiently with adenovirus-green fluorescent protein (Ad-GFP) and untransfected cells were used as a negative and blank control group, respectively. The cell adhesion rate between cell and extracellular matrix (ECM) was determined by cell adhesion assay. To investigate whether Ang-1 could reinforce gastric carcinoma metastasis, we performed migration and invasion assays in BGC-823 cells. The mRNA and protein expression of integrin beta1, CD44V6, uPA and MMP-2 were detected by reverse transcription polymerase chain reaction and Western blotting, respectively. The expression of integrin beta1 and CD44V6 was measured by immunohistochemistry. RESULTS: BGC-823 cells were transfected successfully. The adhesion rate increased significantly in the Ad-Ang-1 group (P < 0.05). The Ad-Ang-1-transfected group had a significant increase in migration and invasion compared with that of the mock-transfected and Ad-GFP groups. The mRNA and protein expression of integrin beta1, CD44V6, uPA and MMP-2 in the Ad-Ang-1 group was higher than that in the Ad-GFP and blank control groups (P < 0.05). Compared with mock-transfected and Ad-GFP groups, integrin beta1 and CD44V6 expression intensity greatly increased (P < 0.05). CONCLUSION: Transfection of Ang-1 into human gastric cancer cell line BGC-823 can significantly increase expression of integrin beta1 and CD44V6, by which cell adhesion and metastasis to the ECM are promoted.