Pseudomonas aeruginosa infection is a severe acute suppurative ulcer that engulfs virtually the entire tissue in a short period and leads to devastating destruction. Antibiotic therapy is a common approach for the prophylaxis and treatment of P. aeruginosa infection. However, it is often associated with serious side effects, complications, and multidrug resistance. Therefore, it has been a long-standing challenge to explore safe and effective methods for controlling P. aeruginosa infection. Herein, tannin-coordinated nanozyme composite-based hybrid hydrogels (TCNH) are developed and characterized for the prophylactic treatment of P. aeruginosa and multidrug-resistant P. aeruginosa infections using mouse keratitis as the animal model. The TCNH eye drops are constructed by photoinitiated free radical polymerization of acetylated gelatin solution containing self-synthesized tannin-coordinated Co3O4/Ag nanozyme composite. The as-prepared TCNH displays good dispersibility, peroxidase-like activity and in vitro/in vivo biocompatibility. The nanozyme composite in TCNH seems to penetrate the interior of bacteria and exhibited significant broad-spectrum antibacterial activity owing to its intrinsic and nanozymic catalytic properties. Furthermore, TCNH eye drops can be successfully applied to treat P. aeruginosa and multidrug-resistant P. aeruginosa keratitis. The findings of this study reveal the potential of tannin-coordinated nanozyme composite-based hybrid hydrogel eye drops for treating infectious diseases.The online version contains supplementary material available at 10.1186/s12951-022-01653-w. Bacterial infections, one of the main problems that threaten public health, are spreading all over the world –3. As co2O2 into highly oxidized hydroxyl radicals (·OH), and effectively inhibit the growth of drug-resistant bacteria [Nanozymes including metals, metal oxides, carbon-based materials, and single-atom nanocatalysts are emerging as biomimic nanomaterials –12. Withbacteria –15. At pbacteria , 16. Howbacteria –19. Currbacteria .Staphylococcus aureus, MDR Escherichia coli, Helicobacter pylori, Streptococcus pneumoniae and Serratia induced infections [Pseudomonas aeruginosa in vitro [P. aeruginosa or drug-resistant P. aeruginosa infectious in vivo, which affect a wide range of patients with traumatized corneas, burns, catheters, cystic fibrosis, and other lung diseases [Nanozymes have been employed to treat bacterial infection induced by various common and drug-resistant bacteria such as MRSA fections –25. Currin vitro , 23, 26.diseases , 28. In diseases , 29, 30.P. aeruginosa infections.Gelatin hydrogels, possessing water-rich gel networks, are prepared by crosslinking of natural biological collagen hydrolysates of the extracellular matrix and have been receiving intensive attention in the fields of regenerative medicine, tissue engineering, and drug delivery due to their merits such as high hydrophilicity, high swelling degree, excellent biocompatibility, good optical property, and low immune response . PhotoinP. aeruginosa keratitis is a special bacterial infection in the cornea that rapidly destroys the entire cornea and induces severe complications including corneal perforation, anophthalmia, and blindness [P. aeruginosa keratitis. In this study, we aimed to construct tannin-coordinated nanozyme-based hybrid hydrogel (TCNH) eye drops for prophylactic treatment of P. aeruginosa keratitis , o-phenylenediamine (OPD) and gelatin were obtained from Macklin Biochemical Co., Ltd. . Acrylic anhydride was purchased from Sigma . Hydrogen peroxide , aqueous ammonia, acetic acid and ethanol were the products of Tianjin Kermel Chemical Plant . 2,2-Dimethoxy-2-phenylacetophenone (DMPA), the live/dead (SYTO 9/PI) bacterial viability kit and PE anti-mouse CD45 (Cat# 12-0451-82) were received from Thermo Fisher Scientific . The lactate dehydrogenase (LDH) cytotoxicity assay kit was obtained from Beyotime . 