The aim of this study is to prepare hybrid polymer–ceramic dental materials for chairside computer-aided design/computer-aided manufacturing (CAD/CAM) applications. The hybrid polymer–ceramic materials were fabricated via infiltrating polymerizable monomer mixtures into sintered hydroxyapatite/bioactive glass (HA/BAG) ceramic blocks and thermo-curing. The microstructure was observed by scanning electron microscopy and an energy-dispersive spectrometer. The phase structure was analyzed by X-ray diffraction. The composition ratio was analyzed by a thermogravimetric analyzer. The hardness was measured by a Vickers hardness tester. The flexural strength, flexural modulus, and compressive strength were measured and calculated by a universal testing machine. The growth of human gingival fibroblasts was evaluated by a 3--2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and immunofluorescence staining. The results showed that the sintering temperature and BAG content affected the mechanical properties of the hybrid polymer–ceramic materials. The X-ray diffraction analysis showed that high-temperature sintering promoted the partial conversion of HA to β-tricalcium phosphate. The values of the hardness, flexural strength, flexural modulus, and compressive strength of all the hybrid polymer–ceramic materials were 0.89–3.51 GPa, 57.61–118.05 MPa, 20.26–39.77 GPa, and 60.36–390.46 MPa, respectively. The mechanical properties of the hybrid polymer–ceramic materials were similar to natural teeth. As a trade-off between flexural strength and hardness, hybrid polymer–ceramic material with 20 wt.% BAG sintered at 1000 °C was the best material. In vitro experiments confirmed the biocompatibility of the hybrid polymer–ceramic material. Therefore, the hybrid polymer–ceramic material is expected to become a new type of dental restoration material. Teeth defects, dentition defects, and missing teeth caused by caries, periodontal disease, and trauma have promoted the development of dentally restorative materials. In recent years, chairside computer-aided design/computer-aided manufacturing (CAD/CAM) has become an increasingly popular technology for personalized restorations. The machinable dental materials should possess good machinability, high strength, high cutting efficiency, and the ability to be easily polished .Hydroxyapatite (HA) ceramics, biphasic calcium phosphate (BCP) ceramics, and bioactive glass (BAG) ceramics have been widely used in the biomedical field. BCP is composed of HA and β-tricalcium phosphate (β-TCP). BCP ceramics have proven to be adequately biocompatible, bioactive, and osteoconductive materials . CompareHybrid polymer–ceramic materials are a new type of CAD/CAM machinable material. They combine the advantages of both polymer and ceramics. Owing to their unique dual network structure, the stiffness, damage tolerance, flexibility, and fracture toughness of hybrid polymer–ceramic materials are superior to porous ceramics . CompareFew previous experiments have been conducted on hybrid polymer–HA/BAG ceramic materials. Thus, the aim of this study is to fabricate hybrid polymer–ceramic composites composed of polymer and HA/BAG. The mechanical and biocompatible properties of the obtained composites were characterized to demonstrate their applicability and superiority in dental restorative materials.2, 36 mol% CaO and 5 mol% P2O5. Hydroxyapatite  was the main raw material for ceramics. Deionized water was obtained from a deionized water purifier . Sodium carboxymethyl cellulose  and polyethylene glycol  were used as binder and dispersant, respectively. Bisphenol-A-glycidyl dimethacrylate , triethylene glycol dimethacrylate , and dibenzoyl peroxide  were components of the polymerizable monomer mixtures.The bioactive glass  used was composed of 59 mol% SiOn wt.%  of BAG (5 µm), 2.5 wt.% CMC-Na, and 2.5 wt.% PEG. The solid–liquid ratio was 1:3.5. The preparation of slurries referred to the articles of Tancred [Slurries were prepared by mixing deionized water with HA (5–20 µm),  Tancred  and Chen Tancred . Pure BAThe polymerizable monomer mixtures were formulated with Bis-GMA and TEGDMA in a mass ratio of 1:1. A 1 wt.% BPO was used as the thermal initiator. They were stirred with a magnetic stirrer for 24 h until transforming into a homogeneous and low viscous liquid. The ceramic blocks were immersed in the monomer mixtures and placed in a vacuum-drying oven with a vacuum degree of −0.1 MPa for 3 days. Compared with normal atmospheric pressure, vacuum capillary action accelerated the complete infiltration of the monomer mixtures into the ceramic blocks. Finally, they were cured by hot isostatic pressing at 100 °C under a pressure of 150 MPa. Under the same conditions, the monomer mixtures were cured into pure resins as a control group. The process flow is shown in n = 2), (25 ± 2) mm × (2 ± 0.1) mm × (2 ± 0.1) mm (n = 10) and (3 ± 0.1) mm × (3 ± 0.1) mm × (5 ± 0.1) mm (n = 10). After their cutting and polishing with 500 and 2000 grit SiC abrasive paper, cured pure resins, ceramic blocks, and cured hybrid polymer–ceramic blocks used in vitro experiments were (3 ± 0.1) mm × (3 ± 0.1) mm × (1 ± 0.1) mm (n = 12).Cured hybrid polymer–ceramic blocks were cut to the appropriate dimensions by a cutting machine  equipped with an electroplated diamond saw blade. The spindle rotation speed of the cutting machine was 1500 rpm, and the feed rate was 2 mm/min. The test specimens were polished with 500, 2000, and 4000 grit silicon carbide (SiC) abrasive papers in turn. Finally, the dimensions of the cubic specimens were (25 ± 2) mm × (6 ± 0.1) mm × (3 ± 0.1) mm . Elemental species and content analysis were differentiated by an energy dispersive spectrometer . Crystal structure analysis was performed using an X-ray diffractometer .2 atmosphere at heating rate of 10 °C/min.Proportion of polymers and ceramics in hybrid polymer–ceramic composites was analyzed by a thermogravimetric analyzer  under OThe surface roughness of (3 ± 0.1) mm × (3 ± 0.1) mm × (1 ± 0.1) mm specimens for in vitro experiments was tested by a 3D digital microscope . Arithmetic mean deviation (Pa) and root mean square deviation (Pq) were calculated by the system.n = 5). Porosity was calculated by Equation (1):1 is the mass of the dry ceramic blocks in the air; m2 is the mass of saturated ceramic blocks in the water; m3 is the mass of saturated ceramic blocks in the air.The porosity of the sintered ceramics was measured by the Archimedes drainage method. The shrinkage rate was calculated by using the volume of the ceramics before and after sintering. The ceramic specimens were not cut and polished. The dimensions of specimens for both tests were (25 ± 2) mm × (10 ± 2) mm × (10 ± 2) mm (n = 2). A total of 10 points were randomly selected to apply the load on each cubic specimen surface. The mechanical properties of natural human teeth . The dimensions of specimens were (25 ± 2) mm × (6 ± 0.1) mm × (3 ± 0.1) mm . According to ISO 10477: 2018 and ISO 4049: 2019 standards of dental restorative materials, 10 cubic specimens were prepared for each of the groups with the size of (25 ± 2) mm × (2 ± 0.1) mm × (2 ± 0.1) mm. The following formulas were used: for flexural strength (Equation (2))Tests were carried out on a universal testing machine . 10 cubic specimens were prepared for each group with the size of (3 ± 0.1) mm × (3 ± 0.1) mm × (5 ± 0.1) mm. A (3 ± 0.1) mm × (3 ± 0.1) mm surface area was loaded with pressure. The compressive strength of the specimens was evaluated according to Equation (4):2 at 37 °C. Cells were passaged using 0.25% trypsin . HGFs were seeded at 5 × 103 cells/well in 96-well cell culture plates containing the cubic specimens. The specimens  were cleaned with ethanol and sterilized under ultraviolet light for 30 min. Their dimensions were (3 ± 0.1) mm × (3 ± 0.1) mm × (1 ± 0.1) mm (n = 12). The same culture medium without the specimen was used as a negative control. The growth status of cells was observed after 48 h and 72 h. After 72 h cell culture, 10 mL of MTT was added to each well and was incubated at 37 °C for 4 h. After that, the medium was removed. A total of 150 μL of DMSO was added and shaken for 10 min. The optical density (OD) was measured at a wavelength of 570 nm with a microplate reader . Results were expressed as a percentage of the negative control values.To examine the cell viability of human gingival fibroblasts  on the hybrid polymer–ceramic materials, direct contact experiment was performed by a 3--2,5-diphenyl tetrazolium bromide  colorimetric assay. HGFs were seeded in cell culture medium. The cell culture medium contained Dulbecco’s modified Eagle medium , 10% fetal bovine serum , and 1% penicillin-streptomycin . HGFs were cultured in an incubator with 5% COHGFs grown for 72 h were fixed with 4% paraformaldehyde  for 15 min. Cells were dual stained with phalloidin  Co., Ltd., Shanghai, China) and 4,6-Diamidino-2-phenyindole dilactate . A solution containing anti-fluorescence quencher  was added dropwise. The extension of labeled cells was observed by laser confocal microscopy .The results were statistically analyzed by SPSS software  and compared using One-Way ANOVA and Tukey’s honestly significant difference. Normality test using Shapiro–Wilk test. The significance level was 0.05.Comparing When the sintering temperature increased, the diffraction peak of HA gradually weakened, the content of HA decreased, and the content of tricalcium phosphate (TCP) increased c–e. Thern = 12). The materials were cured pure resin, ceramic with 20 wt.% BAG sintered at 1000 °C, and hybrid polymer–ceramic material with 20 wt.% BAG sintered at 1000 °C  mm × (3 ± 0.1) mm × (1 ± 0.1) mm  adhered and proliferated well on the cured pure resin, HA/BAG ceramics, and hybrid polymer–ceramic materials after the 48 h culture. They had a typical elongated fusiform morphology. Over time, the number and coverage of the HGFs after the 72 h culture increased, and the arrangement was compact . No obviEnamel and dentin are considered orthotropic due to their microstructural features . In ordeThe sintering temperature significantly affects the mechanical properties of hybrid polymer–ceramic materials. An the experiment showed, with the increase in the temperature (1150 °C→1350 °C), the microhardness and flexural strength of the HA/BAG ceramics increased first and then decreased . For hyb2 and Si are key components for the surface activity and biological properties of BAG. They play important roles in the decomposition of HA [2 had little difference with respect to the amount of HA converted into TCP. Padilla’s experiment confirmed that in 20 wt.