Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").

Widespread macromolecular interaction perturbations in human genetic disorders.

How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.

AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

Signaling Transduction Pathways and G-Protein-Coupled Receptors in Different Stages of the Embryonic Diapause Termination Process in Artemia.

Artemia is a widely distributed small aquatic crustacean, renowned for its ability to enter a state of embryonic diapause. The embryonic diapause termination (EDT) is closely linked to environmental cues, but the precise underlying mechanisms remain elusive. In this study, ATAC-seq and RNA-seq sequencing techniques were employed to explore the gene expression profiles in Artemia cysts 30 min after EDT. These profiles were compared with those during diapause and 5 h after EDT. The regulatory mechanisms governing the EDT process were analyzed through Gene Ontology (GO) enrichment analysis of differentially expressed genes. Furthermore, the active G-protein-coupled receptors (GPCRs) were identified through structural analysis. The results unveiled that the signaling transduction during EDT primarily hinges on GPCRs and the cell surface receptor signaling pathway, but distinct genes are involved across different stages. Hormone-mediated signaling pathways and the tachykinin receptor signaling pathway exhibited heightened activity in the '0-30 min' group, whereas the Wnt signaling pathway manifested its function solely in the '30 min-5 h' group. These results imply a complete divergence in the mechanisms of signal regulation during these two stages. Moreover, through structural analysis, five GPCRs operating at different stages of EDT were identified. These findings provide valuable insights into the signal regulation mechanisms governing Artemia diapause.

Correlation between d Electrons and the Sweet Spot for the Hydrogen Evolution Reaction: Is Platinum Always the Best Electrocatalyst?

Herein, two Laves intermetallic series, ZrCo1.75M0.25 and NbCo1.75M0.25 (M = Fe, Co, Ni, Ru, Rh, Pd, Os, Ir, and Pt), were synthesized, and their hydrogen evolution reaction (HER) activities were examined to reveal the influence of d electrons to the corresponding HER activities. Owing to the different electronegativity between Zr and Nb (χZr = 1.33; χNb = 1.60), Co and/or M elements receive more electrons in ZrCo1.75M0.25 than that of the Nb one. This leads to the overall weak H adsorption energy (ΔGHad) of ZrCo1.75M0.25 series compared to that of NbCo1.75M0.25 and rationalizes well the superior HER activity of the Rh member compared to that of the Pt one in the ZrCo1.75M0.25 series. Under industrial conditions (333 K, 6.0 M KOH), ZrCo1.75Rh0.25 only requires an overpotential of 110 mV to reach the current density of 500 mA/cm2 and can be operated at high current density over 400 h. This work demonstrates that with a proper combination between elements in intermetallic phases, one can manipulate d electrons of the active metal to be closer to the sweet spot (ΔGHad = 0). The Pt member may no longer exhibit the best HER activity in series, and all elements exhibit the potential to outperform the Pt member in the HER with careful control of the d electron population.

Unlocking the potential of N-acetylcysteine: Improving hepatopancreas inflammation, antioxidant capacity and health in common carp (Cyprinus carpio) via the MAPK/NF-κB/Nrf2 signalling pathway.

N-acetylcysteine (NAC) positively contributes to enhancing animal health, regulating inflammation and reducing stress by participating in the synthesis of cysteine, glutathione, and taurine in the body. The present study aims to investigate the effects of dietary different levels of NAC on the morphology, function and physiological state of hepatopancreas in juvenile common carp (Cyprinus carpio). 450 common carps were randomly divided into 5 groups: N1 (basal diet), N2 (1.5 g/kg NAC diet), N3 (3.0 g/kg NAC diet), N4 (4.5 g/kg NAC diet) and N5 (6.0 g/kg NAC diet), and fed for 8 weeks. The results indicated that dietary 3.0-6.0 g/kg NAC reduced hepatopancreas lipid vacuoles and nuclear translocation, and inhibited apoptosis in common carp. Simultaneously, the activities of hepatopancreas alanine aminotransferase and aspartate aminotransferase progressively increased with rising dietary NAC levels. Dietary NAC enhanced the non-specific immune function of common carp, and exerted anti-inflammatory effects by inhibiting the MAPK/NF-κB signaling pathway. Additionally, dietary 3.0-6.0 g/kg NAC significantly improved the antioxidant capacity of common carp, which was associated with enhanced glutathione metabolism, clearance of ROS and the activation of Nrf2 signaling pathway. In summary, NAC has the potential to alleviate inflammation, mitigate oxidative stress and inhibit apoptosis via the MAPK/NF-κB/Nrf2 signaling pathway, thereby improving hepatopancreas function and health of common carp. The current findings provide a theoretical basis for promoting the application of NAC in aquaculture and ecological cultivation of aquatic animals.