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from Solarbio Life Science . FITC anti-mouse F4/80 (Cat# 123108) and FITC anti-mouse Ly-6C/Ly-6C (Gr-1) (Cat# 108406) were obtained from Biolegend . Anti-Ly6G + Ly6c (ab25377) and anti F4/80 (ab111101) were purchased from Abcam . GTVisionTM I detection system/Mo&Rb (GK500510A) was received from Gene Tech (Shanghai) company limited. Four strains of MDR P. aeruginosa  were kindly provided by Qingdao Municipal Hospital (China). Doubly deionized water was purified by a Unique-R20 purification system .CoCl2·6H2O solution . The mixture was vigorously stirred for 10 min, and heated to 60 °C, followed by the sequential addition of H2O2  solution and CoCl2·6H2O solution . After cooling to room temperature, tannin solution  and silver nitrate aqueous ammonia  were added, and the mixture was further stirred for 16 h. The product was centrifuged at 10,000 rpm for 10 min, extensively washed with ethanol, and dried at 60 °C in a vacuum oven for 4 h. The sample was calcined at 400 °C under nitrogen for 2 h to obtain TCN.TCN was prepared using the following procedure. Aqueous ammonia  was slowly dripped into a CoClTCN (40.0 mg), 10% DMPA ethanol solution (10.0 µL), and 5% acrylated gelatin solution (10.0 mL) were mixed in a 50 mL round bottom flask. The mixture was stirred at room temperature for 15 min, and irradiated under ultraviolet light (365 nm) for 30 min to obtain TCNH.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the as-prepared samples were recorded on a Gemini SEM 300 scanning electron microscope  and a JEOL 2000 EX electronic microscope , respectively. High angle annular dark-field scanning TEM (HAADF-STEM) and energy dispersive X-ray spectroscopy (EDS) mapping images were collected on a FEI Talos F200X TEM electron microscope at 200 kV . The X-ray photoelectron spectroscopy (XPS) spectra were collected on an ESCALAB 250 photoelectron spectrometer  using Al Ka (1486.6 eV) as the X-ray source. Powder X-ray powder diffraction (PXRD) of the sample was performed on an X-ray powder diffractometer using CuKa radiation. The surface areas of the samples were calculated from nitrogen adsorption/desorption measurements on a Quadrasorb SI4 adsorptometer . Fourier-transform infrared spectroscopy (FT-IR) characterization was performed on a Thermo Nicolet 380 spectrometer using KBr pellets . Hydroxyl radicals were measured using a JEOL JES-FA200 electron spin resonance (ESR) spectrometer.2O2. In brief, H2O2  were added to PBS solution  containing TMB  and TCNH solution  to start the catalytic process. The reaction was monitored by measuring the UV–vis absorbance of oxTMB at 652 nm using a NanoDrop™ One UV–vis spectrophotometer . In addition, pH-dependent (2.0–12.0) and temperature-dependent  experiments were utilized to study the influence of pH and temperature on the catalytic activity of TCNH. All tests were performed in triplicate. OPD was also adopted to characterize the peroxidase-like activity of TCNH with a similar method as TMB for the UV–vis absorbance of the product 2,3-diaminophenazine (DAP) at 450 nm.The peroxidase-like activity of TCNH was evaluated based on the catalytic oxidation reaction of the substrate TMB into oxTMB in the presence of H4 were seeded on 96-well plates, cultured in DMEM/F12 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin, and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Afterwards, TCNH (1.0 μL) was added to the culture (1.0 mL), and 200.0 μL of the mixture was removed and added to the 96-well plates. The cells were incubated for another 24 h. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and 150.0 μL of LDH releasing reagent was added for further incubation for 1 h. Then 120.0 μL of supernatant and 60.0 μL of LDH detection solution were added to another well after centrifugation at 1000 rpm for 5 min and incubated at room temperature in the dark for 30 min. The absorbance was measured at 490 nm. In addition, the cytotoxicity of TCNH in the presence of H2O2 was evaluated with the same method as that of TCNH.Immortalized human corneal epithelial cells (HCECs) and human corneal fibroblasts (HCFs) were adopted to evaluate the cytotoxicity of TCNH using a lactate dehydrogenase (LDH) cytotoxicity assay kit . In brie5 were grown on glass-bottom cell culture dishes for 24 h, and then cultured with diluted TCNH for another 24 h. The staining solution was prepared by mixing LiveDye (1.0 μL) and NucleiDye (1.0 μL) in DMEM/F12 medium (1.0 mL) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. Afterwards, the cells were stained at 37 °C for 30 min and washed twice with PBS (pH 7.0) before visualization by CLSM. Live and dead cells were dyed green and red, respectively. In addition, the effect of TCNH in the presence of H2O2 on the viability