Each H4TCPB, which connects to four DMF molecules through hydrogen bonds, acts as the structural unit. The building units are packed by π–π, lone pair···π, and lone pair···lone pair interactions to form solid-state crystalline materials. H4TCPB·4DMF exhibits distinct blue fluorescent under UV light, while the quantum yield is as high as 89.02% and the fluorescence lifetime is 1.95 ns. The H4TCPB·4DMF nanocrystal exhibits a specific fluorescence quench sensibility to tetracycline (TC), compared with the common chemicals and ions in environmental water. Moreover, the test results can be obtained quickly and are easily visible to the naked eye. The limit of detection for TC is as low as 12 nM in an aqueous solution. Spectral analysis and density functional theory (DFT) theoretical calculations were used to explain the fluorescence quenching mechanism of H4TCPB·4DMF toward TC, which could be attributed to the photoinduced electron transfer occurring from H4TCPB·4DMF to TC. Our work enriches the database of crystalline luminescent materials and provides theoretical support for the design and mechanical studies of organic fluorescent probes.The response performances of the crystalline organic fluorescence probe are highly dependent on the long-range ordered arrangement of crystalline structure. Herein, a novel organic crystalline fluorescent probe with a high quantum yield was established through the rapid self-assembly of 1,2,4,5-Tetrakis (4-carboxyphenyl) benzene (H Fluorescent materials have attracted significant attention owing to their great potential in optoelectronic science, fluorescence sensing, bioimaging, and environmental monitoring ,2,3,4. FTetracycline (TC) is a broad-spectrum antibiotic that is used to treat bacterial infections because of its low price and powerful function ,19,20. A4TCPB), based on a solvent temperature-controlled rapidly crystallized method for detecting TC in water via a photoinduced electron transfer (PET) fluorescence quenching mechanism. H4TCPB has five benzene rings, one at the center and four on the side, resulting from the benzene ring and the relatively fixed X-shaped space stretch; it is partially flexible as the angle between the central benzene ring and the side benzene rings are rotatable before crystallization  simulation.In this work, we prepared a nanocrystalline organic fluorescent probe using an organic molecule, 1,2,4,5-Tetrakis (4-carboxyphenyl) benzene (Hlization . H4TCPB 4TCPB·4DMF have been mentioned in our previous work (HOF-TCPB-298)  is the concentration of the analyte (100 mg/L), and I0 and I are the luminescence intensities in the absence and presence of the analyte [sv value is determined as 64,413 M−1, which is two to four orders of magnitude higher than those of the remaining ions and chemicals ) of H4TCPB·4DMF) is linearly dependent on the concentration of TC in the concentration range of 0 to 0.4 mg/L and 0.4 to 100 mg/L with a slope value (S) of 0.428 and 0.0274, respectively (2) reached 0.9916 in the high concentration range, indicating that the fluorescence probe has a broad detection range. Such a good linear relationship, with the linear correlation reaching 0.9973, allows for the accurate quantification of TC over a range from 0 to 0.4 mg/L. The limit of detection (LOD) of H4TCPB·4DMF toward TC was calculated to be 12 nM based on 3σ/S  to the lowest unoccupied molecular orbital (LUMO) of the acceptor (TC) [4TCPB·4DMF, the DFT calculation was implemented to further prove the PET mechanism. As the diagram in 4TCPB·4DMF and TC are −0.6545 eV and −6.1981 eV, respectively, while the LUMO energy level of H4TCPB·4DMF is 2.1802 eV, which is higher than that of TC (−2.2859 eV). In addition, the functional groups on TC have a strong electron-withdrawing capacity under the combined effect of conjugation and induction [4TCPB·4DMF in the solution toward TC might be due to the PET process from H4TCPB·4DMF to TC. Moreover, the binding of DMF molecules enhances the van der Waals forces around H4TCPB, and enhances the photostability and photochemical activities during the process quenching C [The mechanism of Hsorption . PET is tor (TC) . Based onduction . As a renching C .4TCPB, 98%) was supplied by Jilin Chinese Academy of Science-Yanshen Technology Co., Ltd . Hydrochloric acid  was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai China) N, N-dimethylformamide , ethanol (99.7%), methanol (99.5%), acetone (99.5%), tetrahydrofuran (99.5%) nitric acid , NaNO3 (99%), KNO3 (99%), Ca(NO3)2 (99%), Ti(NO3)4 (99%), Zn(NO3)2 (99%), Co(NO3)2, (99%), and Fe(NO3)3 (99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai China) A wide range of antibiotics and chemicals, i.e., tetracycline, penicillin sodium, salicylic acid, diuron, disulfiram, and norfloxacin, were purchased from Shanghai Maclin Biochemical Technology Co., Ltd. (Shanghai China).1,2,4,5-Tetrakis (4-carboxyphenyl) benzene (HPowder X-ray diffraction (PXRD) data were collected on an UltimaIV diffractometer from Japan Tokyo. The single-crystal X-ray diffraction (SC-XRD) data collection was carried out on a Bruker AXS D8-VENTURE diffractometer  . Morphologies were observed using a scanning electron microscope . Thermogravimetric analysis (TGA) was performed using STA449F5 . An F-4700 fluorescence spectrophotometer  was used for the collection of luminescence and excitation spectra. Decay curves and PL quantum yields were collected on an FLS 1000 spectrofluorometer . The ultraviolet-visible (UV–vis) absorption spectra were collected on a UV 2600  spectrophotometer.4TCPB was dissolved in DMF (1 mL) within a glass vial. Then, the uncapped vial was placed inside a 100 mL beaker containing 10 mL of deionized water. Lastly, the beaker was tightly sealed and allowed to stand at room temperature. After seven days, the colorless sheet crystals of H4TCPB·4DMF could be obtained.Synthesis of Bulk Crystals: Briefly, 10 mg of H4TCPB was dissolved in 5 mL of DMF. The filtered clear solution was heated at 60 °C for 30 min. Then, 5 mL of H2O (room temperature) was added to the above DMF solution dropwise (60 °C). The nanocrystals were separated immediately. The solid products were obtained via centrifugation at 10,000 rpm for 5 min and subsequently purified twice with ethanol.Synthesis of Nanocrystals: First, 100 mg of H4TCPB·4DMF were collected using Edinburgh FLS1000 . The analytes selected for TC sensing in this work were distributed in H2O. The ground powder sample of H4TCPB·4DMF (1 mg) was immersed in 10 mL of deionized water and ultrasonicated for about 10 min to form a stable turbid suspension. Spectra were collected immediately after ultrasonic treatment until the suspension was evenly mixed, recording the excitation and emission spectra of the detection system. Then, the H4TCPB·4DMF suspension with a concentration of 100 mg/L was used as a fluorescence probe to detect TC in the concentration range of 0.2 mg/L to 100 mg/L at their maximum excitation wavelengths. To study the selectivity ability of H4TCPB·4DMF to detect TC, the probe was used for the spectral acquisition of TC. The emission spectra of penicillin sodium (PNC), salicylic acid (BHA), diuron, disulfiram (TETD), norfloxacin (NFX), Na+, K+, Ca2+, Zn2+, Ti4+, Co2+, and Fe3+ (100 mg/L) were collected at the maximum excitation wavelength.The solid excitation spectra, emission spectra, fluorescence lifetime, and quantum yield of H4TCPB·4DMF [We used first principles to perform all spin-polarized DFT to calculate the orbital energy level of HCPB·4DMF . The PerCPB·4DMF . The proCPB·4DMF ,46. We uCPB·4DMF .TC calculations were performed using Gauss 16 software as well as the B3LYP functional and 6-311G(d) basis set. SMD implicit solvation model was used to explain the solvation effect ,49. The 4TCPB·4DMF based on its excellent luminescence properties. It has a strong fluorescence emission and a high quantum yield owing to the highly ordered crystal structure, a large number of hydrogen bonds, as well as π–π, lone pair···π, and lone pair···lone pair interactions. The likely mechanism was elucidated indicating that the PET effect occurs between H4TCPB·4DMF and TC, leading to a fluorescence quenching effect. Hence, H4TCPB·4DMF nanocrystals are good candidates as nanofluorescence probes for TC. This work enriches the library of crystalline organic fluorescent sensors and provides theoretical support for the design and mechanical studies of organic fluorescent sensors.In summary, we successfully assembled a nanocrystalline fluorescent probe of H


Caenorhabditis elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2′ OH and triphosphate moieties of dNTPs but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except that its lack of the canonical sterile alpha motif domain. This striking conservation in structure, function, and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.Mutations in sterile alpha motif domain and histidine–aspartate domain–containing protein 1 (SAMHD1) are found in a neurodevelopmental disorder, Aicardi–Goutières syndrome, and cancers, and SAMHD1, which is a deoxynucleoside triphosphate (dNTP) triphosphorylase, was identified as a myeloid-specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of an SAMHD1 ortholog of  A series of mutations in human sterile alpha motif (SAM) domain and histidine–aspartate (HD) domain–containing protein 1 (SAMHD1) gene were identified in Aicardi–Goutières syndrome (AGS) , 3, 4. ASAMHD1 was also identified as a host restriction factor against lentiviruses such as HIV type 1 (HIV-1), type 2 (HIV-2), and simian immunodeficiency viruses , 7, 8. TThe dNTPase activity of SAMHD1 lies at the C–T catalytic HD domain, and the N–T SAM domain is dispensable for the dNTPase activity of SAMHD1 , 12, 17.A series of recent studies reported that various SAMHD1 mutations are also found in multiple human cancers such as acute myeloid leukemia , 32, chrCaenorhabditis elegans (C. elegans) and Drosophila, which are highly useful genetic platforms to dissect fundamental cellular and molecular function of gene homologs and orthologs, are not available for AGS.SAMHD1 proteins are highly conserved among mammals including mice, and SAMHD1 orthologs can also be found in invertebrates. However, two independent studies with SAMHD1 knockout mice failed to display the AGS phenotypes even though these knockout mice exhibited the elevations of both interferon-response gene expression and cellular dNTP levels in various tissues , 45. WhiC. elegans with RNA knockdown technology reported that the reduced expression of SAMHD1 ortholog, ZK177.8, led to maternal sterility  encodes 567 amino acid protein that contains the dNTPase-related HD domain with a series of highly conserved amino acid residues found in two separate allosteric regulatory sites  as well as dNTPase catalytic site (red) of hSAMHD1. Also, ZK177.8 encodes a conserved threonine residue at its C–T end , which is known to be a phosphorylation site for the negative regulation of the dNTPase activity in hSAMHD1 -fused ZK177.8 protein in Escherichia coli. The overexpressed GST-fused ZK177.8 protein was initially applied to the GST affinity column, and the GST-free ZK177.8 protein was eluted after the treatment with factor Xα protease on-column that cleaves the sequence between GST tag and ZK177.8 protein. The GST-free ZK177.8 protein was further purified by gel filtration chromatography (A). We also constructed and purified a mutant of ZK177.8 containing mutated highly conserved HD residues (H134R/D135N) at the conserved dNTPase catalytic site, which are equivalent to H206 and D207 in hSAMHD1. These purified ZK177.8 proteins exhibited the expected molecular weight, which is smaller than hSAMHD1 because of lack of the SAM domain; however, they