ChRIPK1 caused necroptosis signaling pathway deficiency in Crassostrea hongkongensis.

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 ChRIPK1 and ChTAK1 were cloned from C. hongkongensis through the rapid amplification of cDNA ends (RACE) technology. ChRIPK1 has almost no homology with human RIPK1 and lacks a kinase domain at the N-terminus but has a DD and RHIM domain. ChTAK1 is conserved throughout evolution. qRT‒PCR was used to analyze the mRNA expression patterns of ChRIPK1 in different tissues, developmental stages, and V. coralliilyticus-infected individuals, and both were highly expressed in the mantle and gills, while ChRIPK1 was upregulated in hemocytes and gills after V. coralliilyticus or S. aureus infection, which indicates that ChRIPK1 is involved in immune regulation. Fluorescence assays revealed that ChRIPK1 localized to the cytoplasm of HEK293T cells in a punctiform manner, but the colocalization of ChRIPK1 with ChTAK1 abolished the punctiform morphology. In the dual-luciferase reporter assay, both ChRIPK1 and ChRIPK1-RIHM activated the NF-κB signaling pathway in HEK293T cells, and ChTAK1 activated ChRIPK1 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 ChRIPK1 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.

A Testis-Specific DMRT1 (Double Sex and Mab-3-Related Transcription Factor 1) Plays a Role in Spermatogenesis and Gonadal Development in the Hermaphrodite Boring Giant Clam Tridacna crocea.

The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

Characterization and functional analysis of a caspase 3 gene: Evidence that ChCas 3 participates in the regulation of apoptosis in Crassostrea hongkongensis.

Caspase 3 plays an important role in apoptotic pathways and contributes to maintaining the homeostasis of the immune system in organisms. The structure, functions, and characteristics of caspase 3 have been extensively investigated in many species, but the research is scarce when it comes to bivalves, particularly oysters. In this study, we identified and cloned a previously unknown caspase 3 gene, named ChCas 3, in C. hongkongensis. The full-length cDNA of ChCas 3 was 1562 bp and included a 175 bp 5'-untranslated region (UTR), a 141 bp 3'-UTR and a 1245 bp open reading frame (ORF) that encoded a polypeptide of 415 amino acids. Similar to caspase 3 in other species, ChCas 3 has a pro-domain, a conserved cysteine active site, a large p20 subunit and a small p10 subunit. Our findings demonstrated the expression of ChCas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. ChCas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1-ChCas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of ChCas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of ChCas 3 led to upregulation of the transcriptional activity of NF-κB and p53 reporter genes in HEK293T cells, which indicated that it was involved in innate immune responses. Finally, the apoptosis rate of the haemocytes declined considerably compared with that of the control group after the expression of ChCas 3 was successfully silenced by dsRNA, corroborating its sentinel role in apoptosis. This study provides comprehensive underpinning evidences, affirming caspase 3 crucial role against bacterial infection and apoptosis in C. hongkongensis.

[Determination of 4 polycyclic aromatic hydrocarbons in spicy strips by vacuum concentration coupled with isotope dilution gas chromatography-mass spectrometry].

OBJECTIVE: A method based on isotope internal standard dilution was established for the determination of four polycyclic aromatic hydrocarbons(PAH4) including chrysene, benzo [a] anthracene, benzo [b] fluoranthene and benzo [a] pyrene in spicy strips sold in the markets. METHODS: The hot strips were homogenized and the target compounds were extracted with n-hexane, concentrated in vacuum, saponified and purified by solid phase extraction column, then the pretreated samples were separated by DB-EUPAH column(20 m×0. 18 mm, 0. 14 µm), detected by gas chromatography-mass spectrometry(GC-MS)and quantified by internal standard of isotope. RESULTS: The recoveries of PAH4 in different concentration levels of hot noodles were 91. 0%-103. 5%, and the relative standard deviation(RSD)was 1. 89%-6. 73%(n=6). The detection limit was 0. 30 µg/kg and the quantitative limit was 1. 0 µg/kg. The content of PAH4(sum) in 27 collected samples ranged from 1. 35 µg/kg to 11. 44 µg/kg, and the detection rate was 100%. CONCLUSION: With less solvent consumption, good purification effect and blank control, the method is simple, rapid and accurate and meets the detecting requirements of PAH4 in hot strips.