% BAG ceramic sintered at 1300 °C, HA was significantly reduced and β-TCP was the main phase [The BAG content also influences the mechanical properties of hybrid polymer–ceramic materials. SiOon of HA . The expon of HA  showed tin phase . These ain phase .For commercial machinable dental materials, the hardness of zirconia ceramics such as Lava TMY-TZP (3M ESPE) is about 13 GPa ; the harFlexural strength is meaningful for repairing the masticatory function of defective and missing teeth. Especially for fixed bridge restorations or patients with abnormal occlusal habits, the flexural strength of the materials should be as high as possible . The fleS. sanguinis [Candida albicans [The processing, finishing, and polishing of restorations affects not only the mechanical properties and aesthetics (surface staining and reflectance), but also the health of the soft tissue , and the marginal integrity of the restoration . In thisanguinis , one of albicans , which calbicans . There iAt present, commercial resin-based CAD/CAM materials contain monomers such as bisphenol-A-glycidyl dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), or bisphenol-A-ethoxylated-glycidyl dimethacrylate (Bis-EMA). In our experiment, Bis-GMA and TEGDMA were selected as organic phases. Bis-GMA has the stable chemical structure of an ether bond, which can prevent the hydrolysis of saliva esterase . The smaHuman gingiva consists of overlying epithelial structures and the underlying connective tissue. Human gingival fibroblasts (HGFs) are the most abundant cells in connective tissue and are very sensitive to the surrounding environment . In addiIn order to detect the cytotoxicity of our hybrid polymer–ceramic material, the growth of HGFs was tested by the MTT method and immunofluorescence staining. We expected that the HGFs’ morphology was typically spindle-shaped with many long cell processes that explore the environment and communicate with other cells . The resStreptococcus mutans and Streptococcus salivarius [Nonetheless, the elution of polymer monomers into the oral cavity is a non-linear relationship with time . Monomerlivarius ,64. Ther(1)The sintering temperature and BAG content affect the mechanical properties of hybrid polymer–ceramic materials;(2)The mechanical properties of hybrid polymer–ceramic materials are similar to those of natural teeth;(3)A short-term exposure of human gingival fibroblasts to the hybrid polymer–ceramic material does not cause cytotoxicity.Base on the hybrid polymer–ceramic dental material prepared in this experiment, the following conclusions can be drawn:


The effective segregation coefficient of Er3+ was determined to be 0.97, close to 1, and, thus, the uniform high-quality Er: LGGG crystal can be grown. In addition, the thermal and spectroscopic properties of Er: LGGG were investigated. Based on the measured characteristics, the Er: LGGG crystal has a huge potential for use in the 3.0 µm mid-infrared laser because of its outstanding optical quality, extraordinary thermal conductivity and stable structure.A high-quality Er Among them, Er3+-doped crystals show promise in producing commercial 2.7~3.0 µm lasers because they can be efficiently pumped by laser diodes (LDs) with wavelengths of 795 or 975 nm. In addition, the Er3+-lasers are likely to own advantages of high power due to the large emission cross section. As is known to all, the 3.0 µm MIR laser can also be applied as a pump sources for obtaining far-infrared tunable lasers from optical parametric oscillator systems  dodecahedral site could be suitable for Er-doping. Accordingly, Lu3+ have the advantages of optimizing crystal structure and reducing lattice distortion. However, as far as we know, no Er: (LuxGd1−x)3Ga5O12 (Er: LGGG) co-doped crystals have been reported.Up to now, there are many reported Er as Y2O3 , Lu2O3  oriented GGG seed under a rotation rate of 10–15 rpm and a pulling rate of 0.79 mm/h. Argon atmosphere was applied during growth to prevent the oxidation of Ir crucible. When the crystal was taken out, it needed to be annealed at 1500 °C for 15 h in the air.The Cz method was used to grow the Er: LGGG crystal. The synthesized polycrystalline materials were transferred to an Ir crucible (α radiation for measuring X-ray diffraction (XRD). Laue back-reflection was performed on Back-Reflection Laue apparatus (Multiwire MWL 120 with North star software) to further make an assessment of the crystalline quality. A square sample of 4 × 4 × 1 mm3 with the thickness in [111] direction was used for measurement. The concentrations of Er3+ and Lu3+ were analyzed using the XRF technique .The successfully grown crystal was processed into crystal samples for characterizations. Single crystal powders were placed in BrukerAXS D2 ADVANCE X-ray diffractometer fitted out Cu-K3 and the (111) end-faces were fine polished. Specific heat was analyzed using a differential scanning calorimetry (DSC) 8000 differential scanning calorimeter. The grown crystal was processed into 4 × 4 × 1 mm3 for the test. A same size sample was gilded and measured in a Netzsch Nanoflash model LFA 457 apparatus for thermal diffusivity. The crystal density was measured by the Archimedes method in distilled water at 25 °C. On the basis of the data of specific heat, thermal diffusivity as well as crystal density, the thermal conductivity data were analyzed.The thermal expansion was characterized using a Mettler–Toledo thermal-mechanical analyzer (TMA/SDTA840). The sample dimensions were 4 × 4 × 4 mm3.The absorption spectrum was measured by a Cary 7000 equipment. The absorption cross section was calculated on the basis of the measured absorption spectrum data. The fluorescence spectrum measurements were measured using a Hitachi F4500 spectrophotometer. The fluorescence attenuation curve was measured by single photon counting method, and the fluorescence lifetime was fitted according to the fluorescence attenuation curve. All spectral characteristics were evaluated for the polished (111) plates sized 4 × 4 × 1 mmΦ 36 × 45 mm3, as shown in The Cz method was used to grow high-quality Er: LGGG crystal with maximum dimensions of a = b = c = 12.369 ± 0.002 Å by software FullProf. The value is smaller than of GGG (12.376 Å)  are defined by symmetrical second-rank tensor, and α11 = α22 = α33 for Er: LGGG because its structure symmetry is related to cubic system. The temperature-dependent thermal expansion curve of Er: LGGG crystal is presented in −6 K−1, comparable with 9.03 × 10−6 K−1 of GGG crystal [Thermal expansion coefficient is a vital physical property of crystal. According to the Neumann principle , the the crystal .−1 K−1 at 293 K to 0.47 J g−1 K−1 at 573 K, which is slightly lower than that of GGG crystal [For laser crystal, the specific heat plays a vital part in affecting the optical damage threshold. Generally speaking, the higher specific heat leads to higher laser damage threshold. The scaling relationship between specific heat and temperature is shown in  crystal ,24, maki2 s−1 at 293 K to 0.81 mm2 s−1 at 773 K. Then, the formula for deriving the thermal conductivity κ can be expressed as follows [λ, ρ and Cp are the thermal diffusivity, density and specific heat of the crystal, respectively, at the corresponding temperature. According to Equation (3), the change of thermal conductivity from 5.43 W m−1 k−1 at 293 K to 3.28 W m−1 k−1 at 773 K is shown in −1 k−1 of GGG crystal at room temperature [The thermal diffusivity is a symmetric second-rank tensor. For the cubic system, there is only one independent principal component.  follows :(3)κ=λ⋅ρperature .3+ centered at 381, 524, 654, 790, 966, 1471 (1532) nm were observed, and they can be attributed to the transition from the ground state 4I15/2 to 4G9/2, 5S3/2, 4F9/2, 4I9/2, 4I11/2 and 4I13/2. The absorption cross section σ can be expressed as follows [α is the absorption coefficient, Nc is the concentration of doping ions, A is the absorbance and L is the thickness of the polished crystal. The maximum absorption cross section of Er: LGGG at the peak of 967 nm is 5.28 × 10−21 cm2, and its full width at half maximum (FWHM) is about 11 nm. The 967 nm band overlaps well with the commercially available high-power InGaAs LDs. The absorption cross-sectional area of Er: YSGG crystal is 1.56 × 10−21 cm2 [The absorption cross section of Er: LGGG crystals within the limits of 350–1800 nm is shown in  follows :(4)σ=αNC0−21 cm2 , 3.4 tim4I11/2 to 4I13/2 in Er3+. These phenomena fully demonstrated that Er: LGGG crystal shows a promise for the use in the laser field.4I11/2) laser level was characterized at 2650 nm. In the case of 10 at.% Er: GGG crystal, the fluorescence decay curve fits a single exponential [3+ makes the fluorescence decay curve double exponential, as seen in A1 is 0.813, A2 is 0.150, 4I11/2).The high concentration of Er3+ takes a leading part in the delay of fluorescence intensity. They can realize the rapid pumping of the number of particles in the lower level and recycle the energy by the complex up-conversion and cross-relaxation processes between adjacent ions. The growth of Er-doped crystals with high concentration needs further development.Under the excitation of 970 nm pulsed laser, the fluorescence decay curve of Er: LGGG for the upper  by fitting with the use of single exponential function. The above experimental results proof that the Er: LGGG crystal is a MIR laser material with great potential for applications.In summary, the bulk Er: LGGG crystal with superior quality was successfully grown using the Cz method. The dimension was


Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99 µmol H2 g dry wt−1 and maximum hydrogenase activity of 179.39 ± 8.18 µmol H2 g dry wt−1 min−1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48 h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis  were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.The unicellular halotolerant cyanobacterium  Firstly, A. halophytica was grown in BG11 with Turk Island salt solution for 7 days to accumulate biomass. The seven-day-old cells were harvested by centrifugation at 8,000 × g at 4 C for 10 min before resuspension in various types of single nutrient-depleted media: nitrogen-deprived BG11 supplemented with Turk Island salt solution (BG110), phosphorus-deprived BG11 supplemented with Turk Island salt solution (BG11-P), potassium-deprived BG11 supplemented with Turk Island salt solution (BG11-K), and sulfur-deprived BG11 supplemented with Turk Island salt solution (BG11-S). To remove nitrogen in BG110, NaNO3 was omitted from BG11. In BG11-P, NaH2PO4 was omitted from BG11. To remove potassium from BG11-K, KCl was omitted from Turk Island salt solution. In addition, K2HPO4 was also omitted from BG11 and replaced by the addition of Na2HPO4. In BG11-S, ZnSO4•7H2O and CuSO4•5H2O were omitted from BG11 and replaced by ZnCl2 and CuCl2, respectively. In addition, MgSO4•7H2O was omitted from both BG11 and Turk Island salt solution and replaced by the addition of MgCl2. Thereafter, the cell suspensions were incubated for 24 h before H2 was measured, under anaerobic conditions.H2 measurement was undertaken. H2 measurement was performed using a Gas Chromatograph  with a molecular sieve 5 °A 60/80 mesh packed column and a thermal conductivity detector using argon as the carrier gas  containing 10 mM methyl viologen dichloride hydrate  and 40 mM sodium dithionite . The mixture was incubated under argon atmospheric conditions at 30°C under darkness for 30 min. H2 from headspace was subsequently analyzed by a gas chromatograph.Bidirectional hydrogenase activity was determined by measuring HA. halophytica cells that had been adapted in various media were harvested by centrifugation at 8,000 × g at 4°C for 10 min and resuspended in fresh medium that contained chlorophyll a at a concentration of 300 µg chl a mL−1. Fifty µL of each cell suspension was added to 200 µL of 30% (w/v) KOH. The mixture was then incubated at 100 °C in a water bath for 90 min. Cells were lysed by ultrasonication at 20% pulse for 5 min on ice. To