of cells was surveyed with the abovementioned method.The effect of TCNH on viability of HCECs and HCFs was examined by a live/ dead cell double staining kit. Briefly, cells with a density of 1 * 102O2 was assessed using 8-week-old female C57BL/6J mice. TCNH (4.8 μL) and H2O2  were mixed and dripped onto the mouse corneas once every hour for 8 h. The mice were sacrificed at 48 h post saline and TCNH + H2O2 intervention. The corneas were carefully harvested for further histopathological examination by hematoxylin–eosin (H&E) staining and morphological observation by SEM.The in vivo biocompatibility of TCNH in the presence of HP. aeruginosa  was collected and diluted to 2*109 CFU/mL. Then bacterial suspension (1.0 μL), TCNH (191.0 μL), and H2O2  were mixed and further incubated at 180 rpm at 37 °C for 30 min. The mixture was diluted by gradient dilution, spread onto the LB agar plates, and then cultured at 37 °C overnight to count the bacterial loads. In addition, the mixture was also stained with a live/dead (SYTO 9/PI) bacterial viability kit for fluorescence detection and fixed with 2.5% glutaraldehyde for SEM analysis. Using the abovementioned method, E. coli (25922) and Candida albicans (98001) were adopted to evaluate the broad-spectrum antibacterial performance of TCNH, and the antibacterial activity of TCNH against drug-resistant bacteria was investigated using four strains of MDR P. aeruginosa .The antibacterial activity of TCNH was assessed by the plate cultivation method. In brief, bacterial monocolony and LB culture medium (20.0 mL) were mixed and incubated at 37 °C for 16 h. After centrifugation, 2O2 , LB medium (455.0 μL) and bacterial suspension  were added to 48-well plates, and the mixture was cultured at 37 °C for 48 h. The formed biofilms were washed with PBS (pH 7.0) and stained with 1% CV staining solution for 20 min. The stained biofilms were then washed three times gently to remove unbonded CV and naturally dried. Acetic acid was used to dissolve the attached CV for quantitative analysis of the formed biofilms. The absorbance was measured using a microplate reader at 595 nm.The anti-biofilm activity of TCNH was investigated using the crystal violet (CV) staining method. In brief, TCNH , HThe biofilm was further cultured on a round glass slide with a similar procedure as above. After 48 h, the glass slides were gently removed and washed with PBS (pH 7.0) to remove planktonic bacteria. Then the slides were stained with a live/dead (SYTO 9/PI) bacterial viability kit and observed under a confocal laser scanning microscope (CLSM).9 CFU/mL, 1.0 mL) was incubated with DCFH-DA (10.0 μL) for 30 min and centrifuged at 5000 rpm for 10 min. The sediments were washed with PBS (pH 7.0) 3 times to remove the free DCFH-DA. The collected bacteria were further used for conducting antibacterial experiments using the same procedure as in Biocompatibility Evaluation part. The resulting mixtures were then photographed with a fluorescence microscope .Intracellular ROS levels were determined by fluorescence spectroscopy using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method. Briefly, the bacterial suspension (2*10P. aeruginosa (19660) keratitis model was selected to evaluate the antibacterial efficacy of TCNH in vivo. Mice (n = 32) were anesthetized through intraperitoneal injection of 0.6% pentobarbital (30.0 mg/kg). Afterwards, three 1-mm incisions were created on mouse corneas using a sterile 25-gauge needle and the mice were randomly divided into four groups (n = 8). The wound surface was inoculated with P. aeruginosa , and the mice in the four groups were treated with saline (5.0 μL), H2O2 (5.0 μL), TCNH (5.0 μL), or TCNH + H2O2 (5.0 μL), once every hour for a total of 8 doses. The corneas were observed using a slit lamp at 24 and 48 h to detect the infection progression. After 48 h of the first administration, all mice were sacrificed and the eyeballs were harvested for H&E staining, SEM, flow cytometry, bacterial load counting, and qRT–PCR analysis.All animal experiments were approved by the Shandong Eye Institute ethics committee and were carried out based on the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6J mice  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The P. aeruginosa (21027) was selected to evaluate the antibacterial efficacy of TCNH on MDR P. aeruginosa keratitis