First-Principles Study of Bimetallic Pairs Embedded on Graphene Co-Doped with N and O for N2 Electroreduction.

The electrocatalytic nitrogen reduction reaction (NRR) is considered a viable alternative to the Haber-Bosch process for ammonia synthesis, and the design of highly active and selective catalysts is crucial for the industrialization of the NRR. Dual-atom catalysts (DACs) with dual active sites offer flexible active sites and synergistic effects between atoms, providing more possibilities for the tuning of catalytic performance. In this study, we designed 48 graphene-based DACs with N4O2 coordination (MM'@N4O2-G) using density functional theory. Through a series of screening strategies, we explored the reaction mechanisms of the NRR for eight catalysts in depth and revealed the "acceptance-donation" mechanism between the active sites and the N2 molecules through electronic structure analysis. The study found that the limiting potential of the catalysts exhibited a volcano-shaped relationship with the d-band center of the active sites, indicating that the synergistic effect between the bimetallic components can regulate the d-band center position of the active metal M, thereby controlling the reaction activity. Furthermore, we investigated the selectivity of the eight DACs and identified five potential NRR catalysts. Among them, MoCo@N4O2-G showed the best NRR performance, with a limiting potential of -0.20 V. This study provides theoretical insights for the design and development of efficient NRR electrocatalysts.

Hymenobacter endophyticus sp. nov., isolated from wheat leaf tissue.

A bacterium, designated strain ZK17L-C2T, was isolated from the leaf tissues of wheat (Triticum aestivum) collected in Chengdu, Sichuan Province, PR China. It is aerobic, non-motile, Gram-negative, rod-shaped and red-to-pink in colour. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZK17L-C2T belonged to the genus Hymenobacter and was most closely related to Hymenobacter rigui KCTC 12533T (98.68 %) and Hymenobacter metallilatus 9PBR-2T (98.19 %). Digital DNA-DNA hybridization (dDDH) values between strain ZK17L-C2T and these two type strains were 26.6 and 26.5 %, and average nucleotide identity (ANI) values were 84.9 and 84.8 %, respectively; these values are lower than the proposed and generally accepted species boundaries for dDDH and ANI. The genomic DNA G+C content of strain ZK17L-C2T was 59.4 mol%. It can grow at pH 5.5-7.5 and 15-30 °C, which is different from the closely related type strains. The major fatty acids of strain ZK17L-C2T were iso-C15 : 0, C16 : 0 and C18 : 0. Overall, the results from biochemical, chemical taxonomy and phylogenetic analyses indicate that strain ZK17L-C2T (=CGMCC 1.19373T=KCTC 92184 T) represents a new species of the genus Hymenobacter, for which the name Hymenobacter endophyticus sp. nov. is proposed.

Molecular cloning and characterization of endoplasmic reticulum stress related genes grp78 and atf6α from black seabream (Acanthopagrus schlegelii) and their expressions in response to nutritional regulation.

Glucose-regulated protein 78 (grp78) and activating transcription factor 6α (atf6α) are considered vital endoplasmic reticulum (ER) molecular chaperones and ER stress (ERS) sensors, respectively. In the present study, the full cDNA sequences of these two ERS-related genes were first cloned and characterized from black seabream (Acanthopagrus schlegelii). The grp78 cDNA sequence is 2606 base pair (bp) encoding a protein of 654 amino acids (aa). The atf6α cDNA sequence is 2168 base pair (bp) encoding a protein of 645 aa. The predicted aa sequences of A. schlegelii grp78 and atf6α indicated that the proteins contain all the structural features, which were characteristic of the two genes in other species. Tissues transcript abundance analysis revealed that the mRNAs of grp78 and atf6α were expressed in all measured tissues, but the highest expression of these two genes was all recorded in the gill followed by liver/ brain. Moreover, in vivo experiment found that fish intake of a high lipid diet (HLD) can trigger ERS by activating grp78/Grp78 and atf6α/Atf6α. However, it can be alleviated by dietary betaine supplementation, similar results were also obtained by in vitro experiment using primary hepatocytes of A. schlegelii. These findings will be beneficial for us to evaluate the regulator effects of HLD supplemented with betaine on ERS at the molecular level, and thus provide some novel insights into the functions of betaine in marine fish fed with an HLD.

Isolation, characterization and application of a lytic phage vB_VspM_VS1 against Vibrio splendidus biofilm.

