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N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/ aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.

MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells." The correction states that there was a mistake in Figure 4J,K of the original publication, where an incorrect DNA fragment was used. The authors performed a luciferase analysis with the correct DNA fragment to correct this mistake. The authors assure that this correction does not affect the scientific conclusions of the study. [Extracted from the article]

Biomineralization is one of the key processes that is notably affected in marine calcifiers such as oysters under ocean acidification (OA). Understanding molecular changes in the biomineralization process under OA and its heritability, therefore, is key to developing conservation strategies for protecting ecologically and economically important oyster species. To do this, in this study, we have explicitly chosen the tissue involved in biomineralization (mantle) of an estuarine commercial oyster species, Crassostrea hongkongensis. The primary aim of this study is to understand the influence of DNA methylation over gene expression of mantle tissue under decreased ~pH 7.4, a proxy of OA, and to extrapolate if these molecular changes can be observed in the product of biomineralization—the shell. We grew early juvenile C. hongkongensis, under decreased ~pH 7.4 and control ~pH 8.0 over 4.5 months and studied OA‐induced DNA methylation and gene expression patterns along with shell properties such as microstructure, crystal orientation and hardness. The population of oysters used in this study was found to be moderately resilient to OA at the end of the experiment. The expression of key biomineralization‐related genes such as carbonic anhydrase and alkaline phosphatase remained unaffected; thus, the mechanical properties of the shell (shell growth rate, hardness and crystal orientation) were also maintained without any significant difference between control and OA conditions with signs of severe dissolution. In addition, this study makes three major conclusions: (1) higher expression of Ca2+ binding/signalling‐related genes in the mantle plays a key role in maintaining biomineralization under OA; (2) DNA methylation changes occur in response to OA; however, these methylation changes do not directly control gene expression; and (3) OA would be more of a 'dissolution problem' rather than a 'biomineralization problem' for resilient species that maintain calcification rate with normal shell growth and mechanical properties. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens. [ABSTRACT FROM AUTHOR]

In bacteria, both promoters and 5′-untranslated regions (5′-UTRs) of mRNAs play vital regulatory roles in gene expression. In this study, we identified 1203 active promoter candidates in Bacillus thuringiensis through analysis of the genome-wide TSSs based on the transcriptome data. There were 11 types of σ-factor and 34 types of transcription factor binding sites found in 723 and 1097 active promoter candidates, respectively. Moreover, within the 1203 transcriptional units (TUs), most (52%) of the 5′-UTRs were 10–50 nucleotides in length, 12.8% of the TUs had a long 5′-UTR greater than 100 nucleotides in length, and 16.3% of the TUs were leaderless. We then selected 20 active promoter candidates combined with the corresponding 5′-UTR DNA regions to screen the highly active promoter-5′-UTR DNA region complexes with different characteristics. Our results demonstrate that among the 20 selected complexes, six were able to exert their functions throughout the life cycle, six were specifically induced during the early-stationary phase, and four were specifically activated during the mid-stationary phase. We found a direct corresponding relationship between σ-factor-recognized consensus sequences and complex activity features: the great majority of complexes acting throughout the life cycle possess σA-like consensus sequences; the maximum activities of the σF-, σE-, σG-, and σK-dependent complexes appeared at 10, 14, 16, and 22 h under our experimental conditions, respectively. In particular, complex Phj3 exhibited the strongest activity. Several lines of evidence showed that complex Phj3 possessed three independent promoter regions located at −251∼−98, −113∼−31, and −54∼+14, and that the 5′-UTR +1∼+118 DNA region might be particularly beneficial to both the stability and translation of its downstream mRNA. Moreover, Phj3 successfully overexpressed the active β-galactosidase and turbo-RFP, indicating that Phj3 could be a proper regulatory element for overexpression of proteins in B. thuringiensis. Therefore, our efforts contribute to molecular biology research and the biotechnological application of B. thuringiensis. [ABSTRACT FROM AUTHOR]

Abstract: Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO2 due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spectrophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO2. Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae. [Copyright &y& Elsevier]