precipitate glycogen, 600 µL of absolute ethanol was added into the extract. Each sample was kept on ice for 1 h. Glycogen was collected by centrifugation at 12,000 × g for 5 min at 4 C. Then, each glycogen pellet was washed twice by absolute ethanol and subsequently dried at 60°C for 10 min. Glycogen was resuspended in 300 µL of 100 mM acetate buffer (pH 4.75). For each sample, glycogen was digested into glucose by adding amyloglucosidase from Aspergillus niger  and α-amylase from Aspergillus oryzae  at final concentrations of 4 and 8 units, respectively. The reaction was incubated at 25°C for 1 h. Insoluble membrane fragments were removed by centrifugation at 12,000 × g at 4°C for 5 min. Total sugar in each supernatant was determined by phenol-sulfuric acid assay . Sterile distilled water was used to wash the cells collected on the microfiber filter. Then, the filter containing cells was dried in an oven at 70°C until weight was constant. The dry cell weight was calculated. For glycogen content determination, glycogen extraction and hydrolysis were performed following the previous studied procedure . A. haloid assay . StandarA. halophytica, 100 ml of A. halophytica cell culture was harvested by centrifugation at 8,000 × g at 4°C for 10 min and subsequently resuspended in 1 ml of 10 mM (2-[4-(2-hydroxyethyl) piperazine-1-yl] ethanesulfonic acid (HEPES) buffer (pH 7.5). Cells were lysed using 20% pulse of ultrasonication on ice for 5 min. The cell-free extract was obtained after centrifugation at 8,000 × g at 4°C for 10 min. To assay pyruvate kinase activity, 50 µL of cell-free extract was added into 950 µL of reaction mixture containing 10 mM HEPES buffer, 10 mM MgCl2, 50 mM KCl, 20 mM ADP  , 10 mM PEP  , 5 mM NADH  , and 0.5 U of L-lactate dehydrogenase . The reaction mixture without PEP was used as a negative control. The decrease of NADH was measured spectrophotometrically at a wavelength of 340 nm at 25°C. One unit of pyruvate kinase activity is defined as the amount of enzyme that catalyzes the production of 1.0 µmol pyruvate from the substrate PEP in 1 min. The specific activity of pyruvate kinase was measured as activity per total protein concentration. Protein concentration was determined by Bradford assay . Then, total RNA was qualified and quantified using a NanoDrop 2000/2000c spectrophotometer . Ribosomal RNA (rRNA) was removed by using a Ribo-Zero rRNA removal Kit  following the manufacturer’s instructions. The construction of the next generation sequencing library was conducted by Genewiz (China) according to the standard protocols. The different indices of libraries were multiplexed and loaded on an Illumina HiSeq/Novaseq instrument according to the instructions of the manufacturer . RNA sequencing was performed using the 2 × 150 paired end (PE) configuration. Image analysis and base calling were conducted using the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. All RNA sequencing and alignment processes were conducted by Genewiz (China).Seven-day old All experiments were performed in triplicate. All data are shown as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used for analysis to the 95% confidence level with IBM SPSS version 19 software.A. halophytica cells incubated in BG110 and BG11-K showed clearly higher H2 production than those in BG11, BG11-P, and BG11-S   . In addi1 medium . This st2 production and H2ase activity by A. halophytica with focus on potassium and nitrogen deprivation was investigated. The results showed that during potassium deprivation, a decrease in nitrogen concentration resulted in higher H2ase activity and H2 production by A. halophytica , respectively. The deprivation of both nitrogen and potassium together promoted H2ase activity, and thus resulted in higher H2 production.In this study, the effects of various concentrations of nitrogen and potassium on Hophytica . Similarophytica . The hignditions . This H2A. halophytica cells were adapted in BG11, BG110, BG11-K, and BG110-K media under photoautotrophic conditions with light exposure (18 h/day) for 1, 2, 3, 4, and 5 days. The hydrogenase activity, H2 production, and O2 production of adapted cells were measured after incubation under dark anaerobic condition for 24 h. The result showed that under nitrogen and/or potassium deprivation, A. halophytica cells gave higher H2 production and hydrogenase activity than cells under normal condition  is known to be activated by K+ . It has r 5 days . The depr 5 days . Similar protein .2 is a strong inhibitor of H2ase, the presence of O2 in systems plays an important role in H2 production. In cyanobacteria, O2 is generated via oxygenic photosynthesis and the produced O2 is consumed in cellular respiration via the electron transport chain. To investigate how O2 evolution decreased under nitrogen and potassium deprivation, the photosynthetic O2 evolution rate and respiration rate of A. halophytica cells adapted in BG11, BG110, BG11-K, and BG110-K for 2 days was measured. The results showed that the highest photosynthetic O2 evolution rate of 16.94 ± 0.64 nmol O2 mg dry wt−1 min−1 was found in cells adapted in BG11. Under single or combined nitrogen and potassium deprivation, its photosynthetic O2 evolution rate decreased  was used to indicate the differences between transcript expression in the experimental and control groups. The results showed that genes encoding allophycocyanin and phycobilisome under N and K deprivation were expressed as log2FC apcA, was upregulated by 0.5 and 1.77 of log2FC under N and K deprivation, respectively. In addition, the expression of different genes involved in photosynthesis showed harmonic direction under N and K deprivation. Most of these genes were upregulated by log2FC psbA was upregulated by log2FC of 1.48 and 1.03 under N and K deprivation, respectively. However, the CP47, CP43 chlorophyll apoproteins, and P680 reaction center D2 protein encoded by psbB, psbC, and psbD respectively were downregulated.Gene expression based on the RNA-seq of rbcL was significantly down-regulated by log2FC of −2.46 and −1.83 under N and K deprivation, respectively. All genes in this pathway were differently expressed by log2FC pyk was expressed differentially, as log2FC of 0.26 and −1.77 under N and K deprivation, respectively. Interestingly, most genes involved in glycogen biosynthesis were upregulated. For example, 1,4-alpha-glucan branching enzyme encoded by glgB was upregulated as log2FC of 1.69 and 4.37 under N and K deprivation, respectively. In the oxidative pentose phosphate pathway (OPP), some genes were upregulated under N and K deprivation. For example, rpe encoding ribulose phosphate 3-epimerase was upregulated by 1.23 and 0.52 of log2FC, respectively. In terms of nitrogen assimilation, as expected, ferredoxin-nitrite reductase encoded by nirA and nirB was downregulated as log2FC of −0.94 and −1.76, respectively, under N-deprived conditions. On the contrary, no differences of expression of these genes were found under K-deprived conditions.Under N and K deprivation, genes involved in the Calvin-Basham-Benson (CBB) cycle were downregulated . Ribulos2 under dark anaerobic conditions, especially when they are incubated in nutrient-deprived media H  pathway H compared to that via the glycolytic pathway in cells incubated in both nitrogen- and potassium-deprived conditions. Therefore, the described effects of both nitrogen and potassium deprivation on H2 production may have been synergistic, and accelerated H2 production in A. halophytica under dark anaerobic conditions.Under dark anaerobic condition, cyanobacterial H NAD(P)H  and redu NAD(P)H . The mai pathway . In thisrivation . The spea system . Therefoe to GAP . Under pegulated . Consequpe cells . Moreovepe cells . Prior tncreased . Moreovee medium . This wa2 production by A. halophytica during deprivation of individual nitrogen and potassium, and combined nitrogen and potassium, under dark anaerobic conditions is shown in 2 metabolism by cyanobacteria, NADH, and NADPH are generated mainly by the glycolytic and OPP pathways . In plentiful conditions or in the case of the BG11  were upregulated in both nitrogen- and potassium-deprived cells. However, pyk and most genes that regulated enzymes in the glycolytic pathway were down-regulated in both nitrogen- and potassium-deprived cells. Interestingly, most genes that regulated enzymes in the oxidative pentose phosphate pathway (OPP) were upregulated. Accordingly, the OPP was suggested as a promising pathway for enhancing H2 production under dark anaerobic conditions in both nitrogen- and potassium-deprived A. halophytica cells. This study indicated that combined nitrogen and potassium deprivation in media is a promising strategy to promote sustainable H2 production by the halotolerant cyanobacterium A. halophytica.Under dark anaerobic conditions, the maximum H


MaOpy2 in Metarhizium acridum were systematically elucidated. The results showed that the MaOpy2 disruption significantly reduced fungal tolerances to UV, heat shock and cell-wall-disrupting agents. Bioassays showed that the decreased fungal pathogenicity by topical inoculation mainly resulted from the impaired penetration ability. However, the growth ability of ∆MaOpy2 was enhanced in insect hemolymph. Importantly, MaOpy2 deletion could significantly increase the conidial yield of M. acridum by shifting the conidiation pattern from normal conidiation to microcycle conidiation on the 1/4SDAY medium. Sixty-two differentially expressed genes (DEGs) during the conidiation pattern shift, including 37 up-regulated genes and 25 down-regulated genes in ∆MaOpy2, were identified by RNA-seq. Further analysis revealed that some DEGs were related to conidiation and hyphal development. This study will provide not only the theoretical basis for elucidating the regulation mechanism for improving the conidial yield and quality in M. acridum but also theoretical guidance for the molecular improvement of entomopathogenic fungi.Opy2 is an important membrane-anchored protein upstream of the HOG-MAPK signaling pathway and plays important roles in both the HOG-MAPK and Fus3/Kss1 MAPK. In this study, the roles of  Entomopathogenic fungi can penetrate directly into the hemocoel of the host to utilize its nutrition , which oMetarhizium acridum, microcycle conidiation exhibited a greater potential in large-scale applications than normal conidiation [Two conidiation patterns, the normal conidiation pattern and the microcycle conidaition pattern, are found in most filamentous fungi ,10. Normidiation . HoweverOpy2 plays distinct roles in different species. In Saccharomyces cerevisiae, Opy2 participates in the SHO1 branch of the high osmolarity glycerol (HOG) pathway and is identified as an osmosensor interacting with Ste50 [Candida albicans, CaOpy2 plays a crucial role in cell wall stress tolerance and is essential for the phosphorylation of Cek1 [Metarhizium robertsii, an alternative transcription start site is achieved, and the regulation of the MrOpy2 transcription level affects the fungal lifestyle and contributes to virulence [Magnaporthe oryzae, MoOpy2 plays essential roles in pathogenicity, turgor pressure, appressorium formation, conidiation and hyphal growth [M. acridum, the biological functions of MaOpy2 have not been systematically elucidated.As a cell transmembrane protein in fungi, th