A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection.

During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.

Microscopic Mechanism for Further NO Heterogeneous Reduction by Potassium-Doped Biochar: A DFT Study.

Biomass reburning is an efficient and low-cost way to control nitric oxide (NO), and the abundant potassium (K) element in biomass affects the heterogeneous reaction between NO and biochar. Due to the incomplete simulation of the NO heterogeneous reduction reaction pathway at the molecular level and the unclear catalytic effect of K element in biochar, further research is needed on the possible next reaction and the influencing mechanism of the element. After the products of the existing reaction pathways are referenced, two reasonably simplified biochar structural models are selected as the basic reactants to study the microscopic mechanism for further NO heterogeneous reduction on the biochar surface before and after doping with the K atom based on density functional theory. In studying the two further NO heterogeneous reduction reaction pathways, we find that the carbon monoxide (CO) molecule fragment protrudes from the surface of biochar models with the desorption of N2 at the TS4 transition state, and the two edge types of biochar product models obtained by simulation calculation are Klein edge and ac56 edge observed in the experiment. In studying the catalytic effect of potassium in biochar, we find that the presence of K increases the heat release of adsorption of NO molecules, reduces the energy barrier of the rate-determining step in the nitrogen (N2) generation and desorption process (by 50.88 and 69.97%), and hinders the CO molecule from desorbing from the biochar model surface. Thermodynamic and kinetic analyses also confirm its influence. The study proves that the heterogeneous reduction reaction of four NO molecules on the surface of biochar completes the whole reaction process and provides a basic theoretical basis for the emission of nitrogen oxides (NOx) during biomass reburning.

Potential protective effects of sodium butyrate on glycinin-induced oxidative stress, inflammatory response, and growth inhibition in Cyprinus carpio.

Investigated mitigating effects of sodium butyrate (SB) on the inflammatory response, oxidative stress, and growth inhibition of common carp (Cyprinus carpio) (2.94 ± 0.2 g) are caused by glycinin. Six isonitrogenous and isoenergetic diets were prepared, in which the basal diet was the control diet and the Gly group diet contained 80 g/kg glycinin, while the remaining 4 diets were supplemented with 0.75, 1.50, 2.25, and 3.00 g/kg SB, respectively. The feeding trial lasted for 8 weeks, and the results indicated that supplementing the diet with 1.50-2.25 g/kg of SB significantly improved feed efficiency and alleviated the growth inhibition induced by glycinin. Hepatopancreas and intestinal protease activities and the content of muscle crude protein were significantly decreased by dietary glycinin, but supplement 1.50-2.25 g/kg SB partially reversed this result. SB (1.50-2.25 g/kg) increased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hepatopancreas and reduced the activities of AST and ALT in the serum. Glycinin significantly reduced immune and antioxidant enzyme activities, whereas 1.50-2.25 g/kg SB reversed these adverse effects. Furthermore, compared with the Gly group, supplement 1.50-2.25 g/kg SB eminently up-regulated the TGF-ß and IL-10 mRNA, and down-regulated the IL-1ß, TNF-α, and NF-κB mRNA in hepatopancreas, mid-intestine (MI), and distal intestine (DI). Meanwhile, supplement 1.50-2.25 g/kg SB activated the Keap1-Nrf2-ARE signaling pathway and upregulate CAT, SOD, and HO-1 mRNA expression in hepatopancreas, MI, and DI. Summarily, glycinin induced inflammatory response, and oxidative stress of common carp ultimately decreased the digestive function and growth performance. SB partially mitigated these adverse effects by activating the Keap1-Nrf2-ARE signaling pathway and inhibiting the NF-κB signaling pathway.