SODs are ubiquitous metalloenzymes that can scavenge superoxides in response to various stresses. In the present study, full-length cDNAs of two SOD genes were isolated from Crassostrea hongkongensis (designated ChMnSOD and ChCuZnSOD). The cDNAs are 997 and 918 bp in length with ORFs of 675 and 468 bp (encoding 225 and 156 amino acids), respectively. Sequence analysis revealed a conserved Sod_Fe domain in ChMnSOD, and a Sod_Cu_Zn domain in ChCuZnSOD. Subcellular localization of ChMnSOD is mitochondrial while intracellular expression of ChCuZnSOD is detected. Although their expression overlaps in a wide range of tissues, ChMnSOD mRNA expression is high in gonad while ChCuZnSOD’s is strong in adductor muscle. After infection by Vibrio alginolyticus, ChMnSOD mRNA was up-regulated 5 fold (p < 0.05) at 4 h, but returned to normal level 6 h post-infection. The expression of ChCuZnSOD gene showed a slight delay to the infection challenge and was elevated roughly 4 fold after 8 h (p < 0.05), returning to normal at 12 h post-infection. The elevated transcript levels of the two SOD genes in response to V. alginolyticus infection highlights their important functions in eliminating toxic reactive oxygen species (ROS) and protecting organisms from bacterial invasion in C. hongkongensis. [ABSTRACT FROM AUTHOR]

Extraordinary variation has been found in mitochondrial (mt) genome inheritance, gene content and arrangement among bivalves. However, only few bivalve mt genomes have been comparatively analyzed to infer their evolutionary scenarios. In this study, the complete mt genome of the venerid Paphia euglypta (Bivalvia: Veneridae) was firstly studied and, secondly, it was comparatively analyzed with other venerids (e.g., Venerupis philippinarum and Meretrix petechialis) to better understand the mt genome evolution within a family. Though several common features such as the AT content, codon usage of protein-coding genes, and AT/GC skew are shared by the three venerids, a high level of variability is observed in genome size, gene content, gene order, arrangements and primary sequence of nucleotides or amino acids. Most of the gene rearrangement can be explained by the 'tandem duplication and random loss' model. From the observed rearrangement patterns, we speculate that block interchange between adjacent genes may be common in the evolution of mt genomes in venerids. Furthermore, this study presents several new findings in mt genome annotation of V. philippinarum and M. petechialis, and hence we have reannotated the genome of these two species as: (1) the ORF of the formerly annotated cox2 gene in V. philippinarum is deduced by using a truncated 'T' codon and a second cox2 gene is identified; (2) the trnS-AGN gene is identified and marked in the mt genome of both venerids. Thus, this study demonstrated a high variability of mt genomes in the Veneridae, and showed the importance of comparative mt genome analysis to interpret the evolution of the bivalve mt genome. Une variation extraordinaire a été observée en ce qui a trait à la transmission, au contenu génique et à l'arrangement du génome mitochondrial (mt) chez les bivalves. Cependant, seuls quelques génomes mt ont été comparés chez les bivalves pour en comprendre l'évolution. Dans ce travail, le génome mt complet du Vénéridé Paphia euglypta (Bivalvia : Veneridae) a été étudié et ensuite comparé à ceux d'autres Vénéridés (e.g., Venerupis philippinarum et Meretrix petechialis) afin de mieux comprendre l'évolution du génome mt au sein d'une famille. Bien que plusieurs caractéristiques étaient semblables chez les trois Vénéridés, comme le contenu en AT, l'usage des codons au sein des régions codant pour des protéines et le biais AT/GC, une grande variabilité a été observée en ce qui a trait à la taille du génome, le contenu et l'ordre génique, l'arrangement et la séquence primaire des nucléotides ou des acides aminés. La plupart des réarrangements géniques pouvaient s'expliquer via le modèle de « duplication en tandem suivie d'une perte aléatoire ». À partir des réarrangements observés, les auteurs proposent que l'échange de blocs entre gènes adjacents pourrait être un phénomène répandu dans l'évolution des génomes mt chez les Vénéridés. De plus, cette étude présente plusieurs nouvelles trouvailles en matière d'annotation des génomes mt chez le V. philippinarum et le M. petechialis. Les auteurs ont ainsi produit une nouvelle annotation du génome mt de ces deux espèces avec les modifications suivantes : 1) l'ORF du gène précédemment annoté comme étant cox2 chez le V. philippinarum a été déduit en utilisant un codon « T » tronqué et un second gène cox2 a été identifié ; 2) et le gène trnS-AGN a été identifié et annoté dans les génomes mt des deux espèces. Ainsi, cette étude a montré la grande variabilité des génomes mt chez les Vénéridés et l'importance de l'analyse comparée des génomes mt pour déduire l'évolution du génome mt chez les bivalves. [ABSTRACT FROM AUTHOR]