Ste50 . In Cand of Cek1 . In Metairulence . In Magnl growth . HoweverOpy2 were identified in M. acridum. It was found that MaOpy2 plays important roles in fungal growth, stress tolerance and fungal pathogenicity in M. acridum. Importantly, we present that MaOpy2 disruption resulted in an increased conidial yield through the regulation of the conidiation pattern shift. RNA-seq showed that MaOpy2 governs the conidiation pattern shift by regulating some gene expressions related to hyphae growth and conidiation in M. acridum.Here, the functions of CQMa102 was cultured on 1/4 SDAY  for 15 days. Escherichia coli DH5α  and Agrobacterium tumefaciens AGL1  were adopted for vector construction and fungal transformation, respectively. For MaOpy2 deletion vector construction, the fragments of the up- and downstream of MaOpy2 were amplified with the primer pairs Opy2-LF/Opy2-LR and Opy2-RF/Opy2-RR, which were clonedinto pK2-SM-F and pK2-SM-R to form pK2-SM-MaOpy2-F and pK2-SM-MaOpy2-R. The 5′-flanking region and MaOpy2 coding region (3397 bp) and downstream sequence (1398 bp) were cloned into the plasmid pK2-Nat to yield the pK2-MaOpy2-Nat vector, which was used for MaOpy2 complementation ). Fungal transformation and transformant validation were conducted as reported previously [For gene deletion construction, the wild type (WT) strain eviously . The pri7 conidia/mL) of fungal strains were evenly spread on a 1/4 SDAY medium, and every 2 h, conidial germination was recorded. For the conidial yield, 2 μL suspensions (1 × 107 conidia/mL) were spotted onto 24-well plates, and every 3 days, conidial yields were calculated. Additionally, for fungal sensitivity to heat shock, conidial suspensions (1 × 107 conidia/mL) treated with a water bath at 45 °C for 3 h, 6 h, 9 h and 12 h were spread on a 1/4 SDAY medium and then cultured at 28 °C for 20 h to access the conidial germination rates. Similarly, for the UV-B tolerance assay, the conidial suspensions (1 × 107 conidia/mL) were spread on 1/4 SDAY and exposed to UV-B radiance with a dose of 1350 mW/m2 for 1.5 h, 3.0 h, 4.5 h and 6.0 h and then cultured at 28 °C for 20 h to access the conidial germination. For fungal tolerances to different chemicals, 2 μL of the suspension (1 × 107 conidia/mL) was spotted onto a 1/4 SDAY medium or 1/4 SDAY medium supplied with SDS, calcofluor white (CFW), Congo red (CR), H2O2, NaCl and sorbitol. After a 5-day cultivation, the fungal colonies were photographed, and the diameters of the colonies were recorded to calculate the relative growth inhibition rates. The fungal pathogenicity assays were determined by two methods. For topical inoculation, the suspensions of WT, ∆MaOpy2 and CP prepared in paraffin  were inoculated to the fifth-instar locust. For intrahemocoel injection, the suspensions of the fungal strains prepared in ddH2O (1 × 106 conidia/mL) were injected into locust hemocoel. The negative controls for these two methods were treated with pure paraffin oil and ddH2O, respectively. Every half day, the survival of the locusts was recorded.Conidial germination and conidial yield assays were conducted as described previously . Briefly7 conidia/mL) was inoculated into the autoclaved locust wings and then cultured at 28 °C for different times. The conidial germination and appressorium formation were recorded under a microscope. To stain the neutral lipids, Nile red  was used [6 conidia/mL, 10 μL) were inoculated into 500 μL of locust hemolymp without blood cells, which was incubated on a rotary shaker at 28 °C and 250 rpm for 4 d or 6 d. To measure the gDNA concentrations for the fungal growth in the locust blood, quantitative real-time PCR was used [To determine the appressorium formation and conidial germination, locust wings were used for analyses, as described previously . Brieflywas used ; then, iwas used . The det7 conidia/mL) of each fungal strain was spread evenly onto 1/4 SDAY plates and incubated at 28 °C. About 1 cm2 media was cut to detect the fungal growth by microscopic observation every 2 h under the digital light microscope . The RNA-sequencing was accomplished by the Beijing Genomics Institution . The DEGs were defined with a false discovery rate ≤ 0.005 and a fold change ≥ 2. The RNA-seq data were validated by quantitative reverse-transcription PCR (qRT-PCR).Fungal fresh conidia cultivated on 1/4 SDAY for 15 days were used for the conidial suspensions. An aliquot of 100 μL of the conidial suspension (1 × 10Tukey’s honestly significant difference (HSD) and a one-way ANOVA were applied to access the statistical differences and the phenotypic estimate, respectively.MaOpy2 in M. acridum is 1362 bp with an intron, encoding 434 amino acids. The molecular weight is 45.4 kD and the isoelectric point is 8.07. SMART  was used for the domain prediction, showing that the N-terminal of MaOpy2 has a transmembrane (TM) domain. The sequences of the TM domain were conserved in filamentous fungi, indicating that Opy2 is a conserved membrane-anchor protein  and the complementation strain (CP) (MaOpy2 was reduced significantly (50s) by UV-B irradiation for the WT and CP strains were 4.42 ± 0.88 h and 4.21 ± 0.48 h, respectively, while the IT50 of ∆MaOpy2 was 3.42 ± 0.29 h (p < 0.01). Additionally, the tolerance of ∆MaOpy2 to heat shock was also significantly impaired (p < 0.01). The IT50 of ∆MaOpy2 was 8.65 ± 0.76 h—significantly decreased compared to that of the WT (12.20 ± 0.89 h) and CP strains . The spot assays revealed that the disruption of MaOpy2 rendered the fungi more sensitive to the cell-wall-disturbing agents SDS and CR; however, the deletion of MaOpy2 did not change the fungal tolerances to oxidative (H2O2) and hyperosmotic stresses (NaCl and Sorbitol)  . Southerain (CP) . Conidiaficantly A. The haorbitol) .MaOpy2 decreased fungal pathogenicity compared to WT and CP. The locusts all died at day 7 when inoculated with WT and CP; however, when infected with ∆MaOpy2, the locusts died at day 9 (MaOpy2 strain exhibited significantly longer LT50 (6.19 ± 0.20 d), while the LT50s of the WT and CP strains were 4.70 ± 0.11 d and 4.48 ± 0.26 d, respectively  when compared to those in WT (1.55 ± 0.11 ng/μL) and CP  and CP (57.6%) strains  was significantly decreased compared with that of WT (4.77 ± 0.54) and CP  was significantly reduced when cultured for 12 h  and CP (5.46 ± 0.34 d) strains  and CP (8.44 ± 0.70 h), the GT50 of ∆MaOpy2 was significantly decreased  was upregulated in ∆MaOpy2. A previous study revealed that a pantothenate transporter gene disruption mutant could not form spores [MaOpy2 may regulate the expression of Gin4 to shift the conidiation pattern, thus promoting the conidiation—but this needs to be further explored.To reveal the mechanism of m spores . In somem spores ,35,36. T


Gastric cancer (GC) is one of the most common malignant tumors in China. Circular RNAs (circRNAs) are novel non-coding RNAs with important regulatory roles in cancer progression. IGF2BP3 has been found to play oncogenic roles in various cancers including GC, while the exact mechanism of IGF2BP3 is largely unknown.The expression of IGF2BP3 in GC was evaluated by Western Blot and bioinformatics analysis. CircRNA expression profiles were screened via IGF2BP3 RIP-seq in GC. Sanger sequencing, RNase R digestion, nucleo-plasmic separation and RNA-FISH assays were used to detect the existence and expression of circARID1A. RNA ISH assay was employed to test the expression of circARID1A in paraffin-embedded GC tissues. Moreover, the function of circARID1A on cellular proliferation was assessed by CCK-8, plate colony formation, EdU assays and GC xenograft mouse model in vivo. Furthermore, the location or binding of circARID1A, IGF2BP3 protein and SLC7A5 in GC was evaluated by RNA-FISH/IF or RNA pull-down assays.in vitro and in vivo and circARID1A played an important role in the oncogenic function of IGF2BP3. Mechanistically, circARID1A served as a scaffold to facilitate the interaction between IGF2BP3 and SLC7A5 mRNA, finally increasing SLC7A5 mRNA stability. Additionally, circARID1A was able to directly bind SLC7A5 mRNA through complementary base-pairing and then formed the circARID1A-IGF2BP3-SLC7A5 RNA–protein ternary complex and promoted the proliferation of GC via regulating AKT/mTOR pathway.We identified a novel circRNA, circARID1A, that can bind to IGF2BP3 protein. CircARID1A was significantly upregulated in GC tissues compared with noncancerous tissues and positively correlated with tumor length, tumor volume, and TNM stage. CircARID1A knockdown inhibited the proliferation of GC cells Altogether, our data suggest that circARID1A is involved in the function of IGF2BP3 and GC proliferation, and the circARID1A-IGF2BP3-SLC7A5 axis has the potential to serve as a novel therapeutic target for GC.The online version contains supplementary material available at 10.1186/s13046-022-02466-3. Gastric cancer (GC) is a global public health issue with over 1 million estimated new cases annually and there were 784 000 deaths from GC related diseases in 2018 . For patRNA-binding proteins (RBPs) are essential players in RNA metabolism and regulate RNA splicing, transport, surveillance, decay and translation . RBPs alCircular RNAs (circRNAs) form a novel class of ncRNAs and are expressed during developmental process and in certain disease states –20. CircSLC7A5 (solute carrier family 7 member 5 or LAT1) is a transporter of large branched-chain and aromatic neutral amino acids , 26 and In the present study, we employed RIP-Seq to screen for circRNAs that bind to IGF2BP3 in SGC7901 cells and identified circARID1A (CircBase ID: hsa_circ_0008494) that was derived from the pre-mRNA of the ARID1A gene and was upregulated in GC and directly interacted with IGF2BP3. Functionally, circARID1A promoted the proliferation of GC and mechanistically elevated SLC7A5 expression by enhancing its stability via the formation of a circARID1A-IGF2BP3-SLC7A5 RNA–protein ternary complex in GC. This study provides a novel therapeutic target for the treatment of GC.GC and the corresponding noncancerous tissue samples 21 pairs) were obtained from Southwest Hospital of the Army Medical University. All patients were diagnosed by histopathological examination and were without chemotherapy or radiotherapy prior to surgery. The specimens were collected immediately in EP tubes containing RNA Later  after surgical resection and stored at -80 °C. Clinical characteristics of GC patients were given in Supporting Information Table S pairs we2 atmosphere in DMEM or RPMI 1640 medium  supplemented with 10% fetal bovine serum .The GC-derived cell lines SGC7901, BGC823, and HGC-27 cells or normal gastric epithelial cell GES-1 were obtained from the Army Medical University . AGS and MKN-74 cells were purchased from Meisen . All cells were cultured at 37 °C in a 5% COTotal protein was extracted from cultured cells using RIPA lysis buffer containing protease inhibitors . Cell lysates were separated on SDS–polyacrylamide gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes that were incubated at 4 °C with primary antibodies in 5% nonfat milk in PBS containing Tween-20 (PBST). The antibodies used for this study were used at 1:1000 dilutions in primary antibody dilution buffer (Beyotime) and were as follows: GAPDH and β-actin (Beyotime), SLC7A5 , IGF2BP3 , mTOR, p-mTOR, AKT and p-AKT . Membranes were washed with PBST for three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The blots were visualized using an ECL chemiluminescent reagent .SiRNAs targeting IGF2BP3, circARID1A, and SLC7A5 were synthesized by GenePharma  . For IGF2BP3 transient overexpression, the overall CDS region of IGF2BP3 was inserted into pcDNA3.1( +) for IGF2BP3 overexpression. The vector was transfected into GC cells using a commercial DNA transfection reagent .4/mL using 100 μL/well and incubated for the indicated times (see below). 