Vibrio splendidus is a common pathogen in the ocean that infects Apostichopus japonicus, Atlantic salmon and Crassostrea gigas, leading to a variety of diseases. In this study, a virulent phage vB_VspM_VS1, which infects V. splendidus, was isolated from aquaculture ponds in Dalian, China, and it belongs to the family Straboviridae in the order Caudoviricetes. vB_VspM_VS1 had an adsorption rate of 96% in 15 min, a latent period of 65 min, and a burst size of 140 ± 6 PFU/cell. The complete genome of phage vB_VspM_VS1 consists of a linear double-stranded DNA that is 248,270 bp in length with an average G + C content of 42.5% and 389 putative protein-coding genes; 116 genes have known functions. There are 4 tail fiber genes in the positive and negative strands of the phage vB_VspM_VS1 genome. The protein domain of the phage vB_VspM_VS1 tail fibers was obtained from the Protein Data Bank and the SMART (http://smart.embl.de) database. Bacterial challenge tests revealed that the growth of V. splendidus HS0 was apparently inhibited (OD600 < 0.01) in 12 h at an MOI of 10. In against biofilms, we also showed that the OD570 value of the vB_VspM_VS1-treated group (MOI = 1) decreased significantly to 0.04 ± 0.01 compared with that of the control group (0.48 ± 0.08) at 24 h. This study characterizes the genome of the phage vB_VspM_VS1 that infects the pathogenic bacterium V. splendidus of A. japonicus.

NEDD4 activates mitophagy by interacting with LC3 to restrain reactive oxygen species and apoptosis in Apostichopus japonicus challenged with Vibrio splendidus.

Mitophagy, the selective degradation of damaged mitochondria by autophagy, plays a crucial role in the survival of coelomocytes in Apostichopus japonicus following Vibrio splendidus infection by suppressing the generation of reactive oxygen species (ROS) and attenuating cell apoptosis. A recent study revealed that reducing the expression of the neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4), an enzyme 3 (E3) ubiquitin ligase, significantly affects mitochondrial degradation. Prior to the present study, the functional role of NEDD4 in marine invertebrates was largely unexplored. Therefore, we investigated the role of NEDD4 in the activation of mitophagy, modulation of ROS levels, and induction of apoptosis in A. japonicus infected with V. splendidus. The results demonstrated that V. splendidus infection and lipopolysaccharide (LPS) challenge significantly increased the mRNA levels of NEDD4 in A. japonicus coelomocytes, which was consistent with changes in mitophagy under the same conditions. Knockdown of AjNEDD4 using specific small interfering RNAs (siRNAs) impaired mitophagy and caused accumulation of damaged mitochondria, as observed using transmission electron microscopy (TEM) and confocal microscopy. Furthermore, AjNEDD4 was localized to the mitochondria in both coelomocytes and HEK293T cells. Simultaneously, coelomocytes were treated with the inhibitor indole-3-carbinol (I3C) to confirm the regulatory role of AjNEDD4 in mitophagy. The accumulation of AjNEDD4 in the mitochondria and the level of mitophagy decreased. Subsequent investigations demonstrated that AjNEDD4 interacts directly with the microtubule-associated protein light chain 3 (LC3), a key regulator of autophagy and mitophagy, indicating its involvement in the mitophagy pathway. Moreover, AjNEDD4 interference hindered the interaction between AjNEDD4 and LC3, thereby impairing the engulfment and subsequent clearance of damaged mitochondria. Finally, AjNEDD4 interference led to a significant increase in intracellular ROS levels, followed by increased apoptosis. Collectively, these findings suggest that NEDD4 acts as a crucial regulator of mitophagy in A. japonicus and plays a vital role in maintaining cellular homeostasis following V. splendidus infection. NEDD4 suppresses ROS production and subsequent apoptosis by promoting mitophagy, thereby safeguarding the survival of A. japonicus under pathogenic conditions. Further investigation of the mechanisms underlying NEDD4-mediated mitophagy may provide valuable insights into the development of novel strategies for disease control in aquaculture farms.

Genome-wide identification m6A modified circRNAs revealed their key roles in skin ulceration syndrome disease development in Apostichopus japonicus.