RIPK1/TAK1 are important for programmed cell death, including liver death, necroptosis and apoptosis. However, there have been few published reports on the functions of RIPK1/TAK1 in invertebrates. In this study, full-length Ch RIPK1 and ChTAK1 were cloned from C. hongkongensis through the rapid amplification of cDNA ends (RACE) technology. Ch RIPK1 has almost no homology with human RIPK1 and lacks a kinase domain at the N-terminus but has a DD and RHIM domain. Ch TAK1 is conserved throughout evolution. qRT‒PCR was used to analyze the mRNA expression patterns of Ch RIPK1 in different tissues, developmental stages, and V. coralliilyticus -infected individuals, and both were highly expressed in the mantle and gills, while Ch RIPK1 was upregulated in hemocytes and gills after V. coralliilyticus or S. aureus infection, which indicates that Ch RIPK1 is involved in immune regulation. Fluorescence assays revealed that Ch RIPK1 localized to the cytoplasm of HEK293T cells in a punctiform manner, but the colocalization of Ch RIPK1 with Ch TAK1 abolished the punctiform morphology. In the dual-luciferase reporter assay, both Ch RIPK1 and Ch RIPK1-RIHM activated the NF-κB signaling pathway in HEK293T cells, and Ch TAK1 activated Ch RIPK1 in the NF-κB signaling pathway. The apoptosis rate of the hemocytes was not affected by the necroptosis inhibitor Nec-1 but was significantly decreased, and Ch RIPK1 expression was knocked down in the hemocytes of C. hongkongensis. These findings indicated that ChRIPK1 induces apoptosis but not necroptosis in oysters. This study provides a theoretical basis for further research on the molecular mechanism by which invertebrates regulate the programmed cell death of hemocytes in oysters. • Ch RIPK1 lost a kinase domain at N-terminus but with a DD and RHIM domain. • The ChRIPK1 and ChTAK1 were expressed in different tissues, embryo stages, and changed after pathogens infection. • The Ch RIPK1 was located in the plasma as a punctiform but changed to homogeneous distribution in the plasma when Co-localization with Ch TAK1 in HEK293T cells. • The Ch RIPK1 activates the NF-κB in HEK293T cells and induces apoptosis but not necroptosis in the hemocytes of the oyster. [ABSTRACT FROM AUTHOR]

5-hydroxytryptamine (5-HT) is a secondary metabolite that interacts with a corresponding receptor, mediating various processes in organisms. However, the molecular function and characteristics of 5-HT receptor (5-HTR) in giant clam gonads remain unknown. In this study, we cloned full-length complementary DNA (cDNA) of four putative 5-HTRs from Tridacna crocea , designated Tc5-HTR1A-like, Tc5-HTR1D-like, Tc5-HTR2-like and Tc5-HTR4-like genes. SMART analysis revealed that all four Tc5-HTR-like proteins contained a seven-transmembrane structural domain, spanning from GN and ending at Y. Phylogenetic analysis showed that the four Tc5-HTR-like proteins were clustered into four distinct groups. The results of RT-qPCR showed that Tc5-HTR-like genes were widely expressed in all tested tissues, including gonads, suggesting that the function of Tc5-HTR-like genes involves the regulation of reproduction, including gametogenesis and embryonic development. Notably, the Tc5-HTR1D-like gene is highly expressed in oocytes of hermaphrodite and female stages. Estradiol-17β (E2) induced the upregulation of Tc5-HTR1A-like, Tc5-HTR1D-like and Tc5-HTR2-like genes, as well as downregulation of Tc5-HTR4-like gene in the gonad within 72 h. In situ hybridization showed that the Tc5-HTR1D-like gene had a positive signal in oocytes, indicating its role in oogenesis. The knockdown of the Tc5-HTR1D-like gene resulted in a significant reduction in oocyte growth and the number of oocytes. Whole-mount in situ hybridization and RT-qPCR analyses suggested that the spatiotemporal expression pattern of the Tc5-HTR1D-like gene is related to the physiological activities of embryonic development. Our data demonstrate that Tc5-HTR-like genes, especially Tc5-HTR1D-like gene, are essential for regulating gamete growth in T. crocea. • Tc5-HTR-like genes was the first successfully cloned from Tridacna crocea. • Tc5-HTR-like genes is involved in gonadal and embryonic development in Tridacna crocea. • Tc5-HTR1D-like gene is a female-biased gene. • Oogenesis was affected following knockdown of Tc5-HTR1D-like gene. • E 2 upregulated A, 5-HTR1D , 5-HTR2-like genes, but downregulated Tc5-HTR4-like gene in the gonad. [ABSTRACT FROM AUTHOR]