10 μL of Cell Counting Kit-8 (CCK-8)  reagent was added to each well and the plate was incubated at 37 °C for 2 h in a 5% CO2 atmosphere, the absorbance was measured at 450 nm using a microplate reader (Thermo).Cells were seeded into 96-well plate at 5 × 103 cells per well. Two weeks after seeding, tumor spheres were fixed using 4% paraformaldehyde for 10 min and then stained with 1% crystal violet solution for 10 min at room temperature. The colony spheres were washed 3 × with water and then dried at room temperature. Plate images were captured using an Epson scanner .Cells were removed from monolayers using trypsin digestion and seeded into 12-well plates at 1 × 104 per well. EdU incorporation utilized a Cell-Light EdU Apollo 567 In Vitro Kit . Cells were treated and exposed to EdU solution (50 μM) for 2 h at 37 °C and then processed according to the manufacturer’s instructions. Cells were observed and photographed using a fluorescent inverted microscope .GC cells were treated and seeded into 48-well plate at a density of 3 × 10RIP assays were performed according to the instructions of Magna RIP RNA-Binding Protein Immunoprecipitation Kit . Briefly, cells were lysed with RIP lysis buffer and incubated with antibodies specific for IGF2BP3 or rabbit IgG. Co-precipitated RNAs were purified for sequencing or cDNA synthesis and used as template for indicated gene expression detection by qRT-PCR with specific primers. Total RNA (Input) or antibody (Rabbit IgG) was used as references.For circARID1A stability evaluation, SGC7901 and BGC823 cells were seeded into 12 well plates and treated with actinomycin D (5 μg/mL)  for 0, 3, 6, and 9 h. Cells were lysed in RNAiso Plus  for RNA extraction and qRT-PCR measurements. For mRNA stability assay, SGC7901 and BGC823 cells were seeded into 12 well plates and treated with si-circARID1A or si-IGF2BP3 and then treated with actinomycin D (20 μg/mL) or vehicle for 0, 3, 6, and 9 h. Cells were lysed in RNAiso Plus (Takara) for RNA extraction and further qRT-PCR detection.SGC7901 and BGC823 cells were seeded into 12 well plates and incubated overnight. Cells were used for total RNA extraction and a total of 5 µg RNA was incubated in the presence or absence of RNase R (6 U)  at 37 °C for 10 min, followed by 85 °C for 5 s. After RNase R treatment, the expression of linear ARID1A and circARID1A were measured using qRT-PCR.−ΔΔCt method was used to analyze the relative expression levels of genes in nuclear and cytoplasm [Nuclear and cytoplasmic RNA in SGC7901 and BGC823 cells were isolated using the Paris Kit  according to the manufacturer’s instructions. For circARID1A, ARID1A, and GAPDH, cDNA was synthesized with a PrimeScript RT Master Mix (Takara) and for snoU6 the cDNA was synthesized by stem-loop methods (RiboBio). Quantitative real-time PCR (qRT-PCR) analysis was executed using a SYBR Green Master Mix (Bioground). The 2ytoplasm .Biotin-labeled probes or FAM-labeled probes were synthesized by Genepharma and used to visualize circARID1A or SLC7A5 in situ. Briefly, SGC7901 and BGC823 cells were seeded into μ-Slide 8-well chamber slide . The medium was removed 24 h later and cells were washed 2 × with PBS. Then cells were fixed with 4% paraformaldehyde for 10 min and treated with Triton X-100 (0.5%) at room temperature for 15 min. Cells were washed 2 × with PBS and incubated with denatured probes targeting circARID1A, SLC7A5, 18S rRNA or negative control and the slides were incubated at 37 °C overnight. The next day, nuclei were counterstained with DAPI and images were taken using a laser confocal microscope . The probe sequences for RNA-FISH are listed in Supporting Information Table SGAPDH was used as an internal control for the relative expression of circRNAs and mRNAs. The 2−ΔΔCt method was used to calculate the relative expression of indicated genes and primers are listed in Supporting Information Table STotal RNA was extracted with RNAiso Plus (Takara) following the manufacturer's instructions. RNA was quantified using a Nanophotometer  and cDNA was synthesized using a PrimeScript RT Master Mix (Takara). qRT-PCR was conducted using SYBR Green Master Mix (Bioground) with CFX connect Real-Time PCR System . Human 7) were harvested and lysed in IP lysis buffer on ice for 30 min. Avidin-labeled magnetic beads were incubated with biotin-labeled circARID1A probes or control probes (GenePharma) at 25 °C for 2 h to generate probe-coated beads. The cell lysates were incubated with circARID1A probe or control probe to pull-down circARID1A. The RNA complexes bound to the beads were purification using phenol/chloroform/isoamyl alcohol (25: 24: 1) for real-time PCR measurements. The protein complexes bound to the beads were denatured by heating at 100 °C for 10 min for Western blot analysis. The probe sequences for RNA pull-down are listed in Supporting Information Table SRNA pull-down assay was performed using a Waals RNA Pull down Kit . Briefly, SGC7901 or BGC823 cells  were fixed using 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were washed 2 × with PBS and blocked with 5% BSA for 30 min at 37 °C, and incubated with specific primary antibody at 4 °C overnight. The primary antibody was removed and cells were washed 3 × with PBS and incubated with corresponding secondary antibody for 30 min at 37 °C, followed by staining with DAPI. Fluorescent images were obtained using a Leica SP8 confocal microscope.TMA were produced from 180 paraffin-embedded GC or adjacent tissues by Outdo Biotech . The clinical pathological characteristics including gender, age, survival time, pathological grading, tumor volume, TNM stage, AJCC stage, the expression of PDL1 or CD8 were public data and provided by Outdo Biotech . ISH was employed to detect the expression of circARID1A in GC tissues. The probes targeting the junction of circARID1A were synthesized by Boster  and the ISH Kit was purchased from Boster. Briefly, TMA was dewaxed in xylene and rehydrated with 100, 95, 85 and 75% ethanol and digested with trypsin. The TMA were hybridized with specific digoxin-labeled circARID1A probes overnight. The specimens were incubated with biotin-conjugated anti-digoxin antibody , and incubated with avidin-conjugated peroxidase and stained with NBT/BCIP (Roche) and photographed. circARID1A expression was quantified and analyzed. The positive staining intensity  was multiplied by the percentage of positive staining cells  to calculate the score for ISH staining. The probe sequence is listed in Supporting Information Table S8/UI. SGC7901 cells were infected with lentivirus at a MOI = 50 using polybrene. The transfection efficiency was measured and proliferation of the SGC7901 stable circARID1A knockdown cells was detected. Briefly, 10 male BABL/C nude mice  aged at 3–4 weeks were randomly divided into two groups. SGC7901 cells transfected with sh-NC or sh-circARID1A were cultured and digested with 0.25% trypsin. Cells were washed twice with cold PBS and diluted with PBS at a concentration of 3 × 107/mL. 100 µL of cell suspension was subcutaneously implanted in the upper-right flank of nude mice. One week later, the length and width of the tumor tissues was measured every two days. The volume of the tumor size was calculated by the formula: volume (mm3) = length × width2 / 2. The experiment was terminated one month after xenograft mouse model construction and the animals were sacrificed and tumor weight was measured. The tumor tissues were collected for indicated gene expression evaluation and IHC staining.Lentivirus constructs containing an shRNA targeting circARID1A was packaged by Hanbio  with a virus titer of 106) in 0.1 mL phosphate-buffered saline (PBS). When the tumor had grown to an appropriate volume, the tumor-bearing mice were randomly divided into three groups (n = 4). In vivo treatment with siRNA in the mouse models was performed as previously described [For in vivo rescue assay of circARID1A and SLC7A5, HGC-27 cells were employed to construct xenograft tumor model in nude mice. Briefly, 3-week-old male BALB/C nude mice (Gempharmatech) were subcutaneously injected in the right flank with HGC-27 cells  according to the manufacturer’s instructions. Six hours later, the supernatant was replaced with fresh DMEM containing 10% fetal bovine serum. Cells were collected after 48 h for further RNA sequencing by Sinotech Genomics .SGC7901 cells were seeded to 6-well plate at a density of 5 × 10P < 0.05 was considered to indicate statistical significance.Student’s t test or one-way analysis of variance (ANOVA) was used to compare the differences of continuous variable between two groups or among multiple groups. Kaplan–Meier analysis was employed for survival analysis, and the differences in the survival probabilities were estimated using the log-rank test. ISH score of circARID1A was categorical variable, and was analyzed by Kruskal–Wallis Test. The statistical analyses were performed using SPSS version 22.0  and Prism 8.0 . https://www.proteinatlas.org/) consistently confirmed that IGF2BP3 was up-regulated in GC compared with normal gastric mucosa  for subsequent study  CircRNA Mini Vector. However, no significant overexpression of circARID1A could be found in SGC7901 and BGC823 cells transfected with these overexpressing plasmids  targeting the back-splicing region of circARID1A. The results showed that we successfully manipulated the expression of circARID1A in SGC7901 and BGC823 cells without affecting the mRNA levels of ARID1A  were employed to establish a model of implanted tumor model of human GC. CircARID1A knockdown resulted in decreased sizes of subcutaneously implanted tumors  and found that the nucleotides at positions + 373–516 in circARID1A could bind to SLC7A5 mRNA at + 115–204 and the predicated binding strength was high at -76.54 kcal/mol . Table S10. Characteristics of GC patients for TMA. Table S11. Downregulated genes evaluated by RNA-seq in SGC7901 cells following circARID1A knockdown. Table S12. RNAs interacting with IGF2BP3 in SGC7901 cells evaluated by RIP-seq (Top 20).Additional file 2: Fig. S1 IGF2BP3 expression in GC tissues or cell lines. A Heat map of RBPs dysregulated in STAD of TCGA database. B Representative IHC images of IGF2BP3 expression in normal gastric mucosa or GC tissues from HPA database. C The expression of IGF2BP3 in normal gastric epithelia cell GES-1 and gastric cancer cell lines SGC7901, BGC823, AGS, and MKN74. Fig. S2 The interference efficiency and inhibiting proliferation effect of two designed IGF2BP3 siRNAs. A The interference efficiency of si-IGF2BP3-1 and si-IGF2BP3-2 in SGC7901 and BGC823 cells. B The viability of SGC7901 and BGC823 cells transfected with si-IGF2BP3-1 and si-IGF2BP3-2. C The proliferation of SGC7901 and BGC823 cells transfected with si-IGF2BP3-1 and si-IGF2BP3-2. The P-values were calculated using the two-tailed Student’s