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m6A modification has been found in circRNA, and m6A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m6A levels are largely unknown. Here, we firstly investigated the genome-wide map of m6A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m6A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m6A peak and about 55% of m6A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m6A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m6A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m6A modification was constructed based on conjoint analysis. Our results indicated that several selected m6A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m6A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m6A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m6A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus.

Corrigendum: CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus.

[This corrects the article DOI: 10.3389/fimmu.2021.770055.].

Global N6-methyladenosine methylation analysis reveals the positive correlation between m6A modification and mRNA abundance during Apostichopus japonicus disease development.

N6-methyladenosine (m6A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA), plays important roles in regulation of gene expression for fundamental biological processes and diverse physiological functions, including combating with pathogen infection. Here, we were first profile transcriptome-wide m6A sequencing in four stages of skin ulceration syndrome-diseased Apostichopus japonicus following Vibrio splendidus infection, including Control (healthy), Early (small ulcer), Later (extensive ulcer), and Resistant (no ulcer) groups. Our results revealed that three experimental groups were all extensively methylated by m6A and the proportion of the m6A modified genes were also significantly increased to 28.90% (Early), 27.97% (Later), and 29.98% (Resistant) when compared with Control group (15.15%), indicating m6A modification could be induced by V. splendidus infection. Intriguingly, we discovered a positive correlation between the m6A methylation level and mRNA abundance, indicating a positive regulatory role of m6A in sea cucumber gene expression during V. splendidus infection. Moreover, genes with specific and differentially expressed m6A methylation in Later group were both enriched in cell adhesion, while Early and Resistant groups were both mainly involved in DNA conformation change and chromosome organization when compared with Control, suggesting the higher-methylated m6A might serve as "conformational marker" and associated to the initiation of related anti-disease genes transcription in order to improve disease resistance of sea cucumber. Subsequently, we selected the pivotal genes enriched in cell adhesion pathway and found that the IggFc-binding protein (FcGBP) and Fibrocystin-L both had higher levels of m6A methylation and higher level of mRNA expressions in Later group. Conversely, Fibrinogen C domain-containing protein 1 (F1BCD1) gene presented as an antibacterial role in sea cucumber and showed higher mRNA expression and higher m6A methylation in Resistant group and lower mRNA level in Later group. The levels of m6A methylation and mRNA abundance of FcGBP and F1BCD1 genes indicates disease occurrence or disease resistant were also verified by MeRIP-qPCR. Overall, our study presents the first comprehensive characterize of dynamic m6A methylation modification in the different stages of disease in sea cucumber. These data provide an invaluable resource for future studies of function and biological significance of m6A in mRNA in marine invertebrates.

CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus.

Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3'-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.

BAG2 mediates coelomocyte apoptosis in Vibrio splendidus challenged sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) are closely related to the occurrence, development, and immune response of diseases. BCL2-associated athanogene 2 (BAG2) is a member of the BAG family that functions in diverse cellular processes, including cell death, differentiation, and cell division. In this study, we cloned the cDNA full-length of sea cucumber (Apostichopus japonicus) BAG2 (AjBAG2) and confirmed it is an anti-apoptotic protein in vitro and in vivo during Vibrio splendidus infection. Moreover, we identified a perfect complementarity between miR-375 and the 3'-untranslated region (UTR) sequence of AjBAG2. The miR-375 expression decreased the luciferase activity dose-dependently when co-transfected with the AjBAG2 3'-UTR-luciferase reporter containing the miR-375 target site in epithelioma papulosum cyprini (EPC) cells. This inhibition was partially recovered by a miR-375 specific inhibitor. The mRNA and protein levels of AjBAG2 were opposite to that of coelomocytes in challenged sea cucumber when treated with miR-375 mimics or inhibitors. Additionally, miR-375 expression induced coelomocytes apoptosis and blocked the anti-apoptotic activity of AjBAG2. Our data demonstrated that AjBAG2 is an anti-apoptotic protein during V. splendidus infection and this function can be inhibited by miR-375 in sea cucumbers.

Multi-Level Features Extraction for Discontinuous Target Tracking in Remote Sensing Image Monitoring.

Many techniques have been developed for computer vision in the past years. Features extraction and matching are the basis of many high-level applications. In this paper, we propose a multi-level features extraction for discontinuous target tracking in remote sensing image monitoring. The features of the reference image are pre-extracted at different levels. The first-level features are used to roughly check the candidate targets and other levels are used for refined matching. With Gaussian weight function introduced, the support of matching features is accumulated to make a final decision. Adaptive neighborhood and principal component analysis are used to improve the description of the feature. Experimental results verify the efficiency and accuracy of the proposed method.