Serum amyloid protein (SAA) is known as an acute reactive protein of innate immunity in mammals. However, in invertebrates, the role of SAA in innate immunity is still unclear. In this study, a full-length cDNA of the SAA gene (named TcSAA) was cloned from Tridacna crocea , mollusca. The gene includes a 193 bp 5′ untranslated region (UTR) and a 129 bp 3' UTR sequence, and the open reading frame (ORF) with 393 bp nucleotides encodes a polypeptide of 130 amino acids. Tc SAA contains a typical signal peptide and an SAA functional domain. The mRNA expression of TcSAA was detected in all 12 selected tissues and 7 different developmental stages. Furthermore, the expression of TcSAA was increased quickly in hemocytes after challenge with V. coralliilyticus or LPS. Furthermore, rTc SAA could bind V. coralliilyticus and V. alginolyticus , and the protein could reduce the lethality rate of the clams from 80% to 55% which caused by V. coralliilyticus about 48 h after injection. In summary, these results indicate that Tc SAA may act as a marker for monitoring health and protecting T. crocea. • A full-length cDNA of serum amyloid gene (named as TcSAA) in T. crocea was cloned. • TcSAA is expressed increased radically after V. coralliilyticus and LPS chanllege. • rTc SAA was expressed and found to bind to V. coralliilyticus and V. alginolyticus. • rTc SAA could reduce the lethality rate of the T. crocea which caused by V. coralliilyticus. [ABSTRACT FROM AUTHOR]

Abstract: Lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) is a novel transcription factor responsible for lipopolysaccharide (LPS)-induced transcription of tumor necrosis factor-alpha. Here, we identified and characterized five new LITAF genes in a mollusk, Crassostrea gigas. The complete cDNA sequences of these newly-cloned CgLITAFs each contain one small ORF encoding putative proteins ranging from 67 to 132 amino acids in length. Each CgLITAF, except LITAF2, includes a conserved domain with two motifs, (H)XCXXC and CXXC; LITAF2 lacks the N-terminal CXXC motif. Phylogenetic analysis shows that the six CgLITAFs members (including a previously reported one) cluster into two different mollusk LITAF branches, implying an ancient origin of two LITAF genes that later diversified. CgLITAF members show distinct gene expression patterns with higher expression in digestive gland, gill, and mantle. Except for LITAF4 and LITAF6, CgLITAF expressions can be induced selectively and to various degrees by different Pathogen-Associated Molecular Patterns (PAMPs). Our results strongly demonstrated that the CgLITAF gene family has diversified in function such that each gene plays a distinct and non-redundant role in host defense of C. gigas. [Copyright &y& Elsevier]