t test. *P < 0.05. Fig. S3 The expression of IGF2BP3 in SGC7901 and BGC823 cells after IGF2BP3 knockdown or overexpression. A The expression of IGF2BP3 in SGC7901 and BGC823 cells after IGF2BP3 knockdown by siRNA. B The expression of IGF2BP3 in SGC7901 and BGC823 cells with IGF2BP3 stable overexpression. Fig.S4 Screening of circRNAs that binding to IGF2BP3 protein in GC. A Heat map of circRNAs binding to IGF2BP3 in SGC7901 cells evaluated by RIP-seq. B The expression of circELK4 in GC tissues (GC) and adjacent tissues (NC). The P-value was calculated using the two-tailed Student’s t test. Fig. S5 CircARID1A sequencing results of GC tissues and the adjacent tissues. A CircARID1A sequencing results of GC tissue and the adjacent tissue from patient 1. B CircARID1A sequencing results of GC tissue and the adjacent tissue from patient 2. Fig. S6 Validation of the binding between circARID1A and IGF2BP3 proteins in BGC823 cells. A RIP analysis of circARID1A enrichment pull-downs by GFP in BGC823 cells overexpressing EGFP-tagged IGF2BP3. B-C RIP analyses of circARID1A enrichment pull-downs by IGF2BP3 in BGC823 cells following (B) IGF2BP3 or (C) circARID1A knockdowns. D Enrichment efficiency of biotin tagged circARID1A probes assessed by RNA pull-downs. E Western blot validation of interaction of circARID1A and IGF2BP3 by RNA pull-down in BGC823 cells. The P-values were calculated using the two-tailed Student’s t test. *P < 0.05. Fig. S7 Regulation of circARID1A and IGF2BP3 to each other in GC cells. A The expression of IGF2BP3 at protein level in SGC7901 and BCG823 cells after circARID1A knockdown. B The expression of IGF2BP3 at RNA level in SGC7901 and BCG823 cells after circARID1A knockdown. C The expression of circARID1A in BGC823 or AGS cells after IGF2BP3 knockdown or overexpression. The P-values were calculated using the two-tailed Student’s t test. Fig. S8 The interference efficiency of circARID1A in SGC7901 and BGC823 cells. A The expression of ARID1A in SGC7901 and BGC823 cells treated with si-circARID1A. B The expression of circARID1A in SGC7901 and BGC823 cells treated with si-circARID1A. The P-values were calculated using the two-tailed Student’s t test. *P < 0.05. Fig. S9 The overexpression of circARID1A in GC cells by using different vectors. A The plasmid profile of pLC5-ciR and overexpression of pLC5-ciR-circARID1A in SGC7901 and BGC823 cells. B The plasmid profile of plv-ciR and overexpression of plv-ciR-circARID1A in SGC7901 and BGC823 cells. C The plasmid profile of pcDNA3.1(+) CircRNA Mini Vector and overexpression of pcDNA3.1-circARID1A in SGC7901 and BGC823 cells. D The overexpression of pLC5-ciR-circPDHK1 in SGC7901 and BGC823 cells. Fig. S10 The expression of SLC7A5 protein after IGF2BP3 overexpression in SGC7901 and BGC823 cells. Fig. S11 The expression of SLC7A5 in GC tissues and cell lines and correlation with IGF2BP3 in GC. A The expression of SLC7A5 mRNA in STAD of TCGA database. B The expression of IGF2BP3 in GC cell lines or normal gastric epithelial cells. C Correlation between SLC7A5 mRNA and IGF2BP3 mRNA in STAD of TCGA database. Fig. S12 The effect of circARID1A knockdown on HGC-27 proliferation. A The viability of HGC-27 cells after circARID1A knockdown. B The plate colony formation of HGC-27 cells after circARID1A knockdown. C The proliferation of HGC-27 after circARID1A knockdown. The P-values were calculated using the two-tailed Student’s t test. *P < 0.05.


Prematurity is the leading cause of childhood death under the age of five. The aetiology of preterm birth is multifactorial; however, inflammation and infection are the most common causal factors, supporting a potential role for immunomodulation as a therapeutic strategy. 15-Deoxy-Delta-12,14-prostaglandin J2 (15dPGJ2) is an anti-inflammatory prostaglandin and has been shown to delay lipopolysaccharide (LPS) induced preterm labour in mice and improve pup survival. This study explores the immunomodulatory effect of 15dPGJ2 on the transcription factors NF-κB and AP-1, pro-inflammatory cytokines, and contraction associated proteins in human cultured myocytes, vaginal epithelial cell line (VECs) and primary amnion epithelial cells (AECs).in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph.Cells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1β as an 15dPGJ2 inhibited IL-1β-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1β-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs.We conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth. Preterm birth (PTB) occurs in 5-18% of pregnancies worldwide and is the leading cause of mortality in children under the age of five . There iin vitro and ex vivo activation of the transcription factors NF-κB and AP-1 by Toll like receptor (TLR) agonists  is an endogenous prostaglandin of the J2 series , 24 and in vitro . Additioin vitro . In contell type , 34–36. ell type , 17, 37,Myometrial biopsies and placentas were collected from women undergoing elective caesarean section between 38- and 41-weeks gestational age at Queen Charlotte’s and Chelsea Hospital, Imperial College Healthcare NHS Trust, London. Consent was taken in accordance ethical committee approval from Riverside Research Ethics Committee (Ref 3358) and Hammersmith, Queen Charlotte’s & Chelsea Hospitals Research Ethics Committee (Ref 2002/628) and in accordance with Imperial College NHS Healthcare Trust Research and Development. Written consent was obtained from all subjects. Women undergoing pre-labour elective caesarean section from healthy pregnancies were selected. This was to avoid any confounding effects of labour and gestational age, such as variations in the expression of prostaglandin pathway and immune mediator genes  to rinse off excess blood and then dissected mechanically using two sterile blades to form a fine paste-like texture. Cells were isolated by incubating the tissue for 45 min at 37°C with a sterile enzyme mix of 10 mg of Collagenase 1A, 10 mg of Collagenase XI, and 200 mg of bovine serum albumin (BSA) in 30 mls of 1:1 of Dulbecco’s Modified Eagle’s Medium (DMEM) and F-12 HAM. DMEM containing 10% fetal calf serum (FCS) was added to inactivate the enzymes before the suspension was filtered through a cell strainer and centrifuged at 3000 rpm for 5 min. The pellet was resuspended in DMEM (10% FCS) containing 2 mM/L L-glutamine, 100 U/ml penicillin, and 100 µg/ml of streptomycin and cultured to confluence. Cells up to passage five were used for experiments. A minimum of 3 biological replicates were used per experiment.® CRL2616™) and stored in liquid nitrogen until use. In preparation for experiments, cells were thawed for 1 min at 37°C in a water bath and transferred into pre-warmed DMEM containing 10% FCS. The suspension was centrifuged at 125 x g for 5 min and cells were resuspended in keratinocytes serum free medium (KSFM), bovine pituitary extract (BPE), epidermal growth factor (EGF) and calcium chloride (CaCl2) at 37°C with 5% CO2 and grown to confluence. Confluent cells were trypsinised and plated for experiments. Only passage numbers 3-6 were used for experiments. Experiments were repeated a minimum of three times.The vaginal epithelial cell line VK2/E6E7 was purchased from ATCC , was selected on the basis of previous data demonstrating inhibitory effect on p-p65 in peripheral blood mononuclear cells (PBMCs) ,myocytesThe adherent cells were lysed with whole cell lysis buffer  with 5 µl/mL of protease inhibitor cocktail (Sigma), 5 µl/mL pf phosphatase inhibitor cocktail (Sigma), 1 mM of PMSF and collected using a cell scraper. Cells were incubated on ice for 5 min and centrifuged at 14,000 g for 10 min at 4°C. Supernatant was collected and the protein was quantified using Bio-Rad quantification assay measuring absorbance at 655 nm . Protein was resolved using SDS-PAGE and transferred onto PVDF membranes  at a constant voltage of 300 mA. The membranes were then blocked in 5% (weight/volume) milk in Tris‐buffered saline supplemented with 0·1% Tween 20 for 1 hr at room temperature before incubation with primary and secondary antibodies as shown in ©  according to the manufacturer’s instructions. 2 µg of RNA was added to the mixture of 1 µl of DNAse 10x buffer and 1 µl of DNAse with DEPC treated water made to total volume of 10 µl. The solution was incubated for 15 min at room temperature before adding 1 µl of stop solution. The mixture was heated to 70°C for 10 min. 8 µl of the DNAse treated samples were then added to the remaining 1 μl 10 mM dNTP mix and 1 μl of Oligo DT (0.5 μg/ml), mixed and incubated at 70°C for 10 min. Later, 2 μl 10x M-MLV RT buffer, 1 μl M-MLV RT, 0.5 μl RNase Inhibitor, and 6.5 μl DEPC treated water were then added and the solution was incubated at room temperature for 15 min, 37°C for 50 min, and 90°C for 10 min. The resulting cDNA was diluted 1:5 for the specific amplification of genes of interest, and the fold change of target genes were analysed taking into account the reference gene β actin, Total RNA was isolated from the adherent cells using TrizolSupernatant was collected after pre-treatment with either vehicle control or 15dPGJ2 and 24 hrs of IL-1β stimulation for quantification of PGE2 production by ELISA according to the manufacturer’s guidance . The supernatant was diluted 1:3 with the assay diluent and plated alongside the calibration curve standards . The optical density of each well was read at 450 nm, 540 nm and 570 nm. To calculate PGE2 concentrations, the average readings from the absorbance values of 540 and 570 nm was subtracted from the reading taken from 450 nm.-7 M of oxytocin was added to demonstrate myometrial strip contractility as a marker of viability. The total area under the curve, average area under the curve, average peak amplitude, and the rate of contractions were analysed by comparing to the vehicle control using the Powerlab software V5.5.6 (ADI Instruments) using the peak parameters extension.Myometrial tissue biopsies were dissected at 4°C into 8 strips of approximately 5 mm x 10 mm and mounted onto the thermostatically-controlled isolated organ baths (DMT Myograph 800MS) and stretched to 4 g of tension. The tissue was allowed to equilibrate for 90 min in 4 mls of oxygenated (95% O2 and 5% CO2) modified Kreb’s solution  0.141 g/L, Potassium phosphate monobasic 0.16 g/L, Potassium chloride 0.35 g/L, Sodium chloride 6.9 g/L, Calcium chloride dihydrate 0.373 g/L, Sodium bicarbonate 2.1 g/L) at 37°C, pH 7.4. Strips were treated with a cumulative dose response from 1 µM to 100 µM of 15dPGJ2 after spontaneous contractions were established. At the end of the experiment 102. The crystal dissolving solution was then added into each well for 12 hrs at 37°C with 5% CO2 before reading the plate at 570 nm.Cell morphology was assessed every four hrs during incubations with 15dPGJ2 or vehicle control using standard light microscopy. The release of the LDH into myocyte culture medium using the LDH assay kit  was performed as per the manufacturer’s instructions with Triton-X (1%) as the positive control. Since the morphological appearance of VECs and AECs changed following incubation with 15dPGJ2, cell viability was determined using the MTT assay. Briefly, 50,000 cells were cultured in a 96 wells plate and MTT reagent was added into each well after treatment and incubated for 4 hrs at 37°C with 5% COStatistical analyses were