in mice. Mice (n = 32) were randomly divided into four groups (n = 8): saline, tobramycin (0.3 mg/mL), gentamicin (0.3 mg/mL), and TCNH + H2O2. Other experimental processes were performed with the same procedure as that for P. aeruginosa  keratitis.In addition, The mouse eyeballs in the four groups were collected, placed in 96-well plates, and digested overnight at 4 °C with dispase (15.0 mg/mL). The corneal epithelium and stromal layer, separated under a microscope and placed separately, were digested with trypsin (0.25%) and collagenase (2.0 mg/mL) at 37 °C for 1–2 h, respectively. The suspensions were merged, incubated with antibody mixtures  for 30 min on ice, and further surveyed using a flow cytometer .The harvested eyeballs were fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin and cut into slices (5 μm-thickness). The slices were stained with H&E, and further imaged under a light microscope . For immunohistochemistry, tissue slices were stained with primary antibodies , and subsequently reacted with secondary antibody (GTVisionTM I detection system/Mo&Rb). The hematoxylin was adopted to visualize the nuclei. The staining was analyzed under a light microscope .P < 0.05 was considered statistically significant between the means. The results are presented as the mean ± standard deviation (SD).All tests were performed at least three times. The data were statistically analyzed using Student’s t tests by Prism 8 . A value of *2+, and Co-metal, respectively. In addition, the high-resolution C1s XPS spectrum exhibited the presence of C = C/C–C (284.5 eV), C = O (288.5 eV), and C-O (286.5 eV) groups. The O1s XPS spectrum could be deconvoluted into three peaks at 533.5 eV (C = O), 532.5 (C-O), and 531.5 eV . These results confirmed the coordination of tannin with the Co3O4/Ag composite in TCNs. PXRD was then used to determine the crystallographic structure of the hydrogel, TCN and TCNH , and C-O bonds in TCN, manifesting the immobilization of tannin into cobalt tetroxide [−1) in gelatin hydrogel and CoIII–O vibration (662.7 cm−1) in TCN were present in the spectrum of TCNH, indicating the successful incorporation of TCN into the hydrogel. In addition, ·OH signals were monitored by ESR. As shown in Fig. 2O2 and TCNH + H2O2 groups, but not in the hydrogel + H2O2 group, confirming the generation of ·OH by H2O2 under the catalysis of TCN. These results indicated the successful synthesis of TCN and TCNH.Nitrogen adsorption-desorption experiments were performed to characterize the specific surface area of TCN using the Brunauer-Emmett-Teller model within the relative pressure range of 0.5–1.0, as shown in Fig. etroxide . In addi2+-tannic acid, Fe3+-tannic acid and Ag-tannic acid have been developed for in vivo disease treatments [Noteworthily, several tannic acid modified nanoparticle systems such as Feeatments –45. Comp2O2 as substrates. ·OH was produced by co-incubation of H2O2 with TCNH, which oxidized TMB into ox-TMB and changed the solution color from colorless to blue  and H2O2  and Hs report . Further2O2, TCNH, TCNH + H2O2) groups after 24 h of intervention. Further LDH assays showed that the cell viabilities of HCECs and HCFs in all treated groups remained greater than 90%, demonstrating the nontoxicity of TCNH to cell growth. The in vivo biocompatibility of TCNH in the presence of H2O2 was verified by dripping TCNH + H2O2 onto the corneal surface of mice. The corneas at 48 h post intervention were observed by SEM . As shown in Additional file 10CFU numbers of P. aeruginosa in both the TCNH + H2O2 and H2O2 groups were over 7.0, and there was no obvious difference between the log10CFU numbers in two groups when the H2O2 and TCNH concentrations were set as 100 μM and 0.4 mg/mL, respectively, indicating the poor antibacterial activities of H2O2 and TCNH + H2O2 against P. aeruginosa under these conditions. With the increase in H2O2 concentration from 100 μM to 400 μM, log10CFU number in the TCNH + H2O2 group significantly decreased, while those in the H2O2 group were slightly reduced, indicating the improved antibacterial activity of TCNH + H2O2 at 400 μM. Then we further investigated the effect of TCN concentration in TCNH on log10CFU numbers with H2O2 concentration at 400 μM  and TCN concentration (4.0 mg/mL) were selected for subsequent antibacterial experiments.The Hruginosa 9,660. Asruginosa 