LncRNA FOXC2-AS1 protects cardiomyocytes from doxorubicin-induced cardiotoxicity through activation of WNT1-inducible signaling pathway protein-1.

Doxorubicin (Dox) is an anthracycline antibiotic that has been used to treat different cancers. Dox-induced cardiotoxicity is common in clinical practice, while its mechanism is unknown. It has been proved that lncRNA FOXC2-AS1 may promote doxorubicin resistance and WNT1-inducible signaling pathway protein-1 (WISP1) blocks doxorubicin-induced cardiomyocyte death. Our study aimed to investigate the involvement of lncRNA FOXC2-AS1 and WISP1 in doxorubicin-induced cardiotoxicity and to explore their interactions. In our study we observed that FOXC2-AS1 and WISP1 mRNA were downregulated in heart tissues of mice with Dox-induced cardiotoxicity. FOXC2-AS1 and WISP1 mRNA expression were positively correlated in mice with Dox-induced cardiotoxicity but not in healthy mice. Overexpression of FOXC2-AS1 promoted to viability of mice cardiomyocytes under Dox treatment and also increased the expression level of WISP1. In contrast, WISP1 overexpression showed no significant effect on FOXC2-AS1. We therefore conclude that lncRNA FOXC2-AS1 may upregulate WISP1 to protect cardiomyocytes from doxorubicin-induced cardiotoxicity.

Tumor necrosis factor receptor-associated factor 3 from Anodonta woodiana is an important factor in bivalve immune response to pathogen infection.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional adaptor protein in innate and acquired immune system that plays a key role in the regulation of the RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling pathway in mammals. However, the immune function of TRAF3 homologs in freshwater mollusks is not well understood. In this study, we identified a bivalve TRAF3 gene (AwTRAF3) from Anodonta woodiana and investigated its potential roles during immune challenges. The present AwTRAF3 encoded a polypeptide of 562 amino acids with predicted molecular mass of 64.5 kDa and PI of 7.9. Similar to other reported TRAF3s, AwTRAF3 contained a RING finger domain, two TRAF domains with zinc finger domains, a coiled coli region and a conserved C-terminal meprin and TRAF homology (MATH) domain. Quantitative real-time PCR (qRT-PCR) analysis revealed that AwTRAF3 mRNA was broadly expressed in all of the examined tissues, with high expression in hepatopancreas, gill and heart. In addition, immune challenge experiments directly showed that transcript levels of AwTRAF3 in hepatopancreas were significantly regulated upon bacterial (Vibrio alginolyticus and Staphylococcus aureus) and viral (poly (I:C)) challenges, respectively. Moreover, GFP-tagged AwTRAF3 fusion protein was found to be located primarily in the cytoplasm in HEK293T cells. Altogether, these data provided the first experimental demonstration that freshwater mollusks possess a functional TRAF3 that was involved in the innate defense against bacterial and viral infection.

A molluscan TNF receptor-associated factor 2 (TRAF2) was involved in host defense against immune challenges.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a member of the TRAF superfamily that acted as a key signal transduction protein and has been implicated in inflammatory and apoptosis processes in mammals. However, identification of TRAF2s in invertebrates is very limited and its function, in particular that under immune challenges, is still unknown. In this report, a molluscan TRAF2 gene (referred to as AwTRAF2) was cloned and characterized from the freshwater bivalve, Anodonta woodiana. The open reading frame (ORF) of AwTRAF2 was 1683 bp in length, which encoded a putative 560 amino acid-protein. The deduced AwTRAF2 sequence shared similar structural characteristics and close evolutionary relationship with mollusk TRAF2s. The tissue-specific expression analysis revealed that AwTRAF2 mRNA was broadly expressed in all tested tissues, with high expression in gill and hepatopancreas. In addition, in vivo injection experiments directly showed that AwTRAF2 mRNA levels in hepatopancreas were significantly up-regulated in response to bacterial pathogen (Vibrio alginolyticus and Staphylococcus aureus) and PAMPs (Lipopolysaccharides and Peptidoglycan) challenges. Moreover, fluorescence microscopy observations revealed that AwTRAF2 was mainly located in cytoplasm of HEK293T cells and its overexpression significantly increased the transcriptional activities of the NF-κB-Luc reporter gene in HEK293T cells. Taken together, this study provided the experimental evidence of the presence of a functional TRAF2 in freshwater bivalves, which revealed its involvement in host response to immune challenges in A. woodiana.