appear predominantly as dimers when compared with gel filtration standards (B). In this study, the dimer fractions were pooled for the biochemical characterization.In order to enzymologically characterize the ZK177.8 gene product, the 567 amino acid long full-length ZK177.8 gene was amplified by RT–PCR from cellular RNAs isolated from adult tography A. We alstandards B. In thiA, the dG product was detected in the reaction with ZK177.8 protein. However, when dGTP was incubated with the ZK177.8 HD catalytic site mutant (H134R/D135N), dG product was not detected (B). Next, we tested whether ZK177.8 also used dGTP as an allosteric activator for dNTPase activity as observed with hSAMHD1. As shown in Figure 2C, in the reactions with dATP, dCTP, or dTTP substrate, the respective dN products were observed in the presence of dGTP but not in the absence of dGTP. Next, we employed TLC-based dNTPase assay that can detect the triphosphate product. This assay uses α-32P labeled dTTP substrate, generating the radiolabeled triphosphate (PPPi) product after hydrolysis, which can be separated in TLC. As shown in Figure 2D, the incubation of the radiolabeled dTTP with 1 μM of ZK177.8 or hSAMHD1 in the presence of dGTP generated high levels of the radioactive TP product, compared with no enzyme control reaction that showed no detectable TP level. Next, since hSAMHD1 can also use rGTP for its activator (E). Next, we tested whether dGMP and dGDP can work as A1 allosteric activators of ZK177.8 by using dTTP as a substrate. As shown in Figure 2E, dGMP and dGDP showed much limited capability to activate ZK177.8 to hydrolyze dTTP, compared with dGTP, which was previously observed with hSAMHD1  with ZK177.8 protein (1 μM), and the products were applied to HPLC. As shown in Figure 2detected B. Next, ctivator , we quanctivator E. Next,  hSAMHD1 . These dA, ZK177.8 displayed significantly reduced capability to hydrolyze these chain terminator triphosphates, suggesting that ZK177.8 also requires 3′ OH for its full dNTPase activity. Next, we tested whether ZK177.8 recognizes the base moiety of dNTP substrates. As shown in the reactions with 2′-amino-2′-dATP, O6-methyl dGTP, dUTP, and 2-thio dTTP (B), ZK177.8 hydrolyzed these dNTPs with base modifications with similar efficiency with their natural dNTP partners. Finally, while hSAMHD1 does not degrade rNTPs . These data support that dGTP allosteric activator mediates the formation of the enzymatically active ZK177.8 tetramers.The dGTP binding to both A1 and A2 sites triggers the formation of the enzymatically active tetramers of hSAMHD1 , 22. NexFigure 3E, ZK177.8 protein was expressed in the hSAMHD1 KO THP-1 cells transduced with the pLVX vector expressing both ZK177.8 and mCherry protein but not in the control cells transduced with the empty pLVX vector expressing only mCherry protein. When we measured the dGTP levels in these THP-1 cells after differentiation to the nondividing macrophage stage by PMA (F), the SAMHD1 KO THP-1 macrophages expressing ZK177.8 and mCherry protein displayed reduced dGTP level, compared with the SAMHD1 KO cells that express only mCherry protein, suggesting that ZK177.8 can hydrolyze cellular dNTPs in the PMA-differentiated nondividing THP-1 cells lacking hSAMHD1.Next, we tested whether ZK177.8 protein affects cellular dNTP levels in human cells. For this test, we employed a human monocytic SAMHD1 knockout THP-1 cell line that we previously established by CRISPR–Cas9 . Upon diFigure 3e by PMA F, the SA41–565 hereafter), which was purified employing the same protocol as used for the full-length protein. ZK177.841–565 was crystallized in space group P1. A total of 198,955 unique reflections in the resolution range 1.8 to 42.5 Å were used in the refinement. The final R-factor is 18.2% and Rfree is 20.9% (41–565 (A) with D2 (222) symmetry, which is the active form of the biological assembly. This tetrameric form of active SAMHD1 structure has been observed in all other SAMHD1 orthologs from mouse and pig . First, within each monomer, a dATP molecule occupies its catalytic site. The crystallographic construct used here contains catalytic mutations of H134R/D135N that are equivalent to catalytically inactive mutations of H205R/R206N in hSAMHD1; therefore, the dATP substrates occupying its catalytic sites are at unhydrolyzed state, and no catalytic metal ions are observed in the reported structures (C). Our three-dimensional homology searches using Dali server also returns high structural similarities to other SAMHD1 orthologs, including Mus musculus (mouse) SAMHD1 (mSAMHD1)  and Sus scrofa (pig) SAMHD1 (sSAMHD1)  that has been released to the PDB. Indeed, a careful comparison between our model and other SAMHD1 orthologs reveals that many key residues interacting with the nucleotide are nearly identical (C). For example, the presence of the tyrosine residue near 2′ position of the ribose, which is presumably responsible for the dNTP selection for hydrolysis (C). As expected from sequence alignment (C). Two nucleotides, GTP and dATP, occupy the allosteric site located at the tetramer interface between each monomer (B). Therefore, there are total of eight nucleotides bound at the tetramer interfaces, in the context of a tetramer. This is in accordance with what has been seen in the core hSAMHD1 structures for the allosteric sites (AL), AL1 and AL2 (D). Structurally, such similarity also extends to sSAMHD1 and mSAMHD1, with the exception that there appear to be far fewer nucleotide-interacting residues within mSAMHD1 at the AL2-binding site (D).Our model shows that each monomer of ZK177.8 HD domain consists of a total of three nucleotides bound B. First,ructures C. Our thmSAMHD1)  (ProteinsSAMHD1)   with the notable exception of the helix bundle (residues 207–232) that is present in ZK177.8 but not in hSAMHD1 . The high similarity between these two models is also indicated by the pairwise comparison results from Dali server . Furthermore, the sensitivity against (2′R)-2′-F and (2′R)-2′-OH nucleotides with 2′-OH group in “down” position -2′-OH nucleotides with 2′-OH group in “up” position, such as ara-CTP) has been reported with hSAMHD1, and its leucine residue (Leu150) appears to play a key role here . In ZK17Our structure of ZK177.8 supports that it is also allosterically regulated, as the identified allosteric sites, AL1 and AL2, show similar patterns of regulating residues proximal to the bound nucleotides. Within the first allosteric site, AL1, only nucleotides with guanosine base are allowed (GTP or dGTP). It is monitored by Asp137, Gln142, and Arg145 in hSAMHD1, and our structure of ZK177.8 shows that the base of GTP molecule is also tightly monitored through interactions by Asp46, Gln51, and Arg54. As in hSAMHD1, the ZK177.8 structure also shows that there are no side chains monitoring the type of ribose at AL1, in line with our biochemical analysis where ZK177.8 can be activated not only by dGTP but also by GTP. In hSAMHD1, the AL2 site requires the opposite; no discrimination against the type of base, yet no ribose with 2′-OH is allowed, proposed to be mainly because of Phe157 residue. Our structure of ZK177.8 also shows Tyr66 at its equivalent site, supporting the idea that this residue will also induce steric hindrance to prevent rNTP bindings at AL2 site.in vivo biological roles of ZK177.8 protein will validate its potentials as animal models of these SAMHD1-related human disorders.Overall, this study demonstrates evolutionary conservation between ZK177.8 and hSAMHD1 in their enzyme functions, regulatory mechanisms, and structures as dNTPases. This biochemical study of ZK177.8 protein supports its potentials as animal models for various SAMHD1-related human disorders  and cellular events such as cell proliferation and intracellular dNTP regulation. Further investigations on the C. elegans RNA was extracted from adult C. elegans, and ZK177.8 complementary DNA was reverse transcribed using SuperScript III Reverse Transcriptase (ThermoFisher). The full-length ZK177.8 complementary DNA was cloned into pGEX5x-3  for protein purification, and the N–T HA-tagged ZK177.8 gene was cloned to pLVX-IRES-mCherry (Clontech) for the ZK177.8 expression in human THP-1 cells. The truncated ZK177.8 construct (residues 41–565) used in structural studies was generated by introducing the NdeI restriction enzyme site at the position corresponding to the residues 39 and 40 onto the full-length ZK177.8 by PCR amplification and cloning to pET14b. All catalytically inactive HD mutants (H134R/D135N), both full-length and truncated ZK177.8, were generated by site-directed mutagenesis.Total g for 30 min, followed by sonication in lysis buffer containing 40 mM Tris–HCl at pH 7.5, 250 mM KCl, 5% glycerol, 0.1% Triton X-100, 5 mM beta-mercaptoethanol (β-Me), 0.1 mM PMSF, and 0.5 mM benzamidine. Cleared lysate was obtained by centrifugation at 39,000g and applied to a GSTrap FF column  that had been equilibrated with GST binding buffer containing 50 mM Tris–HCl (pH 7.5), 10% glycerol, 250 mM KCl, and 5 mM β-Me. The column was washed for 20 column volume (CV) with the GST binding buffer, followed by 5 CV wash with increased KCl . The column was re-equilibrated for 5 CV with protease cleavage buffer containing 100 mM NaCl and 2 mM CaCl2, and factor Xa (New England Biolabs) was added (50 U/CV) to the column and allowed to cleave overnight on-column at 4 °C. The protein was then eluted with the protease cleavage buffer. Fractions containing ZK177.8 protein were combined and further purified on Superdex S200 10/300  with gel-filtration buffer containing 50 mM Tris–HCl at pH 7.5, 20% glycerol, 150 mM KCl, 1 mM β-Me, and 0.25 mM EDTA, and the fraction corresponding to the dimer of ZK177.8 (major product) was combined and flash frozen in liquid nitrogen and stored in −80 °C until use. Purity of the purified ceSAMHD1 is determined to be >95%, as judged by SDS-PAGE (The full-length ZK177.8 gene product (ceSAMHD1-pGEX5x-3) used in all the biochemical studies was expressed in Rosetta DE3 cells (Novagen) by inducing with 0.2 mM IPTG at absorbance of 0.5 to 0.8 for 48 h at 16 °C. Cells were harvested by centrifugation at 4000SDS-PAGE . While sThe truncated ZK177.8 (41–565) used in the structural studies was expressed under the same condition as the full-length ZK177.8 protein, and cleared cell lysate was applied to HisTrap crude FF column  that had been equilibrated with nickel (Ni) binding buffer containing 40 mM Tris–HCl (pH 7.5), 10% glycerol, 500 mM NaCl, 5 mM β-Me, and 20 mM imidazole. The column was washed for 15 to 20 CV with the Ni binding buffer, followed by 5 CV wash with increased NaCl . The column was re-equilibrated with the Ni binding buffer, followed by elution with increased imidazole . The fractions containing the truncated ZK177.8 proteins were pooled and further purified on Superdex S200 16/600  with gel-filtration buffer containing 50 mM Tris–HCl (pH 7.5), 20% glycerol, 300 mM NaCl, and 5 mM β-Me. Purified proteins were pooled and flash frozen in liquid nitrogen for storage at –80 °C until use.2. ZK177.8 (1 μM) was incubated with 1 mM dNTP substrates with or without 50 μM of the indicated activator (g for 5 min. Reactions were mixed with dCMP loading control (1 mM) and diluted fivefold with 12% acetonitrile  and analyzed by anion exchange HPLC using a DNAPac PA100 Nucleic Acid Column (Thermo Scientific). The column was equilibrated with buffer containing 25 mM Tris–HCl (pH 8.0) and 0.5% acetonitrile, and products were eluted with increasing amount of ammonium chloride up to 250 mM. Elution of the products was monitored at 254 nm, and peaks were quantified using 32 Karat software (Beckman Coulter). hSAMHD1 reactions were also conducted under