9,660. AsP. aeruginosa  was initially evaluated using the plate cultivation method. Figure 2O2, TCNH, and TCNH + H2O2). The TCNH + H2O2 and TCNH groups exhibited high antibacterial activities as nearly no bacterial colonies were observed after interventions. However, bacteria all overgrew the LB agar plate in the saline and H2O2 groups, and there was no significant difference between two groups. In the dead/live (SYTO 9/PI) double staining assays  in the saline and H2O2 groups, indicating that the bacteria in both groups were in good condition. After intervention with TCNH + H2O2 and TCNH, the numbers of live bacteria in both groups significantly decreased, which was consistent with the plate cultivation results. The incubated mixtures in the four intervention groups were centrifuged and the residues were further observed by SEM. As shown in Fig. P. aeruginosa in the saline treated group presented as a long oval rod with a smooth surface. After treatment with a low concentration of H2O2, the bacteria maintained their rod shape, and rare disruptions were observed on the bacterial surface, suggesting the poor antibacterial activity of H2O2 against P. aeruginosa. However, the bacterial cells were always accompanied by deformation and dents after intervention with TCNH + H2O2 and TCNH, indicating the obvious damage to P. aeruginosa caused by TCNH + H2O2 and TCNH. Then ROS levels in the four groups were detected by the DCFH-DA method , which might explain the process for inhibiting P. aeruginosa using TCNH.The antibacterial property of TCNH against ays Fig. B, P. aerhod Fig. E. It wasTEM Fig. D. The reP. aeruginosa keratitis. As shown in Fig. P. aeruginosa were well formed after intervention with saline and H2O2 solution. However, there was no obvious biofilm formation in the TCNH + H2O2 and TCNH groups, indicating the significant inhibition efficacy of TCNH + H2O2 and TCNH against P. aeruginosa biofilm formation. The biofilms in the four groups were also quantified by CV staining assay  and were demonstrated to be effective in inhibiting the formation of P. aeruginosa biofilms. The hybrid hydrogel can generate highly toxic ·OH in the presence of H2O2 due to its peroxidase-like activity, which could account for the antibacterial and antibiofilm performances of TCNH.Biofilms, complexes of DNA, proteases, and polysaccharides, are self-produced and encapsulated by bacteria. It can prevent the penetration of antibiotics and reduce the efficacy of antibiotics in the treatment of bacterial infection , 37. Thesay Fig. G. ComparE. coli (25922) and C. albicans (98001). The absence of bacterial colonies, the decreased numbers of live bacteria and the deformed bacterial cells clarified the broad-spectrum antibacterial activity of TCNH.In addition, plate cultivation assays Fig. A, dead/lP. aeruginosa infection was surveyed in vivo using mouse keratitis as the animal model. The corneas with three 1-mm incisions were inoculated with P. aeruginosa (19660). Saline, H2O2, TCNH and TCNH + H2O2 were dripped on the mouse corneas. At 24 and 48 h post-operation, the mouse corneas were observed using a slit lamp. As shown in Fig. 2O2, the mouse corneas were clear, and no obvious infection was found, which was different from the severe corneal infections in the saline and H2O2 groups as well as the mild corneal infection in the TCNH group. The corneal samples in the four groups were further subjected to H&E staining. As presented in Fig. 2O2, and TCNH groups. However, no obvious inflammatory cell aggregation was observed in the TCNH + H2O2 group. The corresponding corneal bacterial loads of P. aeruginosa in the four groups were counted. The bacterial number in the TCNH + H2O2 group was significantly lower than those in the saline, H2O2, TCNH groups  and macrophages (0.80% ± 0.04%) than the other groups. Similar phenomenon can also be observed by immunostaining analysis of macrophages and neutrophils in the four groups , there were nearly no MDR bacterial colonies  in the TCNH and TCNH + H2O2 groups according to the plate cultivation results  in the TCNH + H2O2 and TCN groups significantly decreased  keratitis with gentamicin and tobramycin as the control groups. After administration of gentamicin and tobramycin, clear corneas and no obvious inflammation were observed in the P. aeruginosa (19660) treated group, while similar inflammatory cell infiltration and bacterial loads were observed in the saline (21027) and MDR P. aeruginosa (21027) treated groups . Representative photographs of bacterial colonies after intervened by saline, H2O2, TCNH, and TCNH+H2O2. FigureS5. Immunostaining analysis of macrophages and neutrophils in mouse corneas after saline, H2O2, TCNH, and TCNH+H2O2 interventions. Figure S6. In vivo antibacterial activities of gentamicin and tobramycin on P. aeruginosa (19660) keratitis. Representative photographs (A) and hematoxylin–eosin staining images (B) of treated eyes and bacterial loads of treated mouse eyes (C) after gentamicin and tobramycin interventions.