A molluscan extracellular signal-regulated kinase is involved in host response to immune challenges in vivo and in vitro.

Extracellular signal-regulated kinases (ERKs) are a group of highly conserved serine/threonine-specific protein kinases that function as important signaling intermediates in mitogen-activated protein kinase (MAPK) pathways, which are involved in a wide variety of cellular activities, including proliferation, inflammation and cytokine production. However, little is known about the roles of this kinase in mollusk immunity. In this study, we identified a molluscan ERK homolog (ChERK) in the Hong Kong oyster (Crassostrea hongkongensis) and investigated its biological functions. The open reading frame (ORF) of ChERK encoded a polypeptide of 365 amino acids, with a predicted molecular weight of 41.96 kDa and pI of 6.43. The predicted ChERK protein contained typical characteristic motifs of the ERK family, including a dual threonine-glutamate-tyrosine (TEY) phosphorylation motif and an ATRW substrate binding site. Phylogenetic analysis revealed that ChERK belonged to the mollusk cluster and shared a close evolutionary relationship with ERK from Crassostrea gigas. In addition, quantitative real-time PCR analysis revealed that ChERK expression was detected in all of the examined tissues and stages of embryonic development; its transcript level was significantly induced upon challenge with bacterial pathogens (Vibrio alginolyticus and Staphylococcus haemolyticus) in vivo and PAMPs (lipopolysaccharide and peptidoglycan) in vitro. Moreover, ChERK was mainly located in the cytoplasm of HEK293T cells. Taken together, these findings may provide novel insights into the functions of molluscan ERKs, especially their roles in response to immune challenge in oyster.

Cloning, characterization and comparative analysis of four death receptorTNFRs from the oyster Crassostrea hongkongensis.

Apoptosis plays an important role in homeostasis of the immune systems. The tumor necrosis factor receptors (TNFRs) play critical roles in the extrinsic apoptosis pathways and in determining cell fate. In this study, four death receptors (DR) named ChEDAR, ChTNFR27, ChTNFR5, and ChTNFR16 were identified from the oyster Crassostrea hongkongensis. These ChDRs proteins had 382, 396, 414 and 384 amino acids, respectively, with the typical domains of death receptors, such as the signal peptide (SP), transmembrane helix region (TM) and death domains. Phylogenetic analysis showed that the ChDR proteins clustered into three distinct groups, indicating that these subfamilies had common ancestors. mRNA expression of the ChDRs were detected in all 8 of the selected oyster tissues and at different stages of development. Furthermore, expression of all the genes was increased in the hemocytes of oysters challenged with pathogens or air stress. Fluorescence microscopy revealed that the full-length proteins of the ChDRs were located in the plasma membrane of HEK293T cells. Over-expression of the ChDRs activated the NF-κB-Luc reporter in HEK293T cells in a dose-dependent manner. These results indicate that the ChDRs may play important roles in the extrinsic apoptotic pathways in oysters.

Aquaculture Performance and Genetic Diversity of a New [(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀] Variety of the Oyster "South China No. 1" in Beibu Gulf, China.