the same condition as ZK177.8 reaction except the incubation at 37 °C.Reactions were performed in buffer containing 50 mM Tris–HCl (pH 7.5), 50 mM KCl, and 5 mM MgClctivator . Reactio32P]dTTP or [α-32P]dGTP and analyzed as described previously . Samples were stored at −80 °C. Prior to use, samples were resuspended in buffer containing 50 mM Tris–HCl (pH 8.0) and 10 mM MgCl2. dNTP levels in the dNTP extracts were determined using a single nucleotide incorporation assay described previously (dNTPs were extracted from PMA-differentiated THP-1 cells as described previously . Brieflyeviously . Two miceviously .41–565 was buffer exchanged into 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, and 0.5 mM Tris(2-carboxyethyl)phosphine prior to crystallization using Superdex 75. Peak fractions were pooled, concentrated to 4.0 mg/ml, and incubated with 1 mM GTP/10 mM dATP to facilitate stable tetramer formation. Crystallization was carried out using the microbatch under oil method, using a 2:1 paraffin:silicon oil mixture over the protein mixed with crystallization buffer  at an 1:1 ratio. After initial crystals were obtained, large and well-diffracting crystals were obtained after successive streak seeding drops containing protein mixed with the same crystallization buffer. Crystals were transferred from their mother liquor and cryoprotected in paratone oil prior to flash freezing in liquid nitrogen.The purified His-tagged ZK177.84BZB) was used as a search model for molecular replacement employed in Phaser (University of Cambridge)  . The inimbridge) , followembridge) . Bound nt tests in Prism .All measurements in this study were conducted in triplicate (n = 3). Individual values are reported in each figure, and the mean of these replicates is reflected by the bars on the graphs. Error bars in each figure represent standard deviation. All statistical analyses were conducted using two-tailed and unpaired Welch's All data generated and analyzed during this study are included in this chapter.www.wwpdb.ord (PDB ID: 6PWY).The atomic coordinates and structure factors have been deposited in the PDB, This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Sphingosine-1-phosphate (S1P) is a lipid mediator and its binding to the S1P receptor 2 (S1PR2) is reported to regulate cytoskeletal organization. Epidermal growth factor (EGF) has been shown to induce migration and invasion in tumour cells. Since binding of S1P to S1PR2 and EGF to the EGF receptors exhibit some overlapping functionality, this study aimed to determine whether S1PR2 was involved in EGF-induced migration and invasion of oral squamous cell carcinoma (OSCC) lines and to identify any potential crosstalk between the two pathways. Migration was investigated using the scratch wound assay while invasion was studied using the transwell invasion and multicellular tumour spheroid (MCTS) assays. Activity of Rac1, a RhoGTPase, was measured using G-LISA  while S1P production was indirectly measured via the expression of sphingosine kinase (Sphk). S1PR2 inhibition with 10 µM JTE013 reduced EGF-induced migration, invasion and Rac1 activity, however, stimulation of S1PR2 with 10 µM CYM5478 did not enhance the effect of EGF on migration, invasion or Rac1 activity. The data demonstrated a crosstalk between EGF/EGFR and S1P/S1PR2 pathways at the metabolic level. S1PR2 was not involved in EGF production, but EGF promoted S1P production through the upregulation of Sphk1. In conclusion, OSCC lines could not migrate and invade without S1PR2 regulation, even with EGF stimulation. EGF also activated S1PR2 by stimulating S1P production via Sphk1. The potential for S1PR2 to control cellular motility may lead to promising treatments for OSCC patients and potentially prevent or reduce metastasis.The online version contains supplementary material available at 10.1186/s12935-023-02906-w. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates various cellular activities of the immune, cardiovascular and nervous systems . In cancThus far, studies have aimed to determine the function of the S1P receptor subtypes in several different cells. To date, only the function of S1PR1 has been established, and evidence for the function and role of the other receptor subtypes remains limited. S1PR2 has been reported to regulate cell growth, survival, angiogenesis and adhesion , althougEGF is another cytokine that controls a variety of cellular activities –20. In ONew therapies targeting specific molecules related to individual neoplastic properties might be able to fill gaps in current approaches for the management of cases, where conventional treatments have been exhausted. Since the major cause of cancer deaths is distant metastasis, new approaches that prevent invasion have the potential to improve prognosis and outcome.Additionally, although conventional treatment of OSCC includes excision, chemo- and radio-therapy some cases of recurrence may not be suitable for further conventional treatment. In such situations, a targeted medicine specific to molecules controlling the neoplastic behaviour may improve treatment outcomes. Since the major cause of cancer death is distant metastasis, a medicine that prevents invasion potentially improves the prognosis. For this reason, the present study aimed to investigate the involvement of S1PR2 in EGF-induced migration and invasion in three OSCC cell lines as well as to identify any potential crosstalk between S1P/S1PR2 and EGF/EGF receptor signalling.Human recombinant EGF  was prepared to a final concentration of 20 ng/ml in cell culture media as this concentration has been reported to be optimal for activating squamous carcinoma cells .JTE013, pyridin-6-yl]-4--semicarbazide]  is a S1PR2 antagonist while CYM5478, -2-oxoethyl]-5-(trifluoromethyl)-2(1 H)-pyridinone] , is a S1PR2 agonist. For the experimental work, both JTE013 and CYM5478 were dissolved in dimethyl sulfoxide (DMSO)  and freshly diluted in media to 10 µM immediately prior to use.°C in an atmosphere of 5% CO2. Media was replenished every second day until cultures were approximately 70% confluent.Three human OSCC lines, H357 , H400  and H413  , passageOSCC lines were cultured in 6-well plates until confluent. Cultures were treated with 8 µg/ml mitomycin C  for 2 h to inhibit proliferation. Scratch wounds were generated using sterile disposable plastic pipette tips (10 mm diameter) and washed with phosphate buffered saline (PBS)  before being replenished with FBS-free media containing 20 ng/ml EGF, 20 ng/ml EGF with 10 µM JTE013 and 20 ng/ml EGF with 10 µM CYM5478. Four images were captured with a digital camera  (resolution: 1 pixel = 0.58 μm) immediately and at 24 h after generating scratch wounds, using phase contrast microscopy . Images were analysed by measuring the gap area of the wounds using the MRI wound healing tool plugin for ImageJ software . Experim4 cells were added to the tissue culture inserts, while 500 µl of media containing 20% FBS was added to the lower compartment and cultures maintained at 37 °C in 5% CO2 for 48 h. The collagen and cells on the upper surface of the membrane were removed using a cotton swab. Cells on the lower side of the membrane were stained with calcein AM  and visualised using fluorescence microscopy . Ten images were captured with digital camera  (resolution: 1 pixel = 0.24 μm)   and cell counts were performed with experiments performed in triplicate.Thirty microlitres of 0.5 mg/ml rat tail collagen type I  were added to a 24-well transwell insert  and solidified at 37 °C for 30 min. Three hundred microlitres of FBS-free media containing 2.5 × 102  and the maximum invading distance referred to the largest distance between any two points in a set [Tumour spheroids were generated using the hanging drop method for 24 h (approximately 500 cells/spheroid) with each spheroid embedded in 3 mg/ml rat tail collagen type I . Samples were treated with 8 µg/ml mitomycin C for 2 h to inhibit proliferation, then washed three times with PBS before replenishing with media according to the experimental groups including FBS-free media, media containing EGF concentrations, media containing EGF with 10 µM JTE013 and media containing EGF with 10 µM CYM5478 . The tumin a set .OSCC lines were treated with either the S1PR2 antagonist or agonist for 48 h. The levels of EGF and TGF-β1 were measured in the culture supernatants using ELISA  following the manufacturer’s instructions. Three biological replicates were performed.To compare the expression profile of S1PR1-5, three OSCC lines were cultured in T25 flasks until confluent. Total RNA was extracted using the RNeasy Mini Kit . Single stranded cDNA was synthesized from 2 µg of RNA using the Tetro kit . The amplification process was performed using the following conditions: denaturation at 94°C for 5 min; annealing (25 cycles) at 94°C for 20 sec, 60°C for 20 sec and 72°C for 20 sec; and extension at 72°C for 10 min, respectively . The ampCultures were incubated with media containing either 20 ng/ml EGF; or 20 ng/ml EGF plus 10 µM JTE013 or 10 µM CYM5478 for 48 h. Total RNA was extracted using the RNeasy Mini Kit . Single stranded cDNA was synthesized from 2 µg of RNA using the Tetro kit . cDNA was used as a template for quantitative real-time polymerase chain reaction (qRT-PCR) using a LightCycler 480 SYBR Green I Master kit . The amplifying process included 5 min of initial denaturation at 95°C followed by 45 cycles of amplification . The relative changes of RNA levels of Sphk1 and Sphk2 were normalised to the level of the best housekeeping gene identified for each cell line . Primers sequences used were: Sphk1 F, 5’- GCTGCGAAGTTGAGCGAAAA-3’, Sphk1 R, 5’- CCCGCTGGATCCATAACCTC-3’; and Sphk2 F, 5’-CTAGTCGGGGCATCTGGAAA-3’, Sphk2 R, 5’- CTCACTGTCCTGGCCTGAC-3’. Experiments were undertaken with three biological replicates.Rac1 activity was measured using the G-LISA activation assay . OSCC lines were cultured in T25 flasks until reaching 50% confluence. Cultures were serum starved with 0.5% FBS media for 1 day and FBS-free media for 1 more day. S1PR2 was pre-treated with 10 µM JTE013 or 10 µM CYM5478 for 15 min. Cultures were incubated with 20 ng/ml EGF for 2 min. Cultures were lysed and the total protein was diluted to a concentration of 0.3 mg/ml. The G-LISA was performed according to the manufacturer’s protocol with luminescence measured using a Spark® multimode microplate reader .Differences between groups were statistically analysed using ANOVA followed by post-hoc Tukey tests using GraphPad Prism (Version 9.0.0). P-values of less than 0.05 were considered statistically significant.The three OSCC lines expressed S1PR1-5 (Fig. The activation of S1PR2 can reportedly induce migration in a similar manner to EGF , therefoThe effect of S1PR2 on EGF-induced invasion was investigated using a transwell invasion assay, which showed a similar trend to that previously identified Fig. . InvasioThe transwell invasion assay data indicated invasion potential, however this may have only a limited correlation with the tissue invasion that occurs in vivo. Therefore, a MCTS model to assay invasion was also studied Fig. . Three pAfter establishing the requirement for S1PR2 on EGF-induced migration and invasion, the involvement of the Rac1, a RhoGTPase implicated in cytoskeleton transduction, was investigated . NeitherThe data described above indicated that S1PR2 was involved in the motility of H357, H400 and H413 cells. Consequently, the potential crosstalk between S1P/S1PR2 and EGF/EGF receptors signalling was investigated, and the effect of S1PR2 on the production of EGF and TGF-β1, which was reported to transactivate EGF receptors , was detThe crosstalk between these receptors was also investigated by