Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

Introduction: In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. Methods: We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. Result: The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. Conclusion: These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Immunomodulatory effects of decitabine in pearl oyster Pinctada fucata martensii.

Decitabine (DAC), an inhibitor of DNA methyltransferase, is typically used to reverse DNA methylation and is considered an epigenetic modifying drug. DNA methylation is crucial to the regulation of gene expression without altering genetic information. Our previous research showed that the DNA methylation levels of many immune-related genes changed after the pre-grafting condition in pearl production. In the present study, we evaluated the DNA methylation level and analyzed transcriptome, enzyme, and antimicrobial activities after DAC treatment to evaluate the effect of DAC on DNA methylation and immune system of pearl oyster Pinctada fucata martensii. Results showed that DAC significantly decreased the level of global DNA methylation in the hemocytes of the pearl oysters. Transcriptome analysis obtained 577 differentially expressed genes (DEGs) between the control and DAC treatment group. The DEGs were mainly enriched in the following pathways: "Relaxin signaling pathway," "Cytosolic DNA-sensing pathway," "Platelet activation," and "Peroxisome," and related genes were overexpressed after DAC treatment. DAC treatment resulted in a substantial increase in the levels of serum superoxide dismutase, interleukin-17, phenol oxidase, tumor necrosis factor, and antimicrobial activity, compared with the control. These results suggested that DAC can alter DNA methylation level, activate immune-related genes, and improve the level of humoral immunity in pearl oysters, thereby increasing our understanding of the mechanism underlying DNA methylation in immune regulation.

Exploring crucial molecular events in pearl oyster after pre-grafting conditioning by genome-wide bisulfite sequencing for DNA methylation analysis.

Pre-grafting condition is an important method to promote recovery from transplant surgery during pearl production. In the present study, we constructed two DNA methylomes from pearl oysters with and without conditioning to investigate the molecular mechanism of the pearl oyster Pinctada fucata martensii underlying the pre-grafting condition. A total of 4,594,997 and 4,930,813 methyl CG in the control (Con) and pre-grafting group (PT) were detected, resulting in the whole genome methylation profile and methylation pattern in P. f. martensii. Results reveal that the promoter, especially the CpG island-rich region, was more infrequently methylated than the gene function elements in P. f. martensii. A total of 51,957 differently methylated regions (DMRs) between Con and PT were obtained, including 3789 DMR in the promoter and 16,021 in the gene body. Based on gene ontology and pathway enrichment analyses, these DMRs were mainly related to "cellular process", "metabolic process", "Epstein-Barr virus infection", and "Fanconi anemia pathway". The methylation site in the promoter region may be associated with the promoter activity and transcription factor binding. These results help our understanding of the mechanism of pre-grafting condition, thereby providing key information in guiding to improve the conditioning methods for enhanced pearl oyster survival rate after transplantation.

Polyamine Putrescine Regulates Oxidative Stress and Autophagy of Hemocytes Induced by Lipopolysaccharides in Pearl Oyster Pinctada fucata martensii.

The polyamine putrescine (Put) is a ubiquitous small cationic amine. It plays an essential role in controlling the innate immune response. However, little is known about its function in mollusks. In this study, the Put content was observed to increase in the serum of pearl oyster Pinctada fucata martensii after 6 and 24 h of lipopolysaccharide (LPS) stimulation. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased, and nitric oxide synthase was downregulated in the Put group (i.e., combined treatment with Put and LPS) compared with that in the LPS group (i.e., combined treatment with phosphate-buffered saline and LPS). Furthermore, activities of alkaline phosphatase and acid phosphatase were inhibited after 6 h of LPS stimulation. The expression levels of the nuclear factor kappa B, IκB kinase, Janus kinase, and signal transducer and activator of transcription proteins genes were all significantly suppressed at 12 and 24 h in the Put group. Pseudomonas aeruginosa and Bacillus subtilis grew better after being incubated with the serum from the Put group than that from the LPS group. Additionally, the Put treatment remarkably inhibited the autophagy of hemocytes mediated by the AMP-activated protein kinase-mammalian target of rapamycin-Beclin-1 pathway. This study demonstrated that Put can effectively inhibit the inflammatory response induced by LPS in pearl oysters. These results provide useful information for further exploration of the immunoregulatory functions of polyamines in bivalves and contribute to the development of immunosuppressive agents.

Genome-wide DNA Methylation Analysis of Mantle Edge and Mantle Central from Pearl Oyster Pinctada fucata martensii.

DNA methylation is a type of epigenetic modification that alters gene expression without changing the DNA sequence and mediates some cases of phenotypic plasticity. In this study, we identified six DNA methyltransferase (DNMT) genes and two methyl-CpG binding domain protein2 (MBD2) gene from Pinctada fucata martensii. We also analyzed the genome-wide DNA methylation levels of mantle edge (ME) and mantle central (MC) from P. f. martensii via methylated immunoprecipitation sequencing (MeDIP-Seq). Results revealed that both ME and MC had 122 million reads, and had 58,702 and 55,721 peaks, respectively. The obtained methylation patterns of gene elements and repeats showed that the methylation of the protein-coding genes, particularly intron and coding exons (CDSs), was more frequent than that of other genomic elements in the pearl oyster genome. We combined the methylation data with the RNA-seq data of the ME and MC of P. f. martensii and found that promoter, CDS, and intron methylation levels were positively correlated with gene expression levels except the highest gene expression level. We also identified 313 differential methylation genes (DMGs) and annotated 212 of them. These DMGs were significantly enriched in 30 pathways, such as amino acid and protein metabolism, energy metabolism, terpenoid synthesis, and immune-related pathways. This study comprehensively analyzed the methylomes of biomineralization-related tissues and helped enhance our understanding of the regulatory mechanism underlying shell formation.