LincRNA_XR209691.3 could promote Bombyx mori nucleopolyhedrovirus replication by interacting with BmHSP70.

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides and lacking protein coding potential, have been proven to play important roles in viral infection and host immunity. Bombyx mori nucleopolyhedrovirus (BmNPV) is an important pathogen, which causes the silkworm disease and leads to a huge challenge to the sericultural industry. At present, research on the roles of insect lncRNAs in host-virus interaction are relatively few. In this study, we explored the function of lincRNA_XR209691.3 that was significantly up-regulated in the silkworm fat body upon BmNPV infection. Firstly, the subcellular localization experiment confirmed that lincRNA_XR209691.3 was present in both the nucleus and cytoplasm. Enhancing the expression of lincRNA_XR209691.3 in BmN cells could promote the proliferation of BmNPV, while inhibition of lincRNA_XR209691.3 by RNA interference suppresses the proliferation of BmNPV. Combining RNA pull-down and mass spectrometry, we identified the host and BmNPV proteins that could interact with lincRNA_XR209691.3. Next, by using truncation experiment and RNA immunoprecipitation (RIP) assay, it was found that lincRNA_XR209691.3 could bind to the Actin domain of BmHSP70. Subsequently, overexpression of lncRNA_XR209691.3 in BmN cells promoted the expression of BmHSP70, while knockdown of BmHsp70 suppressed the replication of BmNPV. Based on the above results, it is speculated that lincRNA_XR209691.3 could promote the proliferation of BmNPV through interaction with BmHSP70, possibly by improving the stability of BmHSP70 and thereby enhancing the expression of BmHSP70. Our results shed light on the lncRNA function in insect-pathogen interactions and provide a new clue to elucidate the molecular mechanism of BmNPV infection.

Balance between Activity and Stability of Single Metal and Intermetallic Compounds for Electrocatalytic Hydrogen Evolution Reaction.

The higher population of the antibonding state around the Fermi level will result in better activity yet lower stability of HER (Re vs Ru metal). There seems to be a limitation or balance for using a single metal since the bonding scheme of a single metal is relatively simple. Combining Re (strong bonding), Ru (HER active), and Zr metal (corrosion-resistant) grants ternary intermetallic compound ZrRe1.75Ru025, exhibiting excellent HER activity and stability in acidic and alkaline electrolytes. The overpotential at a current density of 10 mA/cm2 (η10) for ZrRe1.75Ru025 is much lower compared to that of ZrRe2. Although the HER activity of ZrRe1.75Ru025 is not comparable to that of ZrRu2, it demonstrates outstanding HER stability, while the current density of ZrRu2 is over ca. 16% after 6 h. This suggests that intermetallic compounds can break the constraint between activity and stability in a single metal for HER, which may be applied in other fields as well.

Direct Electrosynthesis of Metal Nanoparticles on Ti3C2Tx-Mxene during Hydrogen Evolution.

Herein, we propose a simple yet effective method to deposit metal nanoparticles on Ti3C2Tx-MXene via direct electrosynthesis. Without using any reducing reagent or annealing under reducing atmosphere, it allows the conversion of metal salts (e.g., PtCl4, RuCl3·yH2O, IrCl3·zH2O, AgNO3, and CuCl2·2H2O) to metal nanoparticles with a small particle size (ca. 2 nm). Under these circumstances, it was realized that the support effect from Ti3C2Tx-MXene (electron pushing) is quite profound, in which the Ti3C2Tx-MXene support will act as an electron donor to push the electron to Pt nanoparticles and increase the electron density of Pt nanoparticles. It populates the antibonding state of Pt-Pt bonds as well as the adsorbate level that leads to a "weakening" of the ΔGH* in the optimal position. This rationalizes the outstanding activity of Pt/Ti3C2Tx-MXene (5 wt %, η10 = 16 mV) for the hydrogen evolution reaction (HER). In addition, this direct electrosynthesis method grants the growth of two or multiple types of metal nanoparticles on the Ti3C2Tx-MXene substrate that can perform dual or multiple functions as desired. For instance, one can prepare an electrocatalyst with Pt (2.5 wt %) and Ru nanoparticles (2.5 wt %) on the Ti3C2Tx-MXene support from the same synthetic method. This electrocatalyst (Pt_Ru/Ti3C2Tx-MXene) can display good electrocatalytic HER performance in both acid (0.5 M H2SO4) and alkaline electrolytes (1.0 M KOH).