Genome-Wide Identification of Circular RNAs Revealed the Dominant Intergenic Region Circularization Model in Apostichopus japonicus.

Circular RNAs (circRNAs) were recently recognized to act as competing endogenous RNAs and play roles in gene expression regulation. Previous studies in humans and silkworms have shown that circRNAs take part in immune regulation. Here, we conducted coelomocyte circRNA sequencing to explore its immune functions in healthy and skin ulceration syndrome (SUS)-diseased sea cucumbers. A total of 3,592 circRNAs were identified in libraries with diversified circularization patterns compared with animal models. The common intron-pairing-driven circularization models are not popular in sea cucumber genome, which was replaced with intergenic region circularization. The accuracy of these identified circRNAs was further validated by Sanger sequencing and RNase R-treated assays. Expression profile analysis indicated that 117 circRNAs were upregulated and 144 circRNAs were downregulated in SUS-diseased condition, of which 71.6% were intergenic-type circRNAs. The interaction network of differentially expressed circRNAs and microRNAs (miRNAs) was constructed and showed that miR-2008 and miR-31, detected with significantly differential expression in SUS-affected samples in a previous study, were predicted to be regulated by 10 and 11 differentially expressed circRNAs with more than 10 binding sites, respectively. Moreover, seven circRNAs were further validated by quantitative real-time PCR, whose variation trends were consistent with circRNA sequencing. All our results supported that intergenic-type circRNAs might have a dominant function in Apostichopus japonicas immune response by acting as miRNA regulators.

Characterization of NLRP3-like gene from Apostichopus japonicus provides new evidence on inflammation response in invertebrates.

Inflammatory/defensive response after pathogen invasion is considered a local defense reaction in vertebrates. Inflammation response in Apostichopus japonicus was hardly determined due to scarce information available for nucleotide binding domain-like receptor family, pyrin domain-containing (NLRP) family. In the present study, invertebrate NLRP homologue was identified from A. japonicus (designated as AjNLRP3-like) by rapid amplification of cDNA ends. Full-length cDNA of AjNLRP3-like measured 2970 bp with 2265 bp open reading frame encoding a 754-amino acid (aa) residue protein. Structural analysis revealed that AjNLRP3-like processed characteristic domains of pyrin (32-102aa) and NACHT (183-339aa). Multiple sequence alignment and phylogenetic analysis supported that AjNLRP3-like belongs to a new member of NLRP3 protein subfamily. Spatial expression analysis revealed that AjNLRP3-like was ubiquitously expressed in all examined tissues with larger magnitude in coelomocytes. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly upregulated mRNA expression of AjNLRP3-like when compared with the control group. NLRP3-mediated inflammation response depended on release of lysosomal cathepsin B (CTSB) and subsequent activation of high-mobility group box (HMGB) in vertebrates. We investigated expression profiles of AjNLRP3-like and AjHMGB after AjCTSB knock-down and discovered that AjNLRP3-like was depressed by 0.66-fold and 0.47-fold, whereas AjHMGB was depressed by 0.70-fold and 0.50-fold at 24 and 48 h in AjCTSB-silenced group, respectively. Similarly, down-regulation of AjHMGB was also observed after AjNLRP3-like knock-down. This study therefore suggests that A. japonicus feature similar inflammatory events as those in vertebrates, and activation of AjNLRP3-like depends on AjCTSB expression and release of AjHMGB.

Comparative transcriptome analysis of Sinonovacula constricta in gills and hepatopancreas in response to Vibrio parahaemolyticus infection.