Abstract: A novel homologue of IκB was cloned from a hemocyte cDNA of Crassostrea gigas (designed as CgIκB2). The complete cDNA of CgIκB2 includes an open reading frame (ORF) of 1032 bp, and 3′ and 5′untranslated regions (UTR’s) of 141 bp and 279 bp, respectively. The ORF encodes a putative protein of 343 amino acids with a calculated molecular weight of approximately 37.8 kDa. Alignment analysis reveals that CgIκB2 contains a conserved degradation motif and six ankyrin repeats. A phylogenetic analysis suggests that a gene duplication event prior to the gastropod-bivalve divergence resulted in the emergence of two IκB homologues in C. gigas. Distinct maximal expression patterns of CgIκB1 in hemocytes and CgIκB2 in the gonad were observed. CgIκB1 and CgIκB2 expression in response to bacterial challenge is similar and inducible. Moreover, both CgIκB1 and CgIκB2 are able to inhibit NF-κb/Rel activating transcription in S2 or HEK293 cells. Our findings demonstrate that both CgIκB1 and CgIκB2 are involved in immune defense in C. gigas through regulation of NF-κB/Rel activity. [Copyright &y& Elsevier]

Abstract: Mpegs (macrophage expressed genes) encode members of the MACPF (membrane-attack complex/perforin) protein superfamily that play essential roles in innate immunity. In the present study, a homolog of Mpeg1 was identified in Crassostrea gigas and designed Cg-Mpeg1. The complete cDNA of Cg-Mpeg1 is 2781 bp in length, containing an ORF of 2226 bp, which encodes a putative protein of 742 amino acids with a predicted 19-aa hydrophobic signal peptide, an MACPF domain, and a transmembrane domain. Phylogenetic analysis shows that Cg-Mpeg1 is similar to other mollusk MACPF proteins and might originate in an ancient ancestor gene before the divergence of protostomes and deuterostomes. Localization study revealed that Cg-Mpeg1 protein is found primarily in late endosomes. The MACPF domain from Cg-Mpeg1 exhibits significant antibacterial activity to both Gram-negative and positive bacteria. Furthermore, Real-time Quantitative PCR analysis showed that Cg-Mpeg1 is expressed in all tissues detected with highest expression in gill and gonads. Moreover, Mpeg1 mRNA levels are significantly up-regulated following infection with Vibrio alginolyticus. These results highlight that Cg-Mpeg1 plays an essential role in host defense and elimination of pathogens in C. gigas. [Copyright &y& Elsevier]

Abstract: In invertebrates and vertebrates, carbohydrate-binding proteins (lectins) play an important role in innate immunity against microbial invasion. In the present study, we report the cloning of an F-type lectin (designated as PmF-lectin) from pearl oyster (Pinctada martensii) using a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of PmF-lectin contains an open reading frame (ORF) of 579 bp coding for192 amino acids. The deduced polypeptide possesses six conserved residues of the F-lectin family critical for the formation of disulfide bonds (Cys43-Cys143, Cys75-Cys76 and Cys102-Cys119). Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) analyses in adult tissues showed that the PmF-lectin mRNA was abundantly expressed in haemocytes and gill, moderately expressed in the mantle, and rarely expressed in other tissues tested. After challenge with Vibrio alginolyticus, expression of PmF-lectin mRNA in haemocytes was dramatically up-regulated, reaching the highest level (13-fold higher than that of the control group) at 3 h post challenge, and then dropped gradually. These results suggest that PmF-lectin is a member of the F-lectin family and is involved in the innate immune response in pearl oyster. [Copyright &y& Elsevier]