treating the cell lines with the two concentrations of EGF at 1 ng/ml and 20 ng/ml. S1P production was indirectly measured through gene expression analysis for the enzymes Sphk1 and Sphk2 which convert sphingosine into S1P. Sphk1 expression of the three cell lines increased according to the EGF concentration Fig. A. EGF atBoth EGF/EGF receptors and S1P/S1PR2 pathways are known to affect cell motility , 18, 26.Reorganisation of the cytoskeleton is key to cell motility. To examine how both EGF and S1PR2 may act to coordinate and regulate cell motility, downstream signalling of the RhoGTPase, Rac1, was further investigated. Activation of Rac1 was caused by EGF/EGF receptors stimulation rather than via the S1P/S1PR2 axis, however to stimulate Rac1 activity, a signal from S1PR2 was required. This was in agreement with a previous study, reporting the inability to detect Rac1 changes following S1P activation . NotablyNotably, EGF/EGF receptors and S1P/S1PR2 may neither have a separate cascade nor individually regulate Rac1 since the EGF effect on migration, invasion and Rac1 activity did not increase when both receptors were activated. Several studies have postulated mechanisms that explain the crosstalk of metabolism between S1P and EGF , 36, 37.Another proposed model of breast cancer progression  involvesTo evaluate the crosstalk between EGF/ EGF receptors and S1P/S1PR2 pathways, the metabolism of EGF and S1P was measured. This study also determined the production of TGF-β1 as this molecule is reported to have crosstalk with either EGF/EGFR and S1P/S1PR pathways , 42, 43.A limitation of this study is that the effect of S1PR2 was achieved only using chemical reagents. JTE013 is a selective S1PR2 antagonist and has been widely used at concentrations of up to 10 µM in a number of studies , 45–47, In conclusion, S1PR2 and EGF in OSCC cultures appear linked in both function and metabolism. Migration and invasion of OSCC cells were inhibited following S1PR2 suppression, even after EGF stimulation. EGF itself could increase activation of S1PR2 through the upregulation of S1P production. This suggested that S1PR2 inhibitors could enhance currently available EGF receptor inhibitors as therapeutic targets in OSCC patients. Further work should also identify any potential off-target effects of S1PR2 inhibitors and this has also been proposed in other cell types , 51.Below is the link to the electronic supplementary material.Supplementary Material 1


Cardiomyocyte growth and differentiation rely on precise gene expression regulation, with epigenetic modifications emerging as key players in this intricate process. Among these modifications, N6-methyladenosine (m6A) stands out as one of the most prevalent modifications on mRNA, exerting influence over mRNA metabolism and gene expression. However, the specific function of m6A in cardiomyocyte differentiation remains poorly understood.We investigated the relationship between m6A modification and cardiomyocyte differentiation by conducting a comprehensive profiling of m6A dynamics during the transition from pluripotent stem cells to cardiomyocytes. Our findings reveal that while the overall m6A modification level remains relatively stable, the m6A levels of individual genes undergo significant changes throughout cardiomyocyte differentiation. We discovered the correlation between alterations in chromatin accessibility and the binding capabilities of m6A writers, erasers, and readers. The changes in chromatin accessibility influence the recruitment and activity of m6A regulatory proteins, thereby impacting the levels of m6A modification on specific mRNA transcripts.Our data demonstrate that the coordinated dynamics of m6A modification and chromatin accessibility are prominent during the cardiomyocyte differentiation.The online version contains supplementary material available at 10.1186/s13072-023-00506-6. Heart diseases have long been one of the leading causes of death worldwide , 2. HoweEmerging research has highlighted the importance of epigenetic modifications in regulating cardiomyocyte differentiation, including DNA methylation and histone modifications –14. ReceRecent studies have demonstrated that the m6A modification significantly impacts various biological processes, including cell differential, disease development, neurodevelopment, and immunity . PerturbIn this study, we employed a quantitative m6A detection method by performing multiplexed m6A-immunoprecipitation on barcoded and pooled samples . This apTo obtain an unbiased m6A profile map during human cardiomyocyte differentiation, we first induced human pluripotent stem cells to differentiate into cardiomyocytes. We collected sequential samples at different stages: human Embryonic Stem Cells (hESCs) at day 0 (D0), Mesoderm at day 2 (D2), Cardiac Progenitor Cells at day 5 (D5), and fully differentiated Cardiomyocytes at day 15 (D15). The collected samples were then divided into two portions, with one portion used for RNA-seq analysis and the other for MeRIP-seq analysis. For precise and quantitative assay, we employed MeRIP-seq with a multiplexed m6A-immunoprecipitation strategy on barcoded and pooled samples . Each saTo validate the successful differentiation of cardiomyocytes, we analyzed the RNA-seq data and observed that the expression of stage-specific marker genes was highly elevated, consistent with the expected patterns –34 AddiB. MoreovThe multiplexed m6A-immunoprecipitation strategy also allowed us to estimate the overall m6A methylation level of each specific sample . We furtAlthough no obvious overall dynamic changes in m6A modification were observed, we considered whether m6A modifications in certain genes could affect cardiomyocyte differentiation , 36. To Next, we examined whether these altered m6A peaks are associated with cardiomyocyte differentiation. We identified genes that showed larger changes in m6A levels (fold change > 1.2) at least once during the process of cardiomyocyte differentiation and referred to them as dynamic m6A genes. We discovered that the dynamic m6A genes tended to have a high overlap with genes related to the cardiomyocyte differentiation process  Genes with no changes in either chromatin accessibility or m6A levels. (2) Genes with only changes in chromatin accessibility while m6A levels remained constant. (3) Genes with only changes in m6A levels while chromatin accessibility remained constant. (4) Genes with contrasting trends of change in chromatin accessibility and m6A levels. 5) Genes with similar trends of change in both chromatin accessibility and m6A levels.  modification, and DNA modification also influences cardiomyocyte differentiation progress , 41. HenIn summary, our comprehensive investigation into the role of m6A in cardiomyocyte differentiation has provided valuable insights into its regulatory mechanisms, particularly in relation to chromatin accessibility. Our findings contribute to a better understanding of cardiomyocyte differentiation and hold promise for addressing challenges related to cardiovascular diseases in the future.2. Cells were passaged every 3–4 days using 0.5 mM EDTA  in Dulbecco’s phosphate buffered saline (DPBS) without Ca2+ or Mg2+  at 37 ℃. 5 μM Rho kinase inhibitor Y-27632  was added for the first 24 h after passaging. The E8 medium was changed every day.H1 human embryonic stem cells (hESCs) (obtained from the WiCell Research Institute) were grown on Matrigel -coated 6-well plates in E8 medium  at 37 ℃ with 5% COUndifferentiated hESCs cultured in E8 medium were dissociated into single cell suspension by Accutase  and reseeded onto Matrigel-coated 24-well plate at a density of 105 cells/well in E8 medium containing 10 μM Y-27632. When cells reach ~ 80% confluence 2–3 days after plating, CM differentiation was initiated by switching to the differentiation medium named E8 basal + Lip  supplemented with 50 U ml-1 Penicillin–Streptomycin , Chemically Defined Lipid Concentrate , 10.7 μg ml-1 holo-Transferrin human , 71 μg ml-1 L-Ascorbic acid , 14 ng ml-1 Sodium selenite ). 5 μM CHIR99021  or 3 μM IWP2  was added into the cardiac differentiation medium from days 0–1 and days 2–5, respectively. 3 μg ml-1 heparin was added into the cardiac differentiation medium from days 1–7. 20 μg ml-1 Insulin  was added into the cardiac differentiation medium from day 7 onward and renewed every 2–3 days.Cell samples were captured at time points corresponding to stage-specific transitions in cell state including pluripotency stem cell (differentiation Day 0), mesoderm (Day 2), cardiac progenitor cells (Day 5), and cardiomyocytes (Day 15). Cells were dissociated into single cell using Accutase for 5 min at 37 °C, washed twice with ice-cold wash buffer (DPBS containing 2% FBS). Total RNA was extracted using the NucleoZol reagent  and quantified by a NanoDrop spectrophotometer (ThermoFisher). Then mRNA was purified from total RNA using Dynabeads™ mRNA Purification Kit .The MeRIP-seq protocol builds based on some previous protocols. Briefly, RNA samples were first fragmented to 150 nt by Fragmentation Reagent , and then the purified fragmented RNA was end-repaired using T4 Polynucleotide Kinase . Next, each sample was ligated with different 3′ adapters with barcodes that had been adenylated in advance using 5' DNA Adenylation Kit  with T4 RNA ligase 2, truncated KQ . After ligation, Lambda Exonuclease  was used to remove excess adapters. After the purified products were mixed, 1/10 was taken out as input and temporarily stored at − 20 °C, and the remaining products were subjected to IP in the next step. ProteinG beads  were washed twice in reaction buffer , 0.01% Igepal CA-630) and incubated with m6A antibody  at 4 ℃ on rotator for 40–60 min. The pretreated beads were washed twice in reaction buffer and then incubated with samples at 4 ℃ on rotator for 2 h. After the IP incubation, samples were washed with reaction buffer, low-salt buffer , 0.01% Igepal CA-630) and high-salt buffer , 0.01% Igepal CA-630). Each wash step was conducted 3 times at 4 ℃ on rotator. RNA was eluted from beads with buffer RLT  and a small amount of sample could be taken for qPCR. Then 5′ adapters were ligated to samples using T4 RNA Ligase 1 . After ligation, the product was reverse-transcribed using HiScript III 1st Strand cDNA Synthesis Kit  to generate a cDNA, and the library amplification was performed using 2 × KAPA HiFi Hot Start Ready Mix .Following quality controls (performed with FastQC v0.11.5), reads were aligned to the hg19 genome using hisat2 v2.1.0 . Gene exPaired-end reads of MeRIP-seq were demultiplexed into individual samples with fastq-multx v1.4.3 . DemultiReads were mapped with bwa v0.7.17 to hg 19 genome using default parameters. Mapped reads which MAPQ > 30 were kept. Picard-tools v1.129 was use to remove duplicate reads. Peak calling was performed by MACS2 with the parameters “—nomodel -q 0.01 —shift 100 —extsize 200”. Peaks were annotated to genes using ChIPpeakAnno_3.24.2. Quantification was performed by Featurecounts v1.6.2 and DESeq2 v1.24.0.Reads were mapped with bowtie v1.2.1.1  (for reaThe correlation between expression and m6A level and the correlation between accessibility and m6A level were measured by Pearson correlation coefficient. Genes with correlation coefficients in the top 5% and bottom 5% were classified as positively and negatively correlated genes (pos cor genes and neg cor genes), respectively. Genes with correlation coefficients between −0.1 and 0.1 for expression/accessibility and m6A level were classified as not correlated genes (not cor genes).The visualization of GO enrichment was performed by Metascape  and R. PAdditional file1: Figure S1. Expression level changes during cardiomyocyte differentiation. A