The razor clam Sinonovacula constricta is an important economic species in China. However, bacterial pathogenic diseases limits S. constricta farming industry for large-scale production. In this study, de novo transcriptome sequencing was performed on S. constricta gills and hepatopancreas under Vibrio parahaemolyticus challenge for 12 h and 48 h, respectively. Transcripts assembly constructed 18,330 sequences, each of which was 500 bp long and functionally annotated, and 1781 and 490 transcripts were differentially expressed in the gills and hepatopancreas, respectively. Host immune factors that respond to Vibrio infection were then identified. These factors included up-regulated transcripts with function in non-self recognition, signal transduction, immune effectors and anti-apoptosis. The comparison between the differentially expressed transcripts of the gills and hepatopancreas indicated that immune responses had tissue specificity. As an important external barrier between the environment and the clam, ATP-binding cassette transporters and other ion transporters contribute to immune response in gills, while, transcripts in complement system, such as complement 1 q protein, IgGFc-binding protein, and low affinity immunoglobulin epsilon Fc receptor, were more active in hepatopancreas and often not expressed in gill tissues. Eleven genes were selected to be validated by qRT-PCR and the expressions were consistent with the results of RNA-seq. Our study is the first attempt to identify molecular features in different tissues of S. constricta in response to V. parahaemolyticus infection. These findings improved our understanding of bivalve immunity and defense mechanisms and revealed more potential immune-related genes.

Divergent roles of three cytochrome c in CTSB-modulating coelomocyte apoptosis in Apostichopus japonicus.

Cytochrome c plays crucial roles in apoptosis and the immune response. We previously demonstrated that cathepsin B from Apostichopus japonicus (AjCTSB) induces coelomocyte apoptosis. However, the mechanistic explanation and the regulation of this process have not been investigated. In the present study, we identified three cytochrome c cDNAs from A. japonicus (designated Ajcytc1, Ajcytc-1, and Ajcytc-2) using expressed sequence tag- (EST) and RACE-based approaches. The deduced amino acid sequences of the three cytochrome isoforms contained conserved CXXCH motifs, which are involved in binding heme and maintaining proteolytic activity. Time course expression analysis in vitro and in vivo revealed that the three cytochrome isoforms were induced upon pathogen challenge and LPS exposure. More importantly, AjCTSB knockdown by siRNA dramatically increased mitochondrial membrane potential (ΔΨm) in a time-dependent manner based on JC-1 fluorescent probe staining. AjCTSB knockdown also resulted in decreased expression of these three cytochromes 24 h after siAjCTSB transfection. Functional analysis using isoform-specific siRNAs revealed that Ajcytc-1, but not Ajcytc1 or Ajcytc-2, is involved in coelomocyte apoptosis. Moreover, the transcript level of Ajcaspase-3, an apoptosis executioner, was also consistently down-regulated upon silencing of Ajcytc-1 but not Ajcytc1 or Ajcytc-2. Collectively, these results indicate that Ajcytc1, Ajcytc-1, and Ajcytc-2 play distinct roles in mediating the immune response to bacteria according to AjCTSB expression. Moreover, Ajcytc-1 could be released upon dissipation of the ΔΨm, which could further trigger coelomocyte apoptosis through the activation of Ajcaspase-3.

Microsomal glutathione transferase 1 attenuated ROS-induced lipid peroxidation in Apostichopus japonicus.

Microsomal glutathione transferase (mGST) is a membrane bound glutathione transferase in multifunctional detoxification isoenzymes family and also plays crucial roles in innate immunity. In the present study, a novel microsomal GST homology was identified from Apostichopus japonicus (designated as AjmGST1) by RACE approaches. The full-length cDNA of AjmGST1 was of 1296 bp encoded a protein of 169 amino acids residues. Multiple sequence alignment and phylogenetic analysis together supported that AjmGST1 belonged to a new member in invertebrates mGST family. Spatial expression analysis revealed that AjmGST1was ubiquitously expressed in all examined tissues with the larger magnitude in tentacle. Time-course expression of AjmGST1 mRNA in coelomocytes was up-regulated after Vibrio splendidus challenge from 6 h until 72 h with the peak expression in 24 h, compared with that in the control group. Similarly, the induced expression of AjmGST1 expression was also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. The purified recombinant protein of AjmGST1 showed high activity with GST substrate at pH of 7.0 and temperature of 35 °C. Meantime, the recombinant AjmGST1 depressed H2O2-induced MDA production both in vivo and in vitro. All of these results indicated that AjmGST1 was an important regulator in elimination of lipid peroxidation under immune response.

Molecular cloning and functional characterization of theta class glutathione S-transferase from Apostichopus japonicus.