A protein domain-based interactome network for C. elegans early embryogenesis.

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.

A massively parallel barcoded sequencing pipeline enables generation of the first ORFeome and interactome map for rice.

Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 µg/ml) or (ii) CD9 antibody (1, 5 and 25 µg/ml) or (iii) CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

Tetrahedral Imidazolate Frameworks with Auxiliary Ligands (TIF-Ax): Synthetic Strategies and Applications.

Zeolitic imidazolate frameworks (ZIFs) are an important subclass of metal-organic frameworks (MOFs). Recently, we reported a new kind of MOF, namely tetrahedral imidazolate frameworks with auxiliary ligands (TIF-Ax), by adding linear ligands (Hint) into the zinc-imidazolate system. Introducing linear ligands into the M2+-imidazolate system overcomes the limitation of imidazole derivatives. Thanks to the synergistic effect of two different types of ligands, a series of new TIF-Ax with interesting topologies and a special pore environment has been reported, and they have attracted extensive attention in gas adsorption, separation, catalysis, heavy metal ion capture, and so on. In this review, we give a comprehensive overview of TIF-Ax, including their synthesis methods, structural diversity, and multi-field applications. Finally, we also discuss the challenges and perspectives of the rational design and syntheses of new TIF-Ax from the aspects of their composition, solvent, and template. This review provides deep insight into TIF-Ax and a reference for scholars with backgrounds of porous materials, gas separation, and catalysis.

Genome-scale metabolic network model of Eriocheir sinensis icrab4665 and nutritional requirement analysis.

BACKGROUND: Genome-scale metabolic network models (GEMs) provide an efficient platform for the comprehensive analysis the physical and biochemical functions of organisms due to their systematic perspective on the study of metabolic processes. Eriocheir sinensis is an important economic species cultivated on a large scale because it is delicious and nutritious and has a high economic value. Feed improvement is one of the important methods to improve the yield of E. sinensis and control water pollution caused by the inadequate absorption of feed. RESULTS: In this study, a GEM of E. sinensis, icrab4665, was reconstructed based on the transcriptome sequencing, combined with KEGG database, literature and experimental data. The icrab4665 comprised 4665 unigenes, 2060 reactions and 1891 metabolites, which were distributed in 12 metabolic subsystems and 113 metabolic pathways. The model was used to predict the optimal nutrient requirements of E. sinensis in feed, and suggestions for feed improvement were put forward based on the simulation results. The simulation results showed that arginine, methionine, isoleucine and phenylalanine had more active metabolism in E. sinensis. It was suggested that the amount of these essential amino acids should be proportionally higher than that of other amino acids in the feed to ensure the amino acid metabolism of E. sinensis. On the basis of the simulation results, we further suggested increasing the amount of linoleic acid, EPA and DHA in the feed to ensure the intake of essential fatty acids for the growth of E. sinensis and promote the accumulation of cell substances. In addition, the amounts of zinc and selenium in the feed were also suggested to be properly increased to ensure the basic metabolism and growth demand of E. sinensis. CONCLUSION: The largest GEM of E. sinensis was reconstructed and suggestions were provide for the improvement of feed contents based on the model simulation. This study promoted the exploration of feed optimization for aquatic crustaceans from in vivo and in silico. The results provided guidance for improving the feed proportion for E. sinensis, which is of great significance to improve its yield and economic value.

Cellular Lnc_209997 suppresses Bombyx mori nucleopolyhedrovirus replication by targeting miR-275-5p in B. mori.