Heatmap of expression levels during cardiomyocytes differentiation. B Heatmap of expression levels of some key factors during cardiomyocytes differentiation. Figure S2. GO enrichment analysis results of genes under different clusters in Figure Figure S3. Correlation between gene m6A levels and expression levels. A Dot plot show a weak negative correlation between gene m6A levels and expression levels during differentiation. B Comparison of gene expression levels among different m6A levels. Genes were categorized into three groups based on their m6A levels: low (m6A level < 1.5), medium (1.5 < m6A level < 4), and high (m6A level > 4). ** represents p-value<0.01. Figure S4. Correlation analysis between the expression of potential target genes associated with cardiomyocyte differentiation  and the expression levels of m6A readers. The correlation was assessed using the Pearson correlation coefficient. Figure S5.A The correlation between reader expression and m6A does not affect the accessibility of the corresponding gene. B The reader responds differently to genes with different degrees of accessibility. neg/not/pos cor: genes whose m6A levels or accessibility are negatively/not/positively correlated with the expression of m6A-related readers. Figure S6. Comparison of binding levels of key transcription factors with different m6A level in D0 and D15. A Binding levels of key transcription factors in stem cells. B Binding levels of key transcription factors in cardiomyocytes. Genes were categorized into three groups based on their m6A levels: low (m6A level < 1.5), medium (1.5 < m6A level < 4), and high (m6A level > 4). * represents p-value<0.05, Wilcoxon test. Figure S7. GO enrichment analysis results of genes under four groups base on their m6A level and accessibility changes: A genes with discordant changes, B genes with concordant changes, C genes with only m6A changes, and D genes with only accessibility changes


Several experimental methods were applied to characterize these nanocomposites such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and environmental scanning electron microscopy (ESEM). The calculation of NiS crystallites revealed an average size of 8.0 nm. The ESEM and TEM images of S@g-C3N4 showed a 2D sheet structure and NiS-g-C3N4 nanocomposites showed the sheet materials that were broken up during the growth process, revealing more edge sites. The surface areas were 40, 50, 62, and 90 m2/g for S@g-C3N4, 0.5 wt.% NiS, 1.0 wt.% NiS, and 1.5 wt.% NiS, respectively. The pore volume of S@g-C3N4 was 0.18 cm3, which was reduced to 0.11 cm3 in 1.5 wt.% NiS owing to the incorporation of NiS particles into the nanosheet. We found that the in situ polycondensation preparation of S@g-C3N4 and NiS-g-C3N4 nanocomposites increased the porosity of the composites. The average values of the optical energy gap for S@g-C3N4 were 2.60 eV and decreased to 2.50, 2.40, and 2.30 eV as the NiS concentration increased from 0.5 to 1.5 wt.%. All NiS-g-C3N4 nanocomposite catalysts had an emission band that was visible in the 410–540 nm range and the intensity of this peak decreased as the NiS concentration increased from 0.5 to 1.5 wt.%. The hydrogen generation rates increased with increasing content of NiS nanosheet. Moreover, the sample 1.5 wt.% NiS showed the highest production rate of 8654 mL/g·min due to the homogeneous surface organization.The nanocomposites of S@g-C A significant planer network structure is developed by the covalent connections that are formed by each carbon atom with three nitrogen atoms. The sp2 hybridized C and N atoms are organized in a six-member stalked ring; providing the semiconductor properties of g-C3N4. In the optical range of 260–320 nm, g-C3N4 has a significant UV–Vis absorption peak. The π-π* electron transfer for g-C3N4 with s-triazine rings is responsible for the absorption peak, which is located at about 250 nm. Moreover, the n-π* electron transfer involving a lone pair of electrons on nitrogen atoms in the g-C3N4 produced the absorption peak at 320 nm [3N4 doped with heteroatoms  can be tuned, which is advantageous for expanding their applications in bioimaging and biosensing. For catalytic usage, g-C3N4 has enhanced thermal stability because of higher durability and non-volatility up to 600 °C even in the air, according to the thermogravimetric analysis [3N4 in organic solvents resists the processing of this material.Research on graphitic carbon nitride , SP-PA particles generate hydrogen at a rate of 762.4 mL min−1g−1. The remarkable catalytic activity of SP-PA particles is indicated by the computed activation energy of 29.92 kJ mol−1 for NaBH4 hydrolysis. Fanghui Wang et al. [4. The dandelion-like structure of the Co-P/CNTs-Ni foam catalysts was maintained and produced a maximum amount of hydrogen (2430 mL min−1g−1) at a temperature of 298 K. The data showed that NaBH4 hydrolysis has an activation energy of 49.94 kJ mol−1. Another study used calcination and hydrothermal techniques to generate a hybrid g-C3N4-SiO2-N composite [4 content of 0.25 g revealed a maximal hydrogen evolution rate of 11,400 mL min−1g−1. According to calculations, the hydrolysis of NaBH4 has an activation energy of 33.2 kJ mol−1. Cafer Saka [−1g−1. Meanwhile, the activation energy for hydrolysis was 10.59 kJ mol−1. Highly dispersed CoB alloys implanted on MOF-74-derived graphene nanosheets were synthesized using the chemical reduction technique [−1g−1 for the hydrolysis of NaBH4. Furthermore, the investigation of activation energy for hydrolysis was evaluated as 38.8 kJ mol−1.Over the past few years, numerous efforts have focused on the synthesis of g-C nitride . Cyanami nitride . Additio nitride . The abimaterial . The suranolysis . The datn et al.  reportedg et al.  synthesiomposite . The exp3N4. Therefore, researchers proposed many techniques to improve conductivity and surface area by elemental doping, converting into nanosheets, and combining with metal nanoparticles and other carbon nanomaterials. This in turn will suppress the electron–hole recombination. The in situ polycondensation process can produce metal/g-C3N4 nanocomposites. Meanwhile, this synthesis procedure enables the direct formation of nanocomposites as well as microstructure control. Moreover, this technique has excellent aggregate elimination/reduction along with ideal and reproducible properties.Graphitic carbon nitride suffers from low conductivity and a small electroactive surface area. Low carrier mobility and excessive bulk recombination are the main reasons limiting the efficiency of g-C3N4 in order to improve the catalytic activity. Furthermore, NiS nanostructures can be integrated in situ into the g-C3N4 nanosheet to increase the electroactive surface area and electrical conductivity. The in situ polycondensation process using different ratios of nickel chloride and thiourea at 550 °C for 120 min synthesized the NiS-g-C3N4 nanocomposite samples. The structural measurements for these nanocomposites was conducted using XRD, FTIR, and ESEM techniques. Moreover, an extensive study of the methanol hydrolysis of sodium borohydride was completed. Finally, the hydrogen catalytic efficiency of prepared materials was examined at methanolysis of NaBH4. The hydrogen generation rates increased with increasing NiS nanosheet content. Moreover, the sample 1.5 wt.% NiS showed the highest production rate of 8654 mL/g·min.The current study aims to find an appropriate precursor, morphology, exfoliation condition, and fabrication processes for g-CLoba Chemi, Mumbai, India supplied the chemicals . The absolute methanol was provided by Sigma-Aldrich, Darmstadt, Germany. All provided chemicals were directly used without extra purification.3N4 was synthesized by heating 10.0 g of thiourea powder in a porcelain crucible covered with a lid in the muffle furnace at 550 °C (ramping rate = 3.0 °/min) and maintaining the temperature for 2 h. The yellow solid mass in a crucible was then allowed to cool to room temperature. Using an agate mortar, the resultant bulk S@g-C3N4 was crushed into a fine powder. The in situ polycondensation process using different ratios of nickel chloride and thiourea at 550 °C for 120 min synthesized NiS-g-C3N4 nanocomposite samples. In a typical synthesis, 10 g of thiourea powder and 0.5, 1.0 and 1.5 wt.% of nickel chloride were ground in an agate mortar for 30 min. After that, the powder was transferred to porcelain crucibles covered with a lid inserted inside a muffle furnace. The furnace operated at 550 °C for 120 min at a heating rate of 3.0 °C/min. The obtained nanocomposite samples were allowed to cool and then ground.The bulk S@g-Ckα wavelength of 1.54056 Å. Cukα radiation was created by using a Cu source as an X-ray source. The data scans were collected at the 2Theta range 5.0–80° with a count rate of 0.2°/min. The crystal structure was identified by comparing the diffraction pattern of the synthesized nanocomposite with the JCPDS files in the database. FTIR data were collected using a Shimadzu FTIR spectrometer—100 Tracer. The frequency ranges that can be examined are generally in the 4,000,399 cm−1 range. The sensitive characterization instrument ESEM exposes surface morphology and when coupled with an EDX  accessory, determines the elemental composition of materials. High-resolution 3D micrographs of the morphology were provided utilizing the ESEM technique. ESEM images and EDX data were acquired using a Thermo Fisher Quattro environmental scanning electron microscope (ESEM). Transmission electron microscopy (TEM) has become an essential tool in medical, biological, and materials’ research because of its high magnifications. An investigation of TEM microscopy was carried out using a Thermo Fisher Scientific Talos F200i TEM/STEM electron microscope. The most generally used technique for calculating the specific surface area of produced material is the Brunauer–Emmett–Teller (BET) method. The BET technique involves the multi-layer adsorption of chemically inert N2 gas with relative pressure and gas volume adsorbed in cm3/gm. The samples were degassed at 100 °C overnight to remove trapped moisture molecules. The samples were subjected to N2 gas at 77 K to record the adsorption–desorption isotherm on NOVA 4200e surface area analyzer. As a significant characterization tool in the field of photocatalysis, UV–Vis spectroscopy is a non-destructive method for analyzing optical characteristics such as absorbance, reflectance, transmittance, and bandgap energy that are related to the chemical composition of the material. In order to move electrons from their ground state to their excited state for electronic spectroscopy, it is necessary to absorb photons in the UV–visible region of the spectrum. The UV–Vis spectra were recorded on Agilent Cary 60 Spectrophotometer. The photoluminescence (PL) approach has been widely applied in the field of photocatalysis to study surface processes. Photoluminescence (PL) spectra at room temperature were obtained using a Cary Eclipse fluorescence spectrometer with a 350 nm excitation wavelength.X-ray diffraction studies can analyze structural factors such as crystallinity, grain size, strain, phase composition, and structural defects. The XRD spectra were recorded from a Shimadzu XRD 700 instrument utilizing a Cu4 and 10 mL of methanol was added without stirring. The hydrogen gas volume was recorded via the water displacement method. The measurements were completed at a temperature of 30 °C.The hydrogen catalytic efficiency of prepared materials was evaluated as follows. Typically, 20 mg of the nanocomposite sample was mixed with 0.25 g of NaBH3N4 and NiS-g-C3N4 nanocomposite catalysts are presented in 3N4. The small diffraction peak at 13.08° matched a (100) plane with a distance of 0.676 nm [3N4 material and located at 2θ = 27.20°, which occurs because the atomic radius of sulfur is larger than carbon and nitrogen. Further, small diffraction peaks of sulfur were observed in the XRD spectrum of S@g-C3N4 [3N4 at 0.5, 1.0, and 1.5 wt.