Crassostrea hongkongensis is an economically important bivalve found in various parts of the South China Sea. A new interspecific backcross ([(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀]) variety was bred by the South China Sea Institute of Oceanology which named "South China No. 1". This study aims to explore the effects of stocking density on the growth performance of "South China No. 1", compared their growth performance and genetic diversity to C. hongkongensis, and found the best place breeding site for "South China No. 1" in Beibu Gulf. The results showed that stocking a density of 20 oysters/substrate can significantly increase the shell height, shell width, total weight, survival rate, daily shell height gain and daily body mass gain. It was found that the shell height and total weight of "South China No. 1" cultured in Fangchenggang were significantly higher than that of those in Beihai and Qinzhou from September 2018 to November 2018. Similarly, the shell width of oysters in Fangchenggang and Qinzhou was also significantly higher in September 2018, and the interaction between site and stocking density had significant effects on the shell width in March 2018 and November 2018. In addition, the shell height and shell width of "South China No. 1" were significantly higher than that of C. hongkongensis in all three sites. At all three sites, the phytoplankton community structure was mostly dominated by Bacillariophyta. In the Hardy-Weinberg equilibrium test, for the seven populations and ten microsatellites, in 10 of the 70 groups, the segregation distortion was significant. These results suggest that a stocking density of 20 oysters/substrate can promote the shell height, shell width and total weight of "South China No. 1" in Beibu Gulf, China. "South China No. 1" has better growth performance compared with C. hongkongensis. Fangchenggang is a suitable place to cultivate the "South China No. 1" breed according to the total weight and sum of all algal genus abundances. The results of this study can be used as a reference to further understand the stocking density and genetic diversity of the "South China No. 1" breed in Beibu Gulf, China.

High red/far-red ratio promotes root colonization of Serratia plymuthica A21-4 in tomato by root exudates-stimulated chemotaxis and biofilm formation.

Effective colonization on plant roots is a prerequisite for plant growth promoting rhizobacterias (PGPR) to exert beneficial activities. Light is essential for plant growth, development and stress response. However, how light modulates root colonization of PGPR remains unclear. Here, we found that high red/far red (R/FR) light promoted and low R/FR light inhibited the colonization and growth enhancement of Serratia plymuthica A21-4 (S. plymuthica A21-4) on tomato, respectively. Non-targeted metabolomic analysis of root exudates collected from different R/FR ratio treated tomato seedlings with or without S. plymuthica A21-4 inoculation by UPLC-MS/MS showed that 64 primary metabolites in high R/FR light-grown plants significantly increased compared with those determined for low R/FR light-grown plants. Among them, 7 amino acids, 1 organic acid and 1 sugar obviously induced the chemotaxis and biofilm formation of S. plymuthica A21-4 compared to the control. Furthermore, exogenous addition of five artificial root exudate compontents (leucine, methionine, glutamine, 6-aminocaproic acid and melezitose) regained and further increased the colonization ability and growth promoting ability of S. plymuthica A21-4 on tomato under low R/FR light and high R/FR light, respectively, indicating their involvement in high R/FR light-regulated the interaction of tomato root and S. plymuthica A21-4. Taken together, our results, for the first time, clearly demonstrate that high R/FR light-induced root exudates play a key role in chemotaxis, biofilm formation and root colonization of S. plymuthica A21-4. This study can help promote the combined application of light supplementation and PGPR to facilitate crop growth and health in green agricultural production.

Resistance of an intertidal oyster(Saccostrea mordax)to marine heatwaves and the implication for reef building.

Marine heatwaves (MHWs) have a significant impact on intertidal bivalves and the ecosystems they sustain, causing the destruction of organisms' original habitats. Saccostrea mordax mainly inhabits the intertidal zone around the equator, exhibiting potential tolerance to high temperatures and maybe a species suitable for habitat restoration. However, an understanding about the tolerance mechanism of S. mordax to high temperatures is unclear. It is also unknown the extent to which S. mordax can tolerate repeated heatwaves of increasing intensity and frequency. Here, we simulated the effects of two scenarios of MHWs and measured the physiological and biochemical responses and gene expression spectrum of S. mordax. The predicted responses varied greatly across heatwaves, and no heatwave had a significant impact on the survival of S. mordax. Specifically, there were no statistically significant changes apparent in the standard metabolic rate and the activities of enzymes of the oyster during repeated heatwaves. S. mordax exposed to high-intensity heatwaves enhanced their standard metabolic rate to fuel essential physiological maintenance and increasing activity of SOD and expression of HSP70/90. These strategies are presumably at the expense of functions related to immunity and growth, as best exemplified by significant depressions in activities of enzymes (NaK, CaMg, T-ATP, and AKP) and expression levels of genes (Rab, eEF-2, HMGR, Rac1, SGK, Rab8, etc.). The performance status of S. mordax tends to improve by implementing a suite of less energy-costly compensatory mechanisms at various levels of biological organization when re-exposed to heatwaves. The adaptive abilities shown by S. mordax indicate that they can play a crucial role in the restoration of oyster reefs in tropical seas.