X-Linked Emery-Dreifuss muscular dystrophy is caused by mutations in the gene encoding emerin. Emerin is an inner nuclear membrane protein important for repressive chromatin organization at the nuclear periphery. Myogenic differentiation is a tightly regulated process characterized by genomic reorganization leading to coordinated temporal expression of key transcription factors, including MyoD, Pax7, and Myf5. Emerin was shown to interact with repressive histone modification machinery, including HDAC3 and EZH2. Using emerin-null myogenic progenitor cells we established several EDMD-causing emerin mutant lines in the effort to understand how the functional interaction of emerin with HDAC3 regulates histone methyltransferase localization or function to organize repressive chromatin at the nuclear periphery. We found that, in addition to its interaction with HDAC3, emerin interacts with the histone methyltransferases EZH2 and G9a in myogenic progenitor cells. Further, we show enhanced binding of emerin HDAC3-binding mutants S54F and Q133H to EZH2 and G9a. Treatment with small molecule inhibitors of EZH2 and G9a reduced H3K9me2 or H3K27me3 throughout differentiation. EZH2 and G9a inhibitors impaired cell cycle withdrawal, differentiation commitment, and myotube formation in wildtype progenitors, while they had no effect on emerin-null progenitors. Interestingly, these inhibitors exacerbated the impaired differentiation of emerin S54F and Q133H mutant progenitors. Collectively, these results suggest the functional interaction between emerin and HDAC3, EZH2, and G9a are important for myogenic differentiation. X-linked Emery-Dreifuss muscular dystrophy  is caused by mutations in the gene encoding emerin . Emerin In vitro studies showed that emerin binds directly to HDAC3 and activates its catalytic activity  and histone deacetylases (HDAC) 1 and 3 . Emerin activity . Emerin-activity . Similaractivity . Differeactivity . Treatmentiation , demonstntiation . We hypoInterestingly, EDMD1-causing emerin mutants  all failed to bind HDAC3 . MyogeniMsh homeobox 1 (Msx1) has been shown to colocalize with polycomb repressive complex 2 (PRC2) and H3K27me3 at the nuclear periphery in C2C12 cells and primary myoblasts . Emerin Msx1 can also recruit euchromatic histone lysine methyltransferase 2 (EHMT2 or G9a) in C2C12 cells . H3K9me2Collectively, these data support a model whereby emerin modulates repressive chromatin reorganization at the nuclear envelope during transcriptional reprogramming. How this occurs remains unknown. Thus, we set out to better understand the functional interaction between emerin, HDAC3, and EZH2 and G9a, and its role in myogenic differentiation. Here we show emerin interacts with both G9a and EZH2 in myogenic progenitors. We further demonstrate that emerin HDAC3-binding mutants show enhanced binding to G9a and EZH2, resulting in more H3K27me3 and H3K9me2 at the nuclear envelope and impaired differentiation. Using EZH2 and G9a inhibitors, we show these functional interactions are important for myogenic differentiation.