Abstract Interferon regulatory factors (IRF), a family of transcription factors, are involved in the regulation of interferon to response the pathogen infection. Here, three IRF-like genes including Cg IRF1a, Cg IRF1b and Cg IRF8 were identified in the genome of the oyster C. gigas. Among these genes, Cg IRF1a and Cg IRF1b, which are tandemly located in adjacent loci of scaffold 4, share the same domains. Phylogenetic analysis indicated that Cg IRF1a and Cg IRF1b were two paralogs that may originate from duplication of the same ancestral IRF gene. Subcellular localization analysis confirmed the nuclear distribution of Cg IRF1a and Cg IRF1b. Dual-luciferase reporter assay showed that Cg IRF1a significantly activated the ISRE reporter gene, whereas Cg IRF1b did not. Additionally, overexpression of Cg IRF1b could significantly suppress the activation effect of Cg IRF1a, which strongly suggests that Cg IRF1b may serve as a regulator of the IRF signaling pathway. Furthermore, the result of native page revealed that Cg IRF1a would form homologous dimers, and Cg IRF1b would interact with Cg IRF1a to inhibit the activity of the latter. Taken together, one novel regulatory model of IRF signaling pathways has been raised one paralog of IRF has evolved and appears to be a regulator of IRF. Highlights • Three genes from Crassostrea gigas encoding IRFs were identified. • CgIRF1a and CgIRF1b were two paralogs which were tandemly located in adjacent loci and shared the same domains. • CgIRF1a significantly activated the ISRE reporter gene. • CgIRF1b may serve as one regulator of the IRF signaling pathway by effectively inhibiting the activity of Cg IRF1a. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor superfamily (TNFSF) represents a group of multifunctional inflammatory cytokines that have been shown to participate in a variety of pathological and immunological process. However, the functions of these proteins in oyster are still poorly understood. In the present study, an oyster TNF homolog (named Ch TNF) was identified from a cDNA library of Crassostrea hongkongensis . The complete cDNA of Ch TNF was 2457 bp in length containing an open reading frame (ORF) of 1044 bp, a 5′-untranslated region (UTR) of 381 bp and a 3′-UTR of 1032 bp. The deduced Ch TNF protein consisted of 347 amino acids with a characteristic transmembrane domain and a typical TNF homology domain (THD). Quantitative real-time PCR analysis revealed that Ch TNF was broadly expressed in various oyster tissues and different stages of embryonic development. In addition, transcriptional analysis indicated that Ch TNF transcription levels in hemocytes were increased significantly in pathogen challenge groups ( Vibrio alginolyticus and Staphylococcus haemolyticus ) compared to that in the control. Moreover, in vitro PAMP (lipopolysaccharide and peptidoglycan) treatments showed a stimulatory effect on the expression of Ch TNF in the primary cultured hemocytes of C. hongkongensis . Finally, dual-luciferase reporter assays revealed that Ch TNF could activate the NF-κB-Luc reporter in a dose-dependent manner in HEK293T cells. Altogether, these findings may provide valuable information regarding oyster TNFs and its role in innate immunity. [ABSTRACT FROM AUTHOR]

Thymosin beta-4 (Tβ4) is a ubiquitous protein with multiple and diverse intracellular and extracellular functions in vertebrates. In this study, the full-length cDNA of Tβ4 was cloned and identified in Crassostrea hongkongensis , designated as ChTβ4 . The full-length cDNA of ChTβ4 consists of 530 bp with an open reading frame of 126 bp encoding a 41 amino acid polypeptide. SMART analysis indicated that there is one thymosin domain and a highly conserved actin-binding motif (18LKKTET23) in ChTβ4. In vivo injection of recombinant ChTβ4 protein could significantly increase total hemocytes count in oysters, and knockdown of the expression of ChTβ4 resulted in a significant decrease in the circulating hemocytes. Tissue distribution analysis revealed a ubiquitous presence of ChTβ4, with the highest expression in hemocytes. The upregulated transcripts of ChTβ4 in response to bacterial challenge and tissue injury suggest that ChTβ4 is involved in both innate immunity against pathogen infection and wound healing. Moreover, bacteria-clearance experiment showed ChTβ4 could facilitate the clearance of injected bacteria in oysters. In vivo injection with ChTβ4 resulted in reduction of the intracellular ROS in hemocytes, which was associated with increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD), Catalase, and Glutathione Peroxidase (GPX) by pre-treatment with ChTβ4. These results suggest that ChTβ4 is a thymosin beta-4 homolog and plays a vital role in the immune defense of C. hongkongensis . [ABSTRACT FROM AUTHOR]

Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5′ untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3′ UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40 , with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster. [ABSTRACT FROM AUTHOR]