[Chemical adsorption of thiosalicylic acid on silver surface investigated by surface-enhanced Raman scattering].

Surface-enhanced Raman scattering (SERS) of thiosalicylic acid (TSA) with sulfydryl group was investigated on the surface of electrochemically roughed silver electrode. The result shows that SERS enhancement effect was relative to the concentration and pH, and 1 x 10(-3) mol x L(-1) and pH 4 were the optimal condition. While the concentration increased, the enhancement effect decreased quickly because of steric hindrance. S--Ag peak position by the absorption of TSA was basically consistent, but pH significantly affected its intensity. The distribution and mechanism of TSA at different pH were further investigated. It was showed that TSA was adsorbed on the active silver surface via the sulfydryl group without H of neutral C4H4 (COOH)SH molecule. Competitive adsorption of negative valence C4H4 (COO-) SH and OH- may bring non-SERS under the strong base condition. At the same time, the sulfydryl group significantly influenced the carboxyl vibration peak's change and the distribution of electron cloud of benzene ring conjugate system.

Enhanced bioactivity and hydrothermal aging resistance of Y-TZP ceramics for dental implant.

Although yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) ceramics have been widely used as restorative materials due to their high mechanical strength, unique esthetic effect, and good biocompatibility, their general application to implant materials is still limited by their biological inertness and hydrothermal aging phenomenon. Existing studies have attempted to investigate how to enhance the bioactivity or hydrothermal aging resistance of Y-TZP. Still, more studies need to be done on the modification that combines these two aspects. In this study, Y-TZP was prepared by 77S bioactive glass (BG) sol and akermanite (AKT) sol infiltration and microwave sintering, which provided Y-TZP with high bioactivity while maintaining resistance to hydrothermal aging. Results of phase composition evaluation, microstructural characteristics, and mechanical property tests showed that modified Y-TZP specimens exhibited little or no tetragonal-to-monoclinic (t → m) transformation and maintained relatively high mechanical properties after accelerated hydrothermal aging treatment. The in vitro biological behaviors showed that the introduction of 77S BG and AKT significantly promoted cell adhesion, spreading, viability, and proliferation on the surface of modified Y-TZP ceramics. Therefore, this modification could effectively enhance the bioactivity and hydrothermal aging resistance of Y-TZP ceramics for its application in dental implant materials.

Luffa rootstock enhances salt tolerance and improves yield and quality of grafted cucumber plants by reducing sodium transport to the shoot.

Soil salinity severely limits crop yield and quality. Grafting onto tolerant rootstocks is known as an effective means to alleviate salt stress. The present study was planned to find out the potential roles, mechanisms and applications of luffa rootstock to improve salt tolerance of grafted cucumber plants. Here, we screened a highly salt-tolerant luffa rootstock by evaluating the growth, photosynthetic performance, antioxidant defense and the accumulation of Na+ and K+ under salt stress. Reciprocal grafting between cucumber and luffa showed that luffa rootstock significantly improved the salt tolerance of cucumber plants, as evidenced by higher fresh weight, photochemical efficiency (Fv/Fm), and lower relative electrical conductivity (REC), which was closely associated with the decreased accumulation of Na+ and increased the accumulation of K+ in shoots of luffa grafted cucumber seedlings, leading to a lower Na+:K+ ratio in shoot when compared with self-grafted cucumber. Furthermore, grafting with intermediate stock of luffa also sufficiently alleviated cucumber salt stress by reducing Na+ accumulation in shoot and the whole plant but increasing Na+ accumulation in interstock and root under salt stress, fully proving the salt tolerance depending on the capacity of luffa interstock to limit the transport of Na+ from the root to the shoot. More importantly, luffa rootstock improved the growth, yield and quality of grafted cucumber plants grown in pots in solar greenhouse as revealed by increased net photosynthetic rate, plant height, leaf number, yield, Vitamin C and soluble sugar but decreased titratable acid under both salinity and normal conditions. Together, these results, for the first time, clearly demonstrated that luffa,a new highly salt-tolerant rootstock, enhances salt tolerance and improves yield and quality of grafted cucumber plants by reducing sodium transport to the shoot.