%, the peak (002) moved to 27.55°, 27.66° and 27.64°, respectively. The positions of these reflections shifted to higher angles and thus lowered d-values with increase in NiS content. This indicates a structural contraction along the layer-stacking direction, presumably because of a more extended condensation of the carbon nitride chains [XRD is a non-destructive analytical method that provides details of the physical characteristics and crystalline structure of materials. The results of XRD data for the synthesized S@g-C0.676 nm ,27. The S@g-C3N4 . For NiSe chains . Moreovee chains .3N4 nanocomposites is confirmed by the existence of the two phases. Meanwhile, the crystallite size (D) is inversely proportional to the diffraction peak broadening (β) as proposed by the Scherer equation [The reflections of NiS are located at 30.24°, 34.54°, 45.65°, 53.34°, 60.71°, and 62.94° that corresponds to (100), (101), (102), (110), (103), and (200) of hexagonal crystal structure (ICDD No. 50-1791). The synthesis of the NiS-g-Cequation ,32:(1)D=3N4 decreased as the NiS concentration increased from 0.5 to 1.5 wt.%. This reveals the small crystallite domains of S@g-C3N4. The calculation of NiS crystallites reveals an average size of 8.0 nm for 0.5 wt.% NiS, 1.0 wt.% NiS, and 1.5 wt.% NiS. Moreover, the shift of peak position for the (002) plane after the growth of NiS at different content (0.0–1.5 wt.%) confirms the successful formation of nanocomposites.Accordingly, the calculations of b at the diffraction peak with reflection (002) for S@g-C3N4 and NiS-g-C3N4 nanocomposite samples. The vibrations of C-O stretching and C-OH stretching for the S@g-C3N4 sample located at 1010 and 1132 cm−1 [2O adsorption showed two peaks at 3100–3300 cm−1 [−1 observed in the spectrum of S@g-C3N4 correspond to the typical stretching modes of CN heterocycles [2 from the environment was responsible for a weak band at 2336 cm−1 [−1 [132 cm−1 . This re300 cm−1 . The absrocycles . It was −1 suggests the samples consist of triazine or heptazine building blocks [−1 indicate the presence of C–N (sp3) and C-N(-C)-C bonds [3N4 nanocomposites and agree with the XRD investigations.The medium intense sharp band at around 804 cmg blocks . The ban-C bonds . Moreove3N4 and NiS-g-C3N4 nanocomposite samples were investigated by ESEM images. In 3N4 shows that a 2D sheet structure was achieved for g-C3N4 materials. NiS-g-C3N4 nanocomposites showed the sheet materials were broken up during the growth process in 3N4 is less ordered and crystalline by virtue of the broader spectral peaks.The morphology and structure of the synthesized S@g-C3N4 nanocomposite samples confirm the presence of all the elements supposed. 3N4 nanocomposite samples are of high purity.The EDX spectra displayed in 3N4 and 1.5 wt.% NiS nanocomposite samples are shown in 3N4 reveals stack layers that are connected with XRD and ESEM measurements. Moreover, the sheet materials in 1.5 wt.% NiS nanocomposite sample were broken up throughout the growing process.TEM images of S@g-C2 adsorption–desorption isotherm at 77 K for the S@g-C3N4 and NiS-g-C3N4 nanocomposite samples  is an important parameter to estimate the electronic structure of the S@g-C3N4 and NiS-g-C3N4 nanocomposites. optE is calculated by evaluation of the straight lines intercept at zero photon absorption from the plots of (αhν)2 vs. photon energy (hν) shown in The optical energy gap  of the S@g-C3N4 and NiS-g-C3N4 nanocomposites are calculated with help of the following equations using the H2 volume (V), the mass of catalyst (catm), and time of reaction (t) [The rate of hydrogen production greatly determines the efficiency of the catalyst to speed up the reaction. Hydrogen evolution rates (tion (t) ,53:(3)r=3N4 and NiS-g-C3N4 samples. The hydrogen generation rates increased with the increase in the NiS nanosheet content. Moreover, the sample 1.5 wt.% NiS showed the highest production rate of 8654 mL/g·min. The nanocomposite 1.5 wt.% NiS had the highest generation rate among others due to the promising surface design [4 decomposes in methanol into Na+ and 4 will be accelerated. Additionally, this rate of hydrogen evolution (8654 mL/g·min) exceeds the rates for R–TiO2-NH2 (3525 mL/g·min) [2@PAA (5120 mL/g·min) [The hydrogen evolution curves displayed in L/g·min) , SiO2@PAL/g·min) , ZIF-67@L/g·min) , and Ru/L/g·min) .3N4 and NiS-g-C3N4 and was employed for catalytic hydrogen production from the methanolysis of sodium borohydride. The incorporation of NiS during the growth process played a major role in the enhancement of the surface area and porosity of the S@g-C3N4 composites. The 1.5 wt.% NiS sample had the highest surface area of 90 m2/g compared with the 0.5 wt.% NiS and 1.0 wt.% NiS samples. The pore volume of S@g-C3N4 was 0.18 cm3, which was reduced to 0.11 cm3 in 1.5 wt.% NiS owing to the incorporation of NiS particles into the nanosheet. We found that during the in situ polycondensation preparation of S@g-C3N4 and NiS-g-C3N4 nanocomposites increased the porosity of the composites. The average value of the optical energy gap for S@g-C3N4 was 2.60 eV and decreased to 2.30 eV because of the 1.5 wt.% NiS incorporation. The NiS-g-C3N4 catalysts showed an emission band in the 410–540 nm range and the intensity of this peak decreased as the NiS concentration increased from 0.5 to 1.5 wt.%. The hydrogen generation rates increased with the increase in the NiS nanosheet content. The sample 1.5 wt.% NiS showed the highest production rate of 8654 mL/g·min compared with others due to the promising surface design. The large surface area of the nanocomposite sample 1.5 wt.% NiS helps for adsorption of more 4 will be accelerated. All of these results enhance the possibility of using 1.5 wt.% NiS as a promising catalyst for the production of hydrogen from NaBH4 methanolysis.The in situ polycondensation method was implemented for the preparation of S@g-C


Epigenetic reprogramming is involved in luteinizing hormone (LH)-induced ovulation; however, the underlying mechanisms are largely unknown.We here observed a rapid histone deacetylation process between two waves of active transcription mediated by the follicle-stimulating hormone (FSH) and the LH congener human chorionic gonadotropin (hCG), respectively. Analysis of the genome-wide H3K27Ac distribution in hCG-treated granulosa cells revealed that a rapid wave of genome-wide histone deacetylation remodels the chromatin, followed by the establishment of specific histone acetylation for ovulation. HDAC2 phosphorylation activation coincides with histone deacetylation in mouse preovulatory follicles. When HDAC2 was silenced or inhibited, histone acetylation was retained, leading to reduced gene transcription, retarded cumulus expansion, and ovulation defect. HDAC2 phosphorylation was associated with CK2α nuclear translocation, and inhibition of CK2α attenuated HDAC2 phosphorylation, retarded H3K27 deacetylation, and inactivated the ERK1/2 signaling cascade.This study demonstrates that the ovulatory signal erases histone acetylation through activation of CK2α-mediated HDAC2 phosphorylation in granulosa cells, which is an essential prerequisite for subsequent successful ovulation.The online version contains supplementary material available at 10.1186/s13072-023-00485-8. Ovarian follicle growth and ovulation depend on two gonadotropins secreted from the pituitary gland: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) . GranuloLHCGR) [Areg, Ereg, Sult1e1, and Star [KAT3A/B), two lysine acetyltransferases (KATs) [Several signal pathways are involved in ovulatory signal (LH or hCG)-induced ovulation. When LH or hCG binds to the same receptor, that is, LH receptor (encoded by LHCGR) , it actiLHCGR) –7. TheseLHCGR) . This siand Star . We prevs (KATs) , 10. Thes (KATs) –10. LH/hs (KATs) . DespiteCsnk2a1-encoded CK2α, a protein kinase [Histone acetylation is a dynamic epigenetic modification regulating chromatin conformation and transcription. Of the multiple histone acetylation modifications, histone H3 acetylation at lysine 27 (H3K27Ac) is used as a marker for the active promoter and distal enhancer that facilitate active transcription , 13. H3Kn kinase , 21. Phon kinase . CK2α ovn kinase . HoweverWe here demonstrated a genome-wide histone deacetylation process between two active transcription waves separately mediated by FSH-mediated follicle growth and LH-mediated ovulation. LH/hCG promotes activation of HDAC2 phosphorylation to non-selectively deacetylate histones, which is essential for the subsequent establishment of histone acetylation and gene transcription for ovulation. HDAC2 phosphorylation was found to be dependent on CK2α. When CK2α was inhibited or HDAC2 was depleted, H3K27 deacetylation was blocked and ovulation-induced gene expression was attenuated. This study offers evidence that CK2α/HDAC2-mediated histone deacetylation is a key step in chromatin remodeling for subsequent transcriptional switching during LH/hCG-induced ovulation.To investigate the mechanism underlying LH/hCG-triggered ovulation, a standard ovarian stimulating protocol was used to induce ovulation in a mouse model. The mice were injected with pregnant mare serum gonadotrophin  of the common H3K27-enriched peaks were overlapped with promoters, and 37.8% of the peaks with an increased H3K27ac signal are found in the promoter region  was performed to examine the mRNA levels of all HDACs HDAC1–HDAC10) and identified that HDAC2 mRNA levels were the highest in mouse ovaries at different stages Fig. A. The pr–HDAC10 aStudies have demonstrated that HDAC2 phosphorylation at Ser394 (pHDAC2(S394)) is responsible for corepressor cooperation and histone deacetylase activity . HoweverHdac2 into primary cultured GCs and cumulus–oocyte complexes (COCs). We used forskolin (FSK) and phorbol 12-myristate 13-acetate (PMA) to mimic LH- or hCG-induced activation of the cAMP/PKA and PKC signaling pathway in primary cultured ovarian GCs [Hdac2 siRNA transfection, HDAC2 protein levels decreased, and consequently, H3K27Ac could not be efficiently deacetylated  small-interfering RNA (siRNA) sequence or a siRNA targeting mouse rian GCs , 27. At Hdac2 siRNAs and subjected to in vitro maturation. Hdac2 mRNA levels were significantly decreased and the cumulus cell expansion capacity was impaired compared with that in the control group  for 4 h before hCG injection. We then analyzed H3K27Ac, transcription, and ovulation at different timepoints Fig. A. H3K27Aoup Fig. F. Transcoup Fig. G. These Star, Sult1e1, and Ereg, was also reduced , Star, Sultl1e1, and Ptgs2 [Smad4, encoding a binding partner of SMAD2/3, led to defects in ovulation [LH or hCG mediates the transcription of many ovulation-induced genes, such as EGF-like factors  cannot be initiated before the ovulatory signal because HDAC3 blocks the accessibility of the Areg promoter [HDACs are divided into four classes , and cla ovaries . The Xiapromoter . The comCK2α is a kinase phosphorylating HDAC1 and HDAC2 and thus facilitating their functions . In PCOSHdac2 depletion or inhibition leads to inactive transcription of ovulation-specific genes and abnormal cumulus expansion. These results indicate that HDAC2 has a more crucial role in cumulus GC expansion. HDAC2 is a gene repressor, but many studies have shown that the HDAC2 level is associated with the transcription level [Interestingly, after hCG induction, both HDAC2 and pHDAC2 preferentially localized in cumulus cells and mural GCs adjacent to the follicular fluid Fig. C. This pon level , 49. We on level , E2F1 [5on level , STAT3 [on level , and SMAon level . These pThis study demonstrates that, after ovulatory signal induction, non-selective histone deacetylation occurs to reset the chromatin, which is followed by specific histone acetylation for ovulation. HDAC2 phosphorylation activation-mediated histone deacetylation is essential for ovulation. The study provides a better understanding of the physiology of LH/hCG-induced ovulation.Female ICR mice were obtained from the Experimental Animals Center of Zhejiang Academy of Medical Sciences. Mice were housed in specific pathogen free conditions with regular and normal environment. All animal experiments were conducted according to the guidelines of the Animal Care Committee of Zhejiang University.Ovarian stimulation was performed in 23- to 25-day-old female mice via intraperitoneal injection of 5 IU of PMSG  to stimulate follicle growth followed primed with 5 IU of hCG  44–48 h later to induce ovulation.For inhibitor administration in vivo, 23-day-old female mice were injected with saline, 2.16 mg/kg body weight FK228  or 50 mg/kg body weight TBB  dissolved in saline at 44 h post PMSG injection and then with 5 IU of hCG 4 h later. Ovaries were collected 0 h, 1 h, 4 h, 8 h, and 14 h post hCG injection for frozen sectioning, mRNA extraction and western blotting. Meanwhile, MII oocytes were collected for counting at 14 h after hCG.2. To knock down HDAC2 expression, GCs were transfected with Hdac2 siRNAs (si-Hdac2) or negative control siRNA (si-NC) with Lipofectamine™ 3000 Transfection Reagent  in accordance with the manufacturer’s instructions. The sequences of si-Hdac2 are summarized in Additional file Hdac2 siRNAs for 24 h in α-MEM  medium, supplemented with 26 mM sodium bicarbonate, 0.23 mM pyruvate, 3 mg/ml BSA, 2.5 uM milrinone and 1% (v/v) penicillin and streptomycin , and then were cultured in a commercial in vitro maturation medium  for 14 h at 37 ℃ with 5% CO2.To culture primary COCs and GCs, mice at 3 weeks of age were sacrificed 48 h after injection of 5 IU of PMSG, as described previously . COCs anTo mimic the effects of LH, GCs were treated with FSK  and PMA , as described previously , for 30 Proteins were extracted from tissues and cells dissolved in 1 × Laemmli sample buffer , and then resolved by SDS-PAGE after boiling at 95℃ for 10 min. After the proteins were transferred onto polyvinylidene difluoride membranes , the membranes were blocked in 5% defat milk for 1 h at room temperature, and then incubated with primary antibodies overnight at 4 °C. After 3 times washing in TBS/0.1%Tween-20, the membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies  for 1 h at room temperature. The bound proteins were visualized by incubating enhanced chemiluminescence . The primary antibodies are listed in Additional file Gapdh mRNA levels and compared with those in the controls, and all RT-qPCR assays were performed in triplicate. The primers are shown in Additional file Total RNA was isolated from mouse ovaries and GCs with TRIzol Reagent . RNA was extracted from COCs using RNeasy Mini Kit  in accordance with the manufacturer’s protocol. Reverse transcription was conducted with HiScript II Reverse Transcriptase . RT-qPCR was performed with SYBR qPCR Master Mix  on an CFX96 Real-time System . The relative mRNA expression levels were normalized to the endogenous Ovaries were collected, fixed in 4% (w/v) paraformaldehyde and embedded in paraffin under routine procedures. For histology, the ovaries were then sectioned at 5 μm thickness and stained with haematoxylin and eosin (H&E). To perform IF, the tissues were embedded in optimal cutting temperature (OCT) compound and sectioned at 5 μm thickness. The sections were incubated for 1 h with blocking buffer  and 0.3% Triton X-100 (v/v)). After being probed with primary antibodies at 4 °C overnight, the sections were incubated with Alexa Fluor 568-conjugated goat anti-rabbit IgG (H + L) secondary antibodies  for 1 h and counterstained with 4′,6-diamidino-2-phenylindole  for 10 min. The primary antibodies are summarized in Additional file HDAC2 activity was measured with HDAC-Glo™ I/II Assays  according to the manufacturer’s protocol. Briefly, 100,000 cells were mixed with HDAC-Glo™ I/II Reagent at room temperature for 30 min, and HDAC2 activity was determined by luminescence.As H3K27Ac is mainly deposited in GCs of the ovaries, ChIP was performed using whole ovary samples with the SimpleChIP Plus Enzymatic Chromatin IP Kit  in accordance with the manufacturer’s instructions. Briefly, the ovaries were collected 0, 1, and 4 h post-hCG treatment (approximately 25 mg of the ovarian tissues per immunoprecipitation (IP) experiment) and sliced into small pieces in cold PBS containing a protease inhibitor cocktail and cross-linked with a final concentration of 1.5% formaldehyde in PBS at room temperature. After the addition of glycine to stop cross-linking and two washes in PBS, the tissues were disaggregated into single-cell suspensions by using a high-speed tissue homogenizer. The nuclei were prepared using the provided buffer A (ice-cold) with a protease inhibitor cocktail. Chromatin was digested in 0.5 µL of Micrococcal Nuclease  and then sonicated using a Bioruptor apparatus  at a high intensity for 40 cycles of 10-s ON and 10-s OFF on an ice bath. Then, 1/10 dilutions of the lysates were purified for DNA and subjected to electrophoresis and concentration determination. For each sample, 2% of the remaining volume was kept aside as the input. ChIP was performed on approximately 10 µg of the digested and cross-linked chromatin. The chromatin for each timepoint sample was incubated with 1 µg of histone H3 antibody , 1 µg of normal rabbit IgG , or 1 µg of H3K27Ac antibody  overnight at 4 °C and then with 40 µL of protein G magnetic beads per IP incubation for 2 h at 4 °C with rotation. After washing, elution, and reversal of cross-linking, DNA was purified in the provided columns. The libraries were prepared for the ChIP DNA experiments by using the TruSeq DNA LT/HT Sample Prep Kit (Illumina) and subjected to quality control on the Bioanalyzer (Agilent 2200). Illumina sequencing was performed on the Illumina HiSeq 2000 Sequencing system at RiboBio Co., Ltd.http://david.abcc.ncifcrf.gov/home.jsp) using a cut-off P < 0.05. The GO results were visualized using the bioinformatics online website (http://www.bioinformatics.com.cn/).The ChIP-seq reads were aligned to the mouse genome mm10 using Bowtie (Version 2.0) under default parameters. For peak calling, we used MACS2.0 by applying default parameters and significance threshold , as described previously . To visuFor ChIP-qPCR, 25 mg ovaries were used per reaction. ChIP was performed as described above. Then, qPCR was carried out the to determine the enrichment of antibodies of IgG or H3K27Ac using DNA of input and ChIP eluents.t-test. A P-value < 0.05 was considered statistically significant.The RNA-seq data for mouse granulosa cells at 0 h and 4 h after hCG treatment were retrieved from the GEO (accession number: GSE119508), and the upregulated genes after hCG induction were extracted from the supplementary data in their article. The intensities of the WB bands and fluorescence images were analysed with ImageJ software . The RT-qPCR data, WB and IF intensities, HDAC2 activity and oocyte superovulation numbers are presented as the means ± SDs. The results were analysed with GraphPad Prism . Multiple groups were compared using one-way ANOVA without matching or pairing followed by Tukey’s post-test. Two groups were compared by two tailed unpaired Additional file 1.Figure S1. The Dynamics of H3K27Ac Levels during Follicular Growth and Ovulation. Mice were treated with pregnant mare serum gonadotrophin (PMSG) to stimulate follicle growth or human chorionic gonadotropin (hCG) to trigger ovulation. Ovaries were collected at the indicated timepoints for immunofluorescence. The representative images showing the H3K27Ac (red) levels with DAPI (blue) co-stained for visualization of nucleus (n=3). Scale bar=100um. Figure S2. The H3K27Ac ChIP-seq Analysis for H3K27Ac-gain Genes after Ovulation Signal Induction. (A) Pie charts represent the ratio of stable H3K27Ac-enriched peaks, H3K27-loss peaks and H3K27-gain peakss (de novo H3K27Ac-deposited peaks and H3K27Ac-increased peaks). (B) Genome browser snapshot shows Prss56 is one of de novo H3K27Ac-gain genes after hCG induction. (C) Gene Ontology (GO) analysis shows the biological process (BP) of de novo H3K27Ac-gain genes after hCG induction. (D) Bar charts representing the top enriched KEGG pathway of the ovulatory specific genes 4 h post hCG. (E) The HOMER known and de novo motif analysis for the H3K27Ac-gain peaks to predict transcriptional factors. Figure S3. Semiquantitative analysis of protein levels in Fig. 3. The western blot band intensities of Fig. 3B were measured with ImageJ software. HDAC1 and p-HDAC2 protein levels were normalized to Histone H3 levels. Data were expressed as mean±SD. P value was determined by two-way ANOVA followed by Tukey’s post-test. ** P<0.01.Figure S4. Depletion or inhibition of HDAC2 Causes Increased H3K27Ac Levels and Affects COC expansion. (A) Western blot analysis and quantification of HDAC2 and H3K27Ac levels in siNC and siHdac2 group. Primary granulosa cells were transfected of negative control (siNC) or Hdac2 siRNAs (siHdac2) for 36 h followed by treatment of forskolin  and phorbol 12-myristate 13-acetate  for indicated time length. All the protein levels were normalized to Histone H3 levels. The HDAC2 bands’ intensities were compared with siNC and siHdac2 groups in C. The bands’ intensities of H3K27Ac were compared among FSK/PMA non-treated group (0h) and FSK/PMA treated groups . (B) Semiquantitative analysis of p-HDAC2 and H3K27Ac levels in Fig. 4A. The western blot band intensities of Fig. 4A were measured with ImageJ software. All protein levels were normalized to Histone H3 levels. The siHDAC2 bands’ intensities were compared with siNC bands’ intensities. (C) Comparation of COCs expansion areas in Fig. 4B. The areas of COCs expansion in siNC and siHDAC2 groups were measured with ImageJ software. (D) Quantitative analysis of H3K27Ac, H3K9Ac and H4K16Ac levels in Fig. 5B. All the protein levels were normalized to histone H3 levels. The FK228 bands’ intensities were compared with control bands’ intensities. All data in A-D were expressed as mean±SD. P value was determined by two-way ANOVA followed by Tukey’s post-test. ns. means no significance. * P<0.05, ** P<0.01. Figure S5. Inhibition of HDAC1/2 Hinders Cumulus Expansion and Ovulation. Hematoxylin and eosin (HE) staining results showing FK228, a HDAC1/2 inhibitor, blocks cumulus expansion and cause follicle atresia. Mice pretreated PMSG for 44 h were injected with DMSO or FK228, and 4 h later followed by hCG injection to induce ovulation (n=12). Ovaries were collected at 0 h, 4 h and 8 h post-hCG in mice of control and FK228 group. Scale bar, 50 um. Figure S6. Semiquantitative analysis of CK2α, p-HDAC2, H3K27Ac and p-ERK1/2 levels in Fig. 6A and C. The western blot band intensities were measured with ImageJ software. CK2α, p-HDAC2 and H3K27Ac protein levels were normalized to Histone H3 levels and p-ERK1/2 levels were normalized to ERK1/2. The TBB bands’ intensities were compared with control bands’ intensities. Data were expressed as mean±SD. P value was determined by two-way ANOVA followed by Tukey’s post-test. ** P<0.01Additional file 2:Table S1. Oligonucleotides used for qPCR or siRNA. Table S2. Reagents or commercial kits used in this study. Table S3. The DEG list of RNA-seq data (H4 vs H0).


Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome  Mitochondria are energy-supplying organelles that participate in diverse cellular processes and are important determinants for the oocyte maturation and embryo development in mammals . In oocyThe mitochondrial metabolism is related to fusion and fission morphology transitions . MitochoHowever, the effects of YME1L1, which regulates mitochondrial function and morphology during porcine embryo preimplantation development, have not been reported. In the present research, we hypothesized that YME1L1 is crucial for the porcine embryo preimplantation development by maintaining the mitochondrial morphology and the balance of fission and fusion. To evaluate this hypothesis, YME1L1 was knocked down using double-stranded RNA (dsRNA) at the zygote stage and cultured until the blastocyst stage for further analysis.All reagents were purchased from Millipore Sigma , and all manipulations of live embryos were performed on a heating plate at 38.5°C, unless otherwise indicated.2.Porcine ovaries were collected and stored at 37 °C in saline containing 75 mg/mL penicillin G and 50 mg/mL streptomycin sulfate and delivered from a local slaughterhouse  to the laboratory. The diameter of follicles around 3–6 mm was aspirated using a 10 mL disposable syringe with an 18-gauge needle. The porcine COCs with more than two layers cumulus cells were collected and washed with HEPES briefly. Approximately 100 COCs were transported to each well of 4-well plates with IVM medium  and incubated for 44–48 h at 38.5°C and 5% CO2, 0.05 mM MgS04, 0.01% polyvinyl alcohol , and 0.5 mM 4-(2-hydroxyethyl) piperazine-l-ethanesulfonic acid]. To prevent the pseudo-second polar body extrusion, the activated oocytes were treated with 7.5 μg/mL cytochalasin B for 3–6 h. Subsequently, approximately 50 embryos were washed and cultured in one of 4-well plates with IVC medium for microinjection. The embryos were then cultured for 7 days at 38.5°C and 5% CO2. The blastocysts on the 7th day were randomly collected for further experiments. The total cell number of blastocyst was counted under the epifluorescence microscope (Nikon).The COCs with extended cumulus cells were pipetting for around 40–50 times in 1 mg/mL hyaluronidase to remove cumulus cells. MII stage oocytes with first polar body were then selected and parthenogenetically activated by 2 direct-current pulses of 120 V for 60 µs in activation buffer [297 mM mannitol (pH 7.2) supplemented with 0.1 mM CaClYME1L1 and OMA1were amplified from cDNA of porcine COCs using the primers containing the T7 promoter sequence  to synthesize dsRNA according to the manufacturer’s instructions. After 10 h in vitro transcription, dsRNA mixture was treated with DNase I for 15 min to degrade the DNA template, and then purified using Riboclear™ Plus . The purified dsRNA was dissolved in RNase-free water and stored at −80 °C until use.The fragments of sequence . In vitrYME1L1 or OMA1 was microinjected into 1-cell stage embryos after CB treatment using a Nikon Diaphot Eclipse TE300 inverted microscope  connecting an Eppendorf Femto-Jet  and equipped with a Narishige MM0-202N hydraulic 3-D micromanipulator . After microinjection, the embryos were continually cultured in IVC medium for 7 days. The dsRNA of green fluorescent protein (GFP) was microinjected as the control group.For the knock-down experiments, the dsRNA of 2O, real-time qPCR was performed using a pair of primers for ND1  were used for measurement, the standard reaction solution was prepared and placed on ice avoiding the light before use. Next, 5 µL lysate were transported to 96-well plates and equilibrated for 10 s, and 150 µL of the standard reaction solution was mixed with lysate in each well. ATP levels were measured using a lurninometer  for 10 s after 2 s delay. The signal intensity of YME1L1 knock-down group was relative to the control group which was arbitrarily set to one.Mitochondrial ROS were detected using MitoSOX Mitochondrial Superoxide Indicator (Thermo Fisher Scientific), as previously described . Brieflyth day blastocysts were treated with 2.5 µM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraemyl-imidacarbocyanine iodide (JC-1)  in the IVC medium for 30 min at 38.5°C in 5% CO2 and visualized by the epifluorescence microscope (Nikon). The ratio of intensity of red fluorescence of the activated mitochondria (J-aggregates), to green fluorescence of less-activated mitochondria (J-monomers) was calculated as the membrane potential   and visualized by using a confocal microscope . The number of TUNEL-positive blastomeres was counted under the epifluorescence microscope (Nikon). The ratio of TUNEL-positive to the total blastomeres was indicated as apoptosis index.Following the instructions of the zerland) , ten blaThe BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime) was used for the cell proliferation assay. Briefly, the morula stage embryos were incubated with 10 μM EdU for 10 h at 38.5 °C. Next, the blastocysts were fixed with 3.7% paraformaldehyde at room temperature for 30 min and permeabilized with 1% Triton X-100 for 30 min. After washing thrice with PBS/PVA, the blastocysts were incubated in 100 μL of click reaction cocktail and 5 μg/mL of Hoechst 33342 in the dark for 30 min. Images were captured after mounting in PBS/PVA using a confocal microscope . The number of EdU-positive blastomeres was counted under the epifluorescence microscope (Nikon). The ratio of EdU-positive blastomeres to the total number of blastomeres was calculated as the proliferation rate.c was stained following the instruction in the immunofluorescence and confocal microscopy subsection.Ten blastocysts per group were treated with 500 nM MitoTracker Red CMXRos  in IVC medium at 38.5°C for 30 min. After washing thrice with PBS/PVA, blastocysts were fixed, and cytochrome st antibody at 4 °C overnight including anti-YME1L1 , anti- TOM20 , anti-cytochrome c  and anti-cleaved caspase-3  diluted in blocking solution (3% BSA and 0.1% Triton X-100). After washing more than thrice with PBS/PVA, the embryos were incubated with 2nd antibody such as Donkey anti-Mouse IgG, Alexa Fluor™ 488 , Goat anti-Rabbit IgG, Alexa Fluor™ 488  or Donkey anti-Rabbit IgG, Alexa Fluor™ 546  for 1 h at room temperature. After washing thrice with PBS/PVA, the embryos were then mounted onto slides using VECTASHIELD® Antifade Mounting Medium with DAPI  and captured using the confocal microscope (Zeiss LSM 710 Meta). Images were processed using the Zen software .Approximately ten embryos in each group were fixed in 3.7% paraformaldehyde at room temperature for 30 min, permeabilized with 1% Triton X-100 in the PBS/PVA for 30 min, and then blocked in 3% BSA containing 0.1% Triton X-100 in the PBS/PVA for 1 h at room temperature. Subsequently, the embryos were incubated with 1YME1L1 mRNA and 18S rRNA (reference gene) was performed as follows: initial denaturation at 95°C for 3 min, followed by 40 repeats of cycle for the denaturation at 95°C for 15 s, annealing at 60°C for 25 s, and extension at 72°C for 15 s, and a final extension at 72°C for 5 min. The data were quantified using the 2−ΔΔCT method . Reverse transcription was performed using the LeGene Express 1st Strand cDNA Synthesis System . Real-time RT-PCR for the T method .Approximately one hundred embryos in each group were lysis with 10 μL RIPA buffer and 10 μL 2X loading buffer on ice. After boiling the samples for 10–15 min, protein samples were separated by the different concentration of SDS-PAGE gel depends on the size of target protein and transferred onto polyvinylidene fluoride membranes for 1 h under 250 mA. Subsequently, the membranes were blocked with 5% skim milk in TBST buffer at room temperature for 1 h and then incubated with first antibody at 4°C overnight such as anti - YME1L1 , anti - DRP1 , anti - LC3B , anti - OPA1 , anti - OMA1 , anti-cleaved caspase-3  or anti - GAPDH . After washing with TBST buffer for three times, the membranes were treated with secondary antibody for 1 h at room temperature such as horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG . The membranes were then exposed using a UviSoft software  with SuperSignal™ West Femto Maximum Sensitivity Substrate .According to the manufacturer’s protocol, cytosolic and mitochondrial proteins of 500 blastocyst stage embryos were extracted using the Mitochondria/Cytosol Fractionation Kit . The expression of glyceraldehyde 3-phosphate dehydrogenase (GADPH) was measured as the purity of mitochondria fraction by western blot analysis.p < 0.05 was considered statistical significance. SPSS software v.19  was used for all calculations. All of experiments were replicated at least three times.The statistical data analysis was carried out by one-way analysis of variance (ANOVA) or Student’s t-test. All data were presented as the mean ± SEM. To evaluate the subcellular localization of YME1L1 during porcine embryo preimplantation development, YME1L1 and TOM20 were detected using immunofluorescence. The expression of YME1L1 was lower and colocalized with TOM20 in 1–2-cell stage embryos. However, YME1L1 was partially colocalized with TOM20 after the 4-cell stage at a higher level . The mRNYME1L1 knock-down was analyzed at blastocyst stages (p < 0.001). YME1L1 knock-down was evaluated by western blotting and immunofluorescence, which showed that the protein level of YME1L1 was also significantly reduced in YME1L1 KD blastocysts (p < 0.05). The ability of development to blastocyst stage was significantly decreased after YME1L1 knock-down  . These data suggest that defective YME1L1 may disrupt early embryo development.To investigate the role of YME1L1 in porcine embryo preimplantation development, YME1L1 dsRNA (YME1L1 KD) was injected into 1-cell stage embryos, and then cultured in IVC medium for 7 days. The efficiency of t stages . Compare< 0.005) . Additio< 0.005) . YME1L1 p < 0.01, p < 0.05, To evaluate the effect of YME1L1 on the porcine embryo mitochondrial morphology after knock-down, the blastocysts were stained with TOM20 as total mitochondria and MitoTracker Red CMORos as active mitochondria. The intensity of MitoTracker and fragmentation of mitochondrial clusters were analyzed using ImageJ, the MitoTracker signal were highly distributed and colocalized with TOM20 in all control blastomeres. However, the active mitochondria were observed in partial blastomeres with lower intensity in YME1L1 KD group (Additionally, YME1L1 KD induced more OMA1 due to the reduced degradation of OMA1 . YME1L1 p < 0.05, p < 0.001, Based on the changes in the morphology of mitochondria, we next investigated if YME1L1 knock-down can promote the mitochondrial dysfunction in porcine embryos, which can be induced by ROS over generation, low mitochondrial membrane potential, and low ATP levels. Accordingly, mitochondrial ROS were detected at the blastocyst stage. As expected, compared to control blastocysts, mitochondrial ROS levels in YME1L1 KD blastocysts were significantly increased (c is released from the mitochondria (c and the level of cytochrome c in mitochondria fraction at blastocysts stage was evaluated using immunofluorescence and western blotting after YME1L1 knock-down (c and MitoTracker was less in YME1L1 KD blastocysts, as detected by Mander’s overlap coefficient (c by western blot. The cytochrome c in the cytosol remained relatively unchanged between the control and YME1L1 KD groups; however, mitochondrial cytochrome c decreased in the YME1L1 KD groups compared with the control.Previous research has demonstrated that cytochrome chondria . The colock-down . Immunoffficient . As showp < 0.05, As YME1L1 KD induced mitochondrial dysfunction and blastocyst total cell number reduction, the TUNEL assay was performed to evaluate whether apoptosis can be induced by the knock-down of YME1L1. The ratio of TUNEL-positive signal number to total nuclear number was significantly higher in YME1L1 KD blastocysts than in the control group (Because of the poor developmental ability and poor quality of embryos after YME1L1 KD, we investigated whether YME1L1 could regulate embryo proliferation. As shown in Previous work reported that mitochondria are important for the apoptosis, calcium homeostasis, cellular signaling pathways, gene expression, organic compound biosynthesis and thermogenesis . ApproxiLow oocyte mtDNA content has been reported to result in abnormal embryo development in pigs . Therefoc (c promotes the caspase 3 activation, resulting in apoptosis (c release and the level of active caspase 3. We found that YME1L1 knock-down modulated the cytochrome c release (The dynamic organization of mitochondria with a high membrane potential is crucial for embryo development . Therefoc . The relpoptosis . In this release  and indu release . Moreove release . We foun release .c release in porcine blastocysts. Moreover, the TUNEL and activated caspase-3 were induced by the knock-down of YME1L1 during porcine embryo preimplantation development. These results indicate that YME1L1 plays a crucial role in porcine embryo preimplantation development by modulating the oxidative stress, mitochondrial morphology and functions, and apoptosis.Overall, YME1L1 knock-down significantly reduced the blastocyst quality during porcine embryo preimplantation development . YME1L1