Long non-coding RNA (lncRNA) is a type of non-coding RNA molecule, which exceeds 200 nucleotides in length and participates in the regulation of a variety of life activities. Recent studies showed that lncRNAs play important roles in viral infection and host immunity. At present, the researches on insect lncRNAs are relatively few. In this study, we found the expression of Lnc_209997 was significantly down-regulated in silkworm fat body infected with Bombyx mori nucleopolyhedrosis virus (BmNPV). Inhibition of Lnc_209997 promoted BmNPV replication. Enhancing the expression of Lnc_209997 inhibited the proliferation of BmNPV. miR-275-5p was up-regulated in silkworm fat body infected with BmNPV. Dual luciferase reporter gene system confirmed the interaction between Lnc_209997 and miR-275-5p. Over-expression of Lnc_209997 inhibited the expression of miR-275-5p, while inhibition of Lnc_209997 enhanced the expression of miR-275-5p. Further, over-expression of miR-275-5p can facilitate the replication of BmNPV. These results suggested that BmNPV could increase the expression of miR-275-5p by inhibiting cellular Lnc_209997 expression to promote their own proliferation. Our results are helpful for better understanding the role of lncRNAs in BmNPV infection, and provide insights into elucidating the molecular mechanism of interaction between Bombyx mori and virus.

Transcriptome of miRNA during inhibition of Bombyx mori nuclear polyhedrosis virus by geldanamycin in BmN cells.

Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of several viruses that cause great harm to the sericulture industry, and its pathogenic mechanism is still being explored. Geldanamycin (GA), a kind of HSP90 inhibitor, has been verified to suppress BmNPV proliferation. However, the molecular mechanism by which GA inhibits BmNPV is unclear. MicroRNAs (miRNAs) have been shown to play a key role in regulating virus proliferation and host-pathogen interactions. In this study, BmN cells infected with BmNPV were treated by GA and DMSO for 72 h, respectively, then transcriptome analysis of miRNA was performed from the GA group and the control group. As a result, a total of 29 miRNAs were differentially expressed (DE), with 13 upregulated and 16 downregulated. Using bioinformatics analysis, it was found that the target genes of DEmiRNAs were involved in ubiquitin-mediated proteolysis, phagosome, proteasome, endocytosis pathways, and so on. Six DEmiRNAs were verified by quantitative reverse-transcription polymerase chain reaction. DElong noncoding RNA (DElncRNA)-DEmiRNA-messenger RNA (mRNA) regulatory networks involved in apoptosis and immune pathways were constructed in GA-treated BmN cells, which included 12 DEmiRNA, 132 DElncRNA, and 69 mRNAs. This regulatory network enriched the functional role of miRNA in the BmNPV-silkworm interactions and improved our understanding of the molecular mechanism of HSP90 inhibitors on BmNPV proliferation.

Vitrification of bovine germinal vesicle oocytes significantly decreased the methylation level of their in vitro derived MII oocytes.

CONTEXT: The vitrification of oocytes is important for the conservation of animals, and the effect of vitrification on methylation patterns of bovine oocytes remains unclear. AIMS: This article aims to investigate the effect of vitrification on the DNA methylation patterns on vitrified GV oocytes and their in vitro derived MII oocytes. METHODS: 5-MeC staining and single-cell whole genome bisulphite sequencing (SC-WGBS) were utilised to analyse fresh GV oocytes (F_GV group), MII oocytes (F_MII group), vitrified GV oocytes (V_GV group) and their in vitro derived MII oocytes (V_MII group). KEY RESULTS: Results of both 5-MeC staining and SC-WGBS showed that no significant difference was found between the F_GV group and the V_GV group, while the methylation level of the V_MII group was significantly lower than that of the F_MII group. Moreover, supplementation of 2µM resveratrol (Res) in IVM medium significantly improved maturation and development ability of vitrified GV oocytes by restoring their DNA methylation levels. CONCLUSION: In conclusion, vitrification of bovine GV oocytes significantly decreased the DNA methylation level of their in vitro derived MII oocytes, and 2µM Res improved their development ability by restoring DNA methylation level. IMPLICATIONS: Our results provide an efficient approach to improve the maturation and fertilisation ability of vitrified GV oocytes.