Comparison of biochemical composition, nutritional quality, and metals concentrations between males and females of three different Crassostrea sp.

Gametogenesis can significantly affect the biochemical composition of oysters, but little research on the difference between sexes. Therefore, we conducted the first in-depth study on the composition differences between males and females of three different Crassostrea sp.. The results showed that females had higher glycogen, lipid, Cu and Zn contents than males, while males had higher protein and taurine contents than females at maturity, which might be related to special meiosis pattern of eggs and less energy was required for female gametogenesis. In addition, both males and females had well-balanced essential amino acid compositions. The omega-3: omega-6 (n-3: n-6) ratio of males was significantly higher than that of females, indicating that the nutritional quality of males was higher. These results provide a reliable and refined theoretical and research basis for revealing the nutritional quality, extracting beneficial ingredients, and developing functional food of Crassostrea sp., and provide data support for the sex-regulated breeding of oysters.

A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.

Red and blue light function antagonistically to regulate cadmium tolerance by modulating the photosynthesis,antioxidant defense system and Cd uptake in cucumber(Cucumis sativus L.).

Cadmium (Cd) is highly toxic to both plants and humans.Light plays crucial roles in plant growth, development and stress responses, but how light functions in plant Cd response remain unclear.Here,we found that Cd treatment significantly induced the expression of PHYB but not PHYA and CRY1 in leaves and roots of cucumber. Correspondingly,compared with white light (W) during Cd stress,red light(R) increased Cd sensitivity,whereas blue light (B) enhanced Cd tolerance as evidenced by decreased Cd-induced chlorosis, growth inhibition, photosynthesis inhibition and chloroplast ultrastructure damage.Furthermore,B markedly increased the transcripts and activities of the antioxidant enzymes including ascorbate peroxidase (APX),catalase (CAT),superoxide dismutase (SOD) and glutathione reductase (GR),as well as glutathione (GSH) content and GSH1 expression, resulting in hydrogen peroxide (H2O2) and superoxide (O2.-) reduction,but R treatment showed the opposite trend. Moreover, R and B markedly up-regulated and down-regulated the expression levels of Cd uptake and transport genes including IRT1, NRAMP1 and HMA3, leading to more and less Cd accumulation than the W-treated plants in both shoots and roots, respectively under Cd stress. Collectively, our data clearly demonstrate that R and B function antagonistically to regulate Cd tolerance in cucumber via modulating the photosynthesis, antioxidant defense system and Cd uptake, providing a novel light quality control strategy to enhance crop Cd tolerance and food safety.

MDM2 is involved in the regulation of p53 expression in the immune response of oyster Crassostrea hongkongensis.

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 ChMDM2 and Chp53), and their mRNA expression patterns in tissues and developmental stages were analyzed. Multiple sequence alignment analysis and phylogenetic analysis of ChMDM2 and Chp53 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 ChMDM2 was decreased, while the relative expression of Chp53 was significantly increased in the hemocytes and gills. Furthermore, fluorescence from ChMDM2-EGFP and Chp53-Red were found to be distributed in the nucleus of HEK293T cells. Besides, dual-luciferase reporter assays showed that ChMDM2 antagonized with Chp53 and participates in p53 signaling pathway. In addition, the interaction between ChMDM2 and Chp53 was confirmed strongly by Co-immunoprecipitation assays. Furthermore, the results of RNAi showed that ChMDM2 and Chp53 participated in apoptosis which induced infection of V. coralliilyticus. Taken together, our results characterized the features of ChMDM2 and Chp53, which played a critical role in apoptosis of C. hongkongensis.

Sequencing and analysis of the complete mitochondrial genome of Hippopus porcellanus.

In this study, the complete mitochondrial genome of Hippopus porcellanus was reported. The whole mitochondrial genome was 21,565bp in length with a typical mitochondrial genomic structure including 13 protein-coding genes, 23 transfer RNA genes, 2 ribosomal RNA genes and 1 control region (D-loop). Mitogenome base composition was biased toward A + T content, at 60.3%. A phylogenetic tree based on complete mitogenome sequences revealed that, H. porcellanus is closely related to H. hippopus, both of which belong to the genus Hippopus.