Glutathione S-transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST-θ) by RACE approaches. The full-length cDNA of AjGST-θ was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST-θ processed the characteristic N-terminal GSH-binding site (G-site) and the C-terminal hydrophobic substrate binding site (H-site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST-θ belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST-θ was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up-regulate the mRNA expression of AjGST-θ when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST-θ showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST-θ protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST-θ could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST-θ might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus.

miR-137 modulates coelomocyte apoptosis by targeting 14-3-3ζ in the sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) have emerged as key regulators in the host immune response and play a pivotal role in host-pathogen interactions by suppressing the transcriptional and post-transcriptional expression of target genes. miR-137, a well-documented tumor repressor, was previously found by high-throughput sequencing to be differentially expressed in diseased specimens of the sea cucumber Apostichopus japonicus. In this study, we identified 14-3-3ζ protein (Aj14-3-3ζ) as a novel target of miR-137 using isobaric tags for relative and absolute quantification (iTRAQ) and transcriptome screening. Expression analysis indicated that consistently depressed expression profiles of miR-137 and Aj14-3-3ζ were detected in both LPS-exposed primary coelomocytes and Vibrio splendidus-challenged sea cucumbers, suggesting a positive regulatory interaction. Consistently, miR-137 overexpression or inhibition in vitro and in vivo showed no effect on Aj14-3-3ζ mRNA levels, but the concentration of Aj14-3-3ζ protein was induced or repressed, respectively. Moreover, siRNA-mediated Aj14-3-3ζ knockdown in vivo decreased both mRNA and protein expression levels of Aj14-3-3ζ and significantly promoted coelomocyte apoptosis as assessed by flow cytometry, consistent with miR-137 inhibition. Overall, these results enhance our understanding of miR-137 regulatory roles in sea cucumber pathogenesis.

Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus.

Cathepsin B (CTSB), a member of lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and also plays crucial roles in host immune defense against pathogen infection in vertebrates. However, the function of CTSB within the innate immune system of invertebrates, particularly in marine echinoderms, has been poorly documented. In this study, the immune function of CTSB in Apostichopus japonicus (designated as AjCTSB), a commercially important and disease vulnerable aquaculture specie, was investigated by integrated molecular and protein approaches. A 2153 bp cDNA representing the full-length of AjCTSB was cloned via overlapping ESTs and RACE fragments. AjCTSB contained an open reading frame of 999 bp encoding a secreted protein of 332 amino acid residues with a predicted molecular mass of 36.8 kDa. The deduced amino acid of AjCTSB shared a typical activity center containing three conserved amino acid residues (Cys108, His277 and Asn297). Phylogenetic tree analysis also supported that AjCTSB was a new member of CTSB family with clustering firstly with invertebrate CTSBs. Quantitative real time PCR analysis revealed that AjCTSB was ubiquitously expressed in all examined tissues with the highest levels in intestine. The Vibrio splendidus challenged sea cucumber and LPS-exposed coelomocytes could both significantly boost the expression of AjCTSB. Moreover, the purified recombinant AjCTSB exhibited dose-dependent CTSB activities at the concentration ranged from 0 to 0.24 µg µL-1. Further functional analysis indicated that coelomocytes apoptosis was significantly inhibited by 0.16-fold in vivo and the apoptosis execution Ajcaspase 3 was extremely reduced in Apostichopus japonicus coelomocytes treated with specific AjCTSB siRNA. Collectively, all these results suggested that AjCTSB was an important immune factor and might be served as apoptosis enhancers in pathogen challenged sea cucumber.

Cloning and comparative analysis the proximal promoter activities of arginase and agmatinase genes in Apostichopus japonicus.

Our previous work demonstrated that Apostichopus japonicus arginase and agmatinase from l-arginine metabolism synergistically compete with NOS under pathogens challenge. Here we conducted a study to further investigate the mechanism in the regulation of arginase and agmatinase genes in l-arginine metabolism using EPC cell system. Luciferase analysis and progressive 5' deletion analysis suggested that Ajagmatinase promoter was a very robust promoter for its transcription, and the core region of Ajarginase promoter was located within -277 bp to -157 bp. Besides, their promoter activities were significantly activated by LPS and l-arginine challenge both in a time- and dose-dependent manners in EPC cells. When different truncated reporter vector and expression vector co-transfection experiment revealed transcription factor NF-κB/Rel and STAT5 could significantly inhibited Ajarginase promoter activity, but not Ajagmatinase. Our findings were provided novel insights into the transcriptional regulation of Ajarginase and Ajagmatinase, and selectively change their expressions might prevent pathogens infection.