−/y mice were obtained from Tatiana Cohen and Terence Partridge . Proliferating myogenic progenitors were counted in a Countess® II FL Automated Cell Counter (ThermoFisher Scientific), plated at 650 cells/cm2, and incubated in proliferative media consisting of high-glucose DMEM (ThermoFisher Scientific) supplemented with 2% chick embryo extract , 20% heat-inactivated FBS , 1% penicillin-streptomycin (ThermoFisher Scientific), 2% L-glutamine (ThermoFisher Scientific) and 20 units/mL γ-interferon (MilliporeSigma) at 33°C and 10% CO2, as previously described . Cell pellets were lysed on ice in modified NEHN buffer . Whole cell lysates were then sonicated on ice with 30 s on/off intervals for 10 min and placed on ice for 5 min. This was repeated three more times. Lysates were spun at 16,300 g at 4°C for 20 min. Insoluble components were pelleted and the soluble fraction was collected. Antibody-bound Dynabeads™ protein G magnetic beads were washed twice with washing buffer and incubated overnight with soluble fractions. Beads were separated from the soluble fraction with a magnet and resuspended in washing buffer for three 5-min washes with rotation. Bound complexes were eluted from beads per manufacturer instructions (ThermoFisher Scientific). Eluted fractions were incubated with 1:1 mixture of 2X NuPage™ LDS sample buffer at 75°C and 500 rpm for 10 min on a hot plate mixer . Samples were loaded and separated by SDS-PAGE at 150 V for 50 min. Gels were transferred to nitrocellulose membranes at 250 mA for 75 min on ice with stirring. Membranes were blocked with 5% non-fat milk (Giant Food Stores) in phosphate buffered saline (pH 7.4) containing 0.1% Tween-20 (PBST) for 1 hour at room temperature followed by incubation with antibodies against emerin , G9a  or EZH2  diluted in 0.5% non-fat milk overnight at 4°C with mild shaking. Blots were washed with PBST at least 3 times for 5 min at room temperature and incubated with HRP-conjugated secondary antibodies against mouse IgG . Membranes were then washed with PBST at least 3 times for 5 min. Blots were incubated with SuperSignal™ West PicoPlus chemiluminescent substrate solution  and visualized. Images were taken on a Bio-Rad ChemiDoc MP imaging system. Densitometry was performed using Bio-Rad Image Lab® software. Ratios of bound to input were measured and plotted. Input and unbound sample lanes represent 2.5% of total. Bound lanes represent 50% of input.Dynabeads™ protein G magnetic beads  were washed once with washing buffer. Beads were then resuspended in antibody binding buffer and incubated with antibodies against emerin , G9a , or EZH2 (Active Motif #39239) overnight at 4°C with rotation. Wildtype and emerin-null progenitors expressing S54F or Q133H EDMD1 mutants were grown in 15 cm dishes coated with 0.01% gelatin in proliferative conditions, as described above. Cells were incubated with 0.05% Trypsin-EDTA  for 5 min at 37°C and 10% CO2. Differentiation samples were plated at 25,000 cells/cm2 and incubated in proliferative media overnight at 33°C and 10% CO2. Coverslips and 96-well dishes were washed three times with phosphate buffered saline , fixed with 3.7% formaldehyde in PBS for 15 min, washed once with PBS for 5 minutes at room temperature, permeabilized with PBS containing 0.2% Triton X-100 for 20 min at room temperature, followed by washing once with PBS for 5 minutes at room temperature. All washing and incubation steps are performed with moderate shaking. Coverslips or 96-well plates were blocked in PBS containing 3% BSA and 0.1% Triton X-100 for 2 h at room temperature, followed by incubation with antibodies against emerin , H3K9me2 , G9a , H3K27me3  or EZH2  overnight at 4°C with shaking. Coverslips were washed three times in PBS at room temperature and incubated with Alexa Fluor™-conjugated secondary antibodies against rabbit or mouse IgG (Invitrogen) for 1 h at room temperature shielded from light. All primary and secondary antibodies were diluted in PBS containing 3% BSA and 0.1% Triton X-100. Coverslips were mounted on slides with ProLong™ Diamond Antifade Mountant with DAPI . Myogenic differentiation was done in 96-well plates because myotubes can more readily detach from coverslips. For immunofluorescence microscopy on differentiating cells, the cells were plated at 25,000/cm2 (differentiation density) and incubated overnight in proliferation media at 33°C and 10% CO2. Wells were washed with PBS and differentiation media was added to each well and the cells were incubated for 24–72 h at 37°C, 5% CO2. Blocking, fixing, washing, and incubating with primary and secondary antibodies was done as described for the coverslips. 