Evolutionary and functional analysis of MyD88 genes in pearl oyster Pinctada fucata martensii.

Myeloid differentiation factor 88 (MyD88) is an adapter protein that links toll-like receptor and interleukin 1 receptor-mediated signal transduction. In this study, we identified 20 MyD88 genes from eight mollusk genomes and found that MyD88 was expanded in bivalves. This expansion tends to be tandem duplication. Phylogenetic analysis suggested that the tandem duplication of MyD88 was formed before bivalve differentiation. All of the identified MyD88 contained both of death domain (DD) and toll/interleukin-1 receptor (TIR) domain, and 13 mollusks MyD88 have low complexity regions (LCRs), which were not found in the MyD88 from humans and zebrafish. The genomic structure showed that most of the mollusk MyD88 (14 of 19) contained five conserved introns, four of which were found in humans and zebrafish. Furthermore, the cDNA full length of PfmMyD88-2 (one of the two identified MyD88 in Pincatada fucata martensii) was obtained with 1591 bp, including 260 bp of 5'UTR, 257 bp of 3'UTR, and 1077 bp of open reading frame encoding 358 amino acids. Quantitative real-time PCR analysis demonstrated that PfmMyD88-2 mRNA was widely expressed in all detected tissues. The highest expression level was in the gills and followed by hepatopancreas and feet. After lipopolysaccharide stimulation, PfmMyD88-2 expression level increased and reached the highest level at 12 h and then gradually declined to the normal level. Over-expression of PfmMyD88-2 in HEK293T increased the luciferase activity of the pNF-κB-Luc reporter. We also identified that PfmmiR-4047 could regulate the expression of PfmMyD88-2. These results help us elucidate the mechanism underlying mollusk immune response.

Ultrasensitive Chemiluminescence Biosensor for Nuclease and Bacterial Determination Based on Hemin-Encapsulated Mesoporous Silica Nanoparticles.

Bacterial determination, emerging as a critical step in the understanding of increasingly serious bacterial contaminations, remains a major challenge. Herein, a novel chemiluminescence biosensor was exploited for the ultrasensitive determination of nuclease activity and bacteria, in which, hemin, the chemiluminescent (CL) tag molecule was encapsulated into ordered mesopores of mesoporous silica nanoparticles with a specific DNA gate. The capped DNA could be specifically switched upon exposure to the DNA nuclease or bacterial lysate and allowed for an increased release of the encapsulated hemin, which therefore resulted in an obviously enhanced CL signal for the luminol-H2O2 system. Attributed to this unique behavior with the linear or sigmoidal relationship between CL intensity and DNA nuclease or bacterial concentration, the as-prepared CL biosensor could detect S1 nuclease activity in the concentration range 0.01-10.0 U with a detection limit of 0.1 mU, and Escherichia coli O157:H7 (E. coli) or Staphylococcus aureus (S. aureus) in the concentration ranges 101 to 109 cfu mL-1. The detection limit of E. coli and S. aureus was calculated to be 3.0 and 2.5 cfu mL-1, respectively, which was comparable or even better than that of previous studies. Thus, this detection method could achieve detectable levels without cell enrichment overnight. Moreover, the proposed biosensing system could be conducted in the homogeneous solution without separation and washing, greatly improving the reaction efficiency and simplifying the procedure. As expected, the novel CL biosensor promised a great potential for simple and convenient detection of nuclease and bacteria in fields such as food bacterial contamination, pharmaceuticals, and clinical analysis.

Molecular switch-modulated fluorescent copper nanoclusters for selective and sensitive detection of histidine and cysteine.

A novel assay for histidine and cysteine has been constructed based on modulation of fluorescent copper nanoclusters (CuNCs) by molecular switches. In our previous work, a dumbbell DNA template with a poly-T (thymine) loop has been developed as an excellent template for the formation of strongly fluorescent CuNCs. Herein, for the first time, we established this biosensor for sensing two amino acids by using dumbbell DNA-templated CuNCs as the single probe. Among 20 natural amino acids, only histidine and cysteine can selectively quench fluorescence emission of CuNCs, because of the specific interaction of these compounds with copper ions. Furthermore, by using nickel ions (Ni2+) and N-ethylmaleimide as the masking agents for histidine and cysteine respectively, an integrated logic gate system was designed by coupling with the fluorescent CuNCs and demonstrated selective and sensitive detection of cysteine and histidine. Under optimal conditions, cysteine can be detected in the concentration ranges of 0.01-10.0 µM with the detection limit (DL) of as low as 98 pM, while histidine can be detected in the ranges of 0.05-40.0 µM with DL of 1.6 nM. In addition, histidine and cysteine can be observed with the naked eye under a hand-held UV lamp (DL, 50 nM), which can be easily adapted to automated high-throughput screening. Finally, the strategy has been successfully utilized for biological fluids. The proposed system can be conducted in homogeneous solution, eliminating the need for organic cosolvents, separation processes of nanomaterials, or any chemical modifications. Overall, the assay provides an alternative method for simultaneous detection of cysteine and histidine by taking the advantages of high speed, no label and enzyme requirement, and good sensitivity and specificity, and will satisfy the great demand for determination of amino acids in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. Graphical abstract.