performed using Kruskal Wallis, one-way analysis of variance (ANOVA), or two-way ANOVA with Dunn’s or Bonferroni’s multiple comparison test depending on the distribution of data and the number of treated groups or tests on respective experiments. All analyses, including tests for normality were carried out using GraphPad v5 . A value of p<0.05 was considered to indicate statistical significance.p<0.01), reflecting activation of NF-κB, and that 15dPGJ2 inhibits this effect (p<0.05),  and 12 (p<0.05) hrs, the effect of which was significantly inhibited by 15dPGJ2 .IL-1β stimulation significantly increased IL-6 mRNA at 8 (p<0.001) (p<0.01 and p<0.05), which was inhibited by 15dPGJ2 . was also inhibited by 15dPGJ2  and AECs (p<0.001) and was significantly inhibited by 15dPGJ2 in both cell types (p<0.001 and p<0.01 respectively) , IL-8 (p<0.001) and TNF-α (p<0.001) was seen with IL-1β stimulation. Pre-treatment of VECs with 15dPGJ2 inhibited IL-1β induced IL-8 and TNF-α , and TNF-α (p<0.001) production in AECs , which was significantly inhibited by 15dPGJ2 pre-incubation in both VECs (p<0.05) (p<0.05). IL-1β stimulation had no significant effect on PGE2 production in either cell types. However, incubation with 15dPGJ2 alone significantly increased the production of PGE2 in VECs cells (p<0.001) and AECs (p<0.05) at 24 hrs  and AECs (p<0.01) levels while no effect was seen with IL-1β stimulation alone. In contrast, IL-1β-induced p-ERK was significantly attenuated by 15dPGJ2  but not ERK.We next examined upstream MAP kinase activation since regulation of AP-1 by MAPK signaling pathways involve p-JNK, p-p38 and p-ERK. In VECs, 15dPGJ2 treatment significantly increased p-JNK  inhibitors TB rates . Several effects –54. For  effects . The lacIn vitro assays show that AP-1 transcriptional activity is increased with exposure to TNF- α , c-Jun N terminal kinases (JNKs), and p-38 mitogen activated protein kinases (p38). Studies have demonstrated variable effects of 15dPGJ2 on the MAP kinases, but most report on activation rather than inhibition. 15dPGJ2 can induce JNK activation (The mechanism for 15dPGJ2 induced activation of AP-1 is unclear. AP-1 regulation by the mitogen-activated protein kinase (MAP) signal transduction pathways occurs tivation , p-p38  and Hammersmith, Queen Charlotte’s & Chelsea Hospitals Research Ethics Committee (Ref 2002/628). The patients/participants provided their written informed consent to participate in this study.LS, ZR, TT, DM, and PB were responsible for the conception and design of the study. ZR, SK, YL, and LS conducted experiments and data analysis. All figures and tables were created by ZR and LS. ZR drafted the manuscript and all authors critically reviewed it. All authors contributed to the article and approved the submitted version.This study was funded by Majlis Amanah Rakyat (MARA) (MARA Ref: 3304082377500), Malaysian Government Agency. LS was funded by a Wellbeing of Women Research Training Fellowship (Grant Ref 148), a National Institute for Health Research (NIHR) Clinical Lectureship and Genesis Research Trust and is currently a Parasol Foundation Clinical Senior Lecturer in Obstetrics. DAM was supported by the Medical Research Council (Grant Ref MR/L009226/1). This work was also supported by the National Institute for Health Research Comprehensive Biomedical Research Centre at Imperial College Healthcare NHS Trust and Imperial College London (Grant Ref P45272). This publication presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of Imperial College, the NHS, the NIHR or the Department of Health.We would like to thank the study participants from Imperial College Healthcare NHS Trust for their contribution. We would also like to thank Xintong Li for her contributions as part of her BSc project at Imperial College London.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MECP2 gene. The epigenetic reader MeCP2 binds to methylated cytosines on the DNA and regulates chromatin organization. We have shown previously that MECP2 Rett syndrome missense mutations are impaired in chromatin binding and heterochromatin reorganization. Here, we performed a proteomics analysis of post-translational modifications of MeCP2 isolated from adult mouse brain. We show that MeCP2 carries various post-translational modifications, among them phosphorylation on S80 and S421, which lead to minor changes in either heterochromatin binding kinetics or clustering. We found that MeCP2 is (di)methylated on several arginines and that this modification alters heterochromatin organization. Interestingly, we identified the Rett syndrome mutation site R106 as a dimethylation site. In addition, co-expression of protein arginine methyltransferases (PRMT)1 and PRMT6 lead to a decrease of heterochromatin clustering. Altogether, we identified and validated novel modifications of MeCP2 in the brain and show that these can modulate its ability to bind as well as reorganize heterochromatin, which may play a role in the pathology of Rett syndrome.Rett syndrome is a human intellectual disability disorder that is associated with mutations in the X-linked  As DNA methylation is mainly found on the less transcriptionally active heterochromatin, MeCP2 is prominently localized in vivo at pericentric chromatin regions, which contain highly methylated major satellite DNA repeats (via these regions (reviewed in (The methyl-CpG-binding protein 2 (MeCP2) is the founding member of the methyl-CpG binding domain (MBD) protein family and specifically binds to methylated CpGs  repeats . In addi repeats , the int repeats . MeCP2 biewed in . Severaliewed in . MeCP2 miewed in . Aside fiewed in .via regions outside of the MBD . Increa the MBD .MECP2 gene were linked to Rett syndrome, a human neurological disorder affecting mainly females, that is associated with intellectual disability among other symptoms . The phcription . Besidescription .in vivo) and determine whether these modifications are involved in MeCP2 chromatin binding and clustering. 23% of the MeCP2 protein is composed of positively charged amino acids and we found only a few modified arginines compared to many modified lysines. In addition, we identified several phosphorylated serine and threonine residues, including the previously reported S80 and S421. We show that arginine methylation and to a much lesser extent also serine phosphorylation affect heterochromatin accumulation and binding kinetics and MeCP2 heterochromatin clustering function. In addition, coexpression of MeCP2 variants and the protein arginine methyltransferase 6 (PRMT6) reveals differences in heterochromatin clustering.In this study, we aimed to identify post-translational modifications of MeCP2 from mouse brain . After centrifugation for 10 min at 1,000 x g, the supernatant was discarded and the pellet resuspended in sucrose buffer to a final sucrose concentration of 2.1 M. The raw nuclei fraction was obtained by ultracentrifugation for 30 min at 50,000 x g. The pellet was resuspended in 0.25 M sucrose solution and centrifuged at 1,000 x g. During the procedure, samples were taken after resuspension of the tissue, after homogenization and after nuclei isolation, fixed with 3.7% formaldehyde in solution for 15 min, dropped on slides, dried and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for microscopic examination of the individual steps.3-month-old C57BL/6 mice  were sacrificed according to the animal care and use regulations , and the organs were collected from the sacrificed animals, washed with PBS and frozen in liquid nitrogen. For nuclei isolation the frozen mouse brains were crushed to powder and homogenized in 0.25 M sucrose solution (20 mM triethanolamine-HCl (pH 7.6), 30 mM KCl, 10 mM MgCl7 mouse brain nuclei in PBS were pelleted by centrifugation at 1,000 x g for 10 min. The nuclei were resuspended in buffer B , 5 mM MgCl2), incubated on ice for 10 min and centrifuged at 1,000 x g for 10 min. The supernatant was discarded and the pellet washed three times by resuspension in 100 µl buffer C (2 mM triethanolamine-HCl (pH 7.6), 0.5 mM MgCl2) and centrifugation for 10 min at 1,000 x g. The pellet was resuspended in 500 µl 1 M NaCl equilibration buffer , followed by sonication 3 × 20 s  with microscopic control after each step. Subsequently, the lysate was diluted using 500 µl equilibration buffer without NaCl and added to the Nickel-Iminodiacetic acid (Ni-IDA) beads for incubation overnight at 4°C with rotation. The beads were washed with 300 mM NaCl equilibration buffer, then with wash buffer . The Ni-IDA beads were then resuspended in Laemmli buffer , 10% glycerol, 0.01% bromophenol blue, 100 mM DTT), incubated at 95°C for 10 min and separated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein enrichment from E. coli BL21 (DE3) was performed using the pTYB1-MeCP2wt plasmid coding for MeCP2 with a C-terminal intein-CBD tag allowing protein binding to chitin beads and subsequent elution by cleavage as described before . First, 10d before .2O (MS grade), the bands of interest were excised, cut to small cubes and dried using a vacuum concentrator. For destaining the gel pieces were covered with destaining solution , incubated at 37°C for 30 min with shaking and the solution was removed. Destaining was repeated at least two times and the gel pieces were dried using a vacuum concentrator. For trypsin digestion the gel pieces were covered with 12.5 ng/μl trypsin  in 40 mM ammonium bicarbonate and incubated at 37°C with shaking overnight. The peptides were eluted by adding elution solution , incubation for 20 min in an ultrasonic bath, transfer of the peptide solution to a new tube and drying using a vacuum concentrator. Samples were resuspended in 20 µl buffer , incubated in an ultrasonic bath for 5 min and transferred to HPLC vials. Subsequent drying of the samples in a vacuum concentrator allowed storage at room temperature in the dark until the measurement.The samples to be analyzed by mass spectrometry were analyzed by SDS-PAGE and the gel was stained with Coomassie staining solution -(18)-hydrate, 10% ethanol p.a., 0.02% CBB-G250 (Coomassie brilliant blue), 2% orthophosphoric acid ) over niThe HPLC-MS/MS measurement was performed with the setup described before . BrieflyMus musculus. A maximum of two missed tryptic cleavages was accepted and methionine oxidation, N-terminal acetylation, N-terminal pyroglutamate, lysine acetylation, lysine ubiquitination, lysine and arginine mono-methylation or di-methylation and serine/threonine/tyrosine phosphorylation were set as variable modifications. To identify all methylation and dimethylation sites, the search was repeated including either only lysine/arginine methylation or dimethylation. The MaxQuant search was run with default parameters having matching between runs enabled.Data analysis was performed using Proteome discoverer 1.3 (Thermo Fisher Scientific) with SEQUEST  and MaxQhttp://n2t.net/addgene:110186; RRID:Addgene_110186)    (Mecp2-EGFP under the control of the TRE3G promoter and flanking the Tet-On® 3G expression cassette under control of the CAG promoter to reduce leaky expression of Mecp2-EGFP. All cDNA sequences for MeCP2 modification site mutants were generated using overlap extension PCR (Mecp2 cDNA sequences (PCR products) were inserted into pmMeCP2G wt to replace WT Mecp2 cDNA through a cloning step using unique restriction sites of SalI and BamHI. The pTYP1-MeCP2wt plasmid used for bacterial expression was a gift from Christopher L. Woodcock (All plasmids used in this study are listed in _110186) , AAVS1-Te_52343) , HSC1-HSe_58540) , pSpCas9e_62988) . The line_62988) . Three Hsion PCR  with theWoodcock .hPRMT1, the cDNA of PRMT1 transcript variant 2 (mPRMT4 cDNA fragment was amplified by PCR from pSG5-HA_PRMT4 (via EcoRI and XhoI into pcDNA3.1. The hPRMT5 transcript variant 1 (NM_006109) sequence was obtained by PCR using hPRMT5-fwd and hPRMT5-rev primers generating BamHI and EcoRI sites, which enabled cloning of PRMT5 into pcDNA3.1.For the cloning of ariant 2  was geneHA_PRMT4  and subs2 in a humidified incubator. Mycoplasma tests were performed regularly and all cell lines are listed in C2C12 mouse myoblast cells , MTF mouse tail fibroblast  MeCP2 -/y cells see  and huma2 in a humidified incubator. 