96-well dishes were incubated with 0.2 μg/ml DAPI in PBS for 5 min and washed twice with PBS.Myogenic progenitors were plated on gelatin-coated coverslips or directly in gelatin-coated 96-well dishes for immunofluorescence microscopy. Proliferative cells were plated on gelatin-coated coverslips and grown for 2 days (30%–40% confluent) at 33°C, 10% COSlides of myogenic progenitors were prepared as described above and confocal images were taken using a Nikon Eclipse Ti2-E Inverted Research Microscope equipped for confocal, brightfield and DIC imaging in conjunction with a Nikon A1rSi Laser Point Scanning Confocal System. All images were taken with a 60X oil-immersion objective (N.A. = 1.4). Nikon Imaging Software (NIS) Elements was used to record images. Each field was subject to the same light conditions and exposure times. At least 30 nuclei were imaged for each trial. Files were analyzed in NIS Elements Viewer software and ImageJ. Localization was measured using 21.1 μm lines that were drawn across similar axes of nuclei using ImageJ software. Fluorescence was quantified along the superimposed line in each field and measurements were plotted on the same graph.2.5 µM G9a inhibitor UNC0638  or 4.0 µM EZH2 inhibitor GSK126  were added to differentiation media upon differentiation induction (t = 0). At 24, 48, or 72 h after induction of differentiation, the cells were lysed in SDS-PAGE sample buffer, separated by SDS-PAGE, and western blotting was done using antibodies against H3K9me2 , H3K27me3 , or γ-tubulin . The levels of H3K9me2 and H3K27me3 were normalized to γ-tubulin expression and analyzed as described above. Validation of inhibitors was performed in all cell lines and two biological replicates were prepared for each treatment at each time point.The ClickIt EdU reaction kit  was used to assess cell cycle withdrawal. EdU was added to media 2 h prior to washing with PBS and fixing with 3.7% formaldehyde in PBS for 15 min. Cells were washed once with PBS, permeabilized with 0.2% Triton X-100 in PBS for 20 min at room temperature, and stained per manufacturer instructions. Nuclei were incubated with 0.2 μg/ml DAPI in PBS for 5 min at room temperature. Images of EdU (Alexa-647) and DAPI (405 nm) were taken using a 40X objective (N.A. = 0.65) on an Evos FL Auto 2 microscope. Differentiation was quantified by monitoring the expression of myosin heavy chain (MyHC) using MyHC antibodies . A minimum of three biological replicates were prepared for each condition and at least three images were taken (random location) for each biological replicate in each field. At least 75 nuclei were imaged for each trial. Images were counted using the cell counter plugin on ImageJ and analyzed in Prism 9 software. Percentages of EdU-positive or MyHC-positive cells were compared to total nuclei (DAPI) for each replicate. Fusion index was determined by merging DAPI and MyHC channels and counting cells containing at least 3 nuclei sharing MyHC-stained cytoplasm.via Msx1, so we tested whether emerin and the EDMD1 mutants could bind to EZH2. H3K9me2 has also been implicated in organizing repressive chromatin at the nuclear envelope, so we also tested if emerin could bind G9a.Our previous data suggests that emerin binding to HDAC3 results in more chromatin deacetylation at the nuclear envelope which could recruit HMTs  to methylation targets and more stably repress nuclear envelope-associated chromatin. Emerin was reported to interact with EZH2 n = 3) or emerin-null progenitors expressing either emerin S54F (n = 3) or Q133H (n = 3) at wildtype levels. A small fraction of endogenous emerin bound G9a (n = 3) and 13.8% and 9.6% binding to S54F (n = 3) and Q133H (n = 3), respectively , whereas 71.8% of S54F (n = 3) and 77.8% of Q133H (n = 2) bound EZH2. Similar results were seen in the reciprocal immunoprecipitation, with 2.4% EZH2 bound to wildtype emerin (n = 3), and 57.9% and 89.4% of EZH2 binding to S54F (n = 3) and Q133H (n = 2), respectively . A line was drawn through each nucleus and relative fluorescence along this line was plotted to monitor spatial distribution of each protein and its associated modification. H3K9me2 . To validate our approach (n = 2), respectively. H3K9me2 levels at 24 h showed a reduction of 35.2% in wildtype, 17.8% in S54F, and 26.7% in Q133H differentiating myogenic progenitors, demonstrating that UNC0638 remains functional throughout the differentiation assay. Treatment with UNC0638 lowered the levels of H3K9me2 at all time points, with the exception of emerin-null cells (n = 2). H3K27me3 levels in wildtype, in S54F, in Q133H, and in emerin-null cells were reduced 71.2% 68.1%, 74.7%, and 71.5% after 72 h of differentiation  and EZH2 (GSK126) to reduce H3K9me2 and H3K27