MDM2 (mouse double-minute) and p53 form a negative feedback loop and play a prominent role in preventing the induction of uncontrolled apoptosis. To better understand their potential roles in oyster Crassostrea hongkongensis , MDM2 and p53 homologs were first isolated and cloned in C. hongkongensis (named Ch MDM2 and Ch p53), and their mRNA expression patterns in tissues and developmental stages were analyzed. Multiple sequence alignment analysis and phylogenetic analysis of Ch MDM2 and Ch p53 displayed a high degree of homology and conservation. In addition, exposure to Vibrio coralliilyticus resulted in DNA damage and apoptosis in the hemocytes of C. hongkongensis , and found that the mRNA expression level of Ch MDM2 was decreased, while the relative expression of Ch p53 was significantly increased in the hemocytes and gills. Furthermore, fluorescence from Ch MDM2-EGFP and Ch p53-Red were found to be distributed in the nucleus of HEK293T cells. Besides, dual-luciferase reporter assays showed that Ch MDM2 antagonized with Ch p53 and participates in p53 signaling pathway. In addition, the interaction between Ch MDM2 and Ch p53 was confirmed strongly by Co-immunoprecipitation assays. Furthermore, the results of RNAi showed that Ch MDM2 and Ch p53 participated in apoptosis which induced infection of V. coralliilyticus. Taken together, our results characterized the features of Ch MDM2 and Ch p53, which played a critical role in apoptosis of C. hongkongensis. • Ch MDM2 and Ch p53 were first isolated and cloned from C. hongkongensis. • Ch MDM2 antagonized with Ch p53 and participates in p53 signaling pathway. • Ch MDM2 and Ch p53 were located in the nucleus of HEK293T cells. • Ch MDM2 and Ch p53 participated in apoptosis during V. coralliilyticus infection. [ABSTRACT FROM AUTHOR]

Abstract: Microsomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster, Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5′ UTR of 39 bp, a 3′ UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx1QRx2H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria. [Copyright &y& Elsevier]

Abstract: Receptor for activated C kinase 1 (RACK1) is involved in superoxide anion generation and play an important role in the immune response. In the study, we cloned the full-length sequence of pearl oyster, Pinctada martensii, RACK1 (designated as PmRACK1) by a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmRACK1 is 1176 bp in length, containing a 5′ UTR of 83 bp, a 3′ UTR of 139, and an open reading frame (ORF) of 954 bp encoding 317 amino acids. Analysis of protein domain features showed that the deduced polypeptide contain seven WD domains characteristic of RACK1 protein family. The tissue distribution of PmRACK1 in unchallenged pearl oysters and temporal expression pattern of PmRACK1 in pearl oysters challenged with bacteria and exposed to 0.1 ppm cadmium were analyzed by quantitative real-time PCR (qRT-PCR). The transcript was detected in all tissues tested, and the expression level was highest in hepatopancreas and lowest in adductor muscle. After challenge with bacteria, expression level of PmRACK1 in haemocytes was gradually decreased until 6 h post challenge, and then up-regulated over time. After exposure to cadmium, its expression level in gill decreased on 1 d post exposure, and then increased as time elapsed, and its expression level in hepatopancreas gradually decreased until 2 d post exposure, and then increased over time. These results suggested that PmRACK1 was involved in oxidative stress response caused by bacteria and cadmium and was a useful biomarker for cadmium exposure. The expression pattern of PmRACK1 in response to bacterial challenge also has a potential link with organism’s immune response. [Copyright &y& Elsevier]

Abstract: Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3′, 5′-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion. [Copyright &y& Elsevier]

Abstract: Tumor necrosis factor (TNF) receptor-associated factor 7 (TRAF7) is one of several adaptor proteins that are critically involved in the activation of TLR-dependent NF-κB signaling. In this report, the first mollusk TRAF7 (designated ChTRAF7) homolog was isolated from Crassostrea hongkongensis by screening a suppression subtractive library. The full-length cDNA, 2290 bp in length, encodes a putative protein of 686 amino acids that contains a RING finger domain, an adjacent zinc finger domain, and seven WD40 repeats. ChTRAF7 is ubiquitously expressed in various tissues including digestive gland, mantle, gill, heart, hemocytes, muscle, and gonads, with the highest expression observed in gonads. Temporal expression of ChTRAF7 following bacterial infection shows that expression of ChTRAF7 in hemocytes decreases from 2 to 12 h post-challenge, and then recovered to the original level after 24 h. These results indicate that ChTRAF7 may play an important role in signal transduction in the immune response of oysters. [Copyright &y& Elsevier]