7 h after transfection, cells were washed with PBS and transcription was induced by adding medium supplemented with 2 µM tetracycline. 20 h after induction, cells were washed with PBS and fixed either with ice cold methanol for 6 min (MTF -/y) or with 3.7% formaldehyde (C2C12) for 15 min. HEK293T cells were transfected using polyethylenimine  as described previously  according to the manufacturer’s instructions. For cotransfections of MeCP2 mutants and PRMTs a plasmid amount ratio of 1:5  was used. After transfection, cells were seeded on gelatin-coated coverslips and grown at 37°C and 5% COeviously .C2C12 cells were transfected with plasmids expressing MeCP2-WT, S80D, or 3L and treated 7 h with 1 μg/ml tetracycline followed by incubation for 20 h. The cells were harvested by trypsinization, washed using PBS, counted, and aliquoted into four tubes with the same cell number. Cells were resuspended in 100 µl lysis buffer . Cells were lysed by syringe treatment , followed by 25 min incubation on ice. The lysate was collected by centrifugation at 4,600 × g for 15 min at 4°C. The pellet was dissolved in 2% SDS in water. Both lysate and pellet were mixed with Laemmli buffer, and boiled at 95°C for 5 min before Western blot analysis.Mouse brain nuclei were lysed in Laemmli buffer (see above), mechanically disrupted and incubated for 5 min at 95°C. The samples were analyzed by SDS-PAGE and transferred to a nitrocellulose membrane using a semi-dry blotting system at 25 V for 35 min. For detection of post-translational modifications, membranes were blocked with 5% BSA in TBS-T (0.1% Tween 20 in TBS) for 1 h and incubated with anti-PTM antibody diluted in TBS-T  or with 3.7% formaldehyde (C2C12) for 15 min and washing with PBS, cells were permeabilized with 0.7% Triton X-100 in PBS for 20 min and washed three times with 0.01% PBST. Cells were either directly stained with DAPI or blocked with 2% BSA in PBST for 20 min and incubated with primary antibody diluted in 2% BSA in PBST for 2 h .Fluorescence and DIC images of transfected C2C12 cells were taken using a Nikon Eclipse TiE2 system with a 40x air Plan Apo Fluorescence images of transfected C2C12 cells for calculation of heterochromatin accumulation were acquired on a Zeiss Axiovert 200 microscope. Image segmentation was performed using an ImageJ macro described previously . First, 2 and roundness coefficient >0.8 that are not touching the edges of the image. Heterochromatin segmentation was also performed using the DAPI channel image, identifying high intensity spots within the nuclei. Spots with a spot-to-region intensity ratio of at least 0.35, an area of at least five px2 and a relative spot intensity of 0.0253 were considered. The nucleus and heterochromatin masks generated based on the DAPI channel image were used to segment the images of the other channels and intensity and morphology properties of nuclei and heterochromatin clusters were measured . Briefly, cell nuclei were segmented based on the DAPI channel image considering nuclei with a size of 110–250 μmured see . MeCP2 iFor the cell cycle analysis, the frequencies of the DAPI sum intensities per nucleus were plotted as histogram. The DAPI intensities of the different samples were normalized as described before . The int2 and 60% humidity. MTF -/y cells were transfected with MeCP2 mutation constructs, seeded on gelatin-coated glass bottom p35 plates followed by induction with 1 µM tetracycline 7 h later and DNA staining with 100 nM SiR-DNA (SiR-Hoechst)  in presence of 5 µM verapamil (included in #SC007). 16 h later heterochromatin clusters were bleached with a 488 nm argon laser at 100% intensity for 2 s and confocal images were taken with a frame size of 256 × 256 with 200 Hz and a pinhole of one AU in time intervals of 1.5 s. For analysis the image series were registration corrected using ImageJ plugin StackReg (correction based on GFP channel) or HyperStackReg (correction based on DNA staining). The mean fluorescence intensities of the (pre- and post-bleach) bleached and unbleached region were background subtracted for each time point. For single normalization, intensities were normalized to the mean of the prebleach intensities. Fluorescence recovery curves were fitted in ImageJ and t-half values and mobile fraction were obtained from the fitted curves. At least 10 cells were analyzed for each construct and the means of the fitted curves, t-half values and mobile fractions were plotted.Live cell imaging for fluorescence recovery after photobleaching (FRAP) experiments was carried out on a confocal microscope Leica SP5 II with a HCX PL APO ×63 oil lambda blue objective equipped with an ACU live cell chamber at 37°C, 5% CO2. Confocal z-stacks were acquired at 30 s intervals for 20 min. Z-stack images were taken first in PBS/EDTA and then in PBS with 0.5% Triton X-100 for 20 min with 30 s intervals. Quantifications were performed using Volocity (PerkinElmer Life Sciences). The mean fluorescence intensity signal at the heterochromatin clusters/cell at each time was calculated and normalized to the mean fluorescence intensity of heterochromatin clusters before Triton X-100 treatment.The protein extractability was measured in live cells. In brief, the C2C12 cells were transfected with plasmids expressing MeCP2-WT, S80D, or 3 L, plated onto µ-Slide eight Well plate , and treated 7 h with 1 μg/ml tetracycline followed by incubation for 20 h. Live-cell imaging was performed on an UltraVIEW VoX spinning disc system (PerkinElmer Life Sciences) mounted on a Nikon Ti microscope equipped with an oil immersion 60 Plan-Apochromat NA 1.45 objective lens at 37°C, 5% COMECP2 mutations identified in Rett syndrome patients affect arginine residues. The arginine methylation sites identified on MeCP2-E2 isoform (starting in exon 2) namely R91, R106, R162, and R167 were selected for further analysis and were validated by manual inspection of the spectra  of MeCP2, we first isolated nuclei from mouse brain tissue using a sucrose buffer in combination with ultracentrifugation. Then, we enriched MeCP2 from mouse brain nuclei using its natural hepta-histidine sequence localized in its C-terminal domain see  for protctra see . The argctra see .As the MeCP2 sequence comprises 13.2% of lysines and 7% of arginines, it is likely that some regions could not be covered in our measurements due to the generation of very short peptides by trypsin which cuts after lysine and arginine. In addition, there is a long sequence without any lysines and arginines in the C-terminus of MeCP2, containing the hepta-histidine sequence and a proline-rich region see  that wasE. coli to confirm the antibody specificity by Western blot  to mimic phosphorylation or alanine (A) to prevent phosphorylation, there are no commonly used substitutions for methylated arginines. Thus, we decided to mutate the arginines identified to be methylated to lysine (K) to retain the positive charge, to glutamine (Q) to sterically mimic a methylated arginine and to leucine (L) to obtain methyl groups similar to a methylated arginine compare . In addiThe MeCP2-GFP plasmids point mutated for modified sites  were traFirst, we analyzed the subcellular localization of the altered MeCP2 proteins and compared them with the wild type MeCP2 protein. Like wild type MeCP2, the MeCP2 proteins with 3K, 3Q, and 3L substitutions  as well To quantitatively analyze heterochromatin accumulation of the MeCP2 mutant constructs, we performed a cellular DNA/chromatin binding assay. C2C12 cells were transfected with the mutant constructs, fixed and counterstained with the DNA dye DAPI. After imaging the cells, the nuclei were segmented semi-manually and heterochromatin compartments were segmented using a self-made macro in ImageJ/Fiji , Supplem. By calcThe quantitative analysis of heterochromatin accumulation revealed a slightly higher accumulation of MeCP2 3K than MeCP2 wild type. MeCP2 3Q showed a lower accumulation than wild type MeCP2 and MeCP2 3L an even lower accumulation compared to the MeCP2 3Q . The hetNext, we wanted to know whether the MeCP2 modification site mutants show differences in heterochromatin binding kinetics. Therefore, MTF-/y cells were transfected with the different constructs and heterochromatin compartments were photobleached using a focused laser microbeam on a confocal microscope. The fluorescence recovery was measured by taking images before and every 1.5 s after photobleaching . Curve fin situ extraction analysis and by lysing the cells with increasing salt concentrations followed by Western blot analysis of the soluble and insoluble fractions  with Mecp2 to test whether the PRMTs affect the MeCP2 heterochromatin clustering function. We confirmed transfected cell viability by cell cycle profiling , we identified only a few of them as modified by methylation. One possible explanation is the removal of the modification during the experimental procedure. Although arginine methylation is considered a rather stable and permanent modification, recent reports argue for the existence of arginine demethylation enzymes chromatin association. Thus, our results contribute to understanding the function of MeCP2 S80 phosphorylation in global heterochromatin binding. MeCP2 S80 plays a role in heterochromatin association, but not in its clustering. In contrast, the serine 421 phosphorylation mimicking mutant S421D showed increased heterochromatin clustering (with lower heterochromatin cluster numbers and larger cluster areas) compared to wild type MeCP2. As S421 phosphorylation was found exclusively in the brain upon neuronal stimulation , suggesting that a high degree of arginine methylation in the cells drastically impairs MeCP2 clustering function. This effect was more pronounced for PRMT6 than for PRMT1, although PRMT1 is considered responsible for the majority of cellular arginine methylation  substituting the arginine with lysine (3K), glutamine (3Q) or leucine (3L). Although none of these substitutions can truly mimic the different arginine (modification) states, lysine retains the positive charge as (methylated) arginine, the polar glutamine should sterically mimic a methylated arginine and the unpolar leucine should mimic the methyl groups of methylated arginine  and glutamine (Q), in very few cases also to glycine (G) and leucine (L). MeCP2 R106W is a frequent Rett syndrome mutation causing severe phenotypes  and the paration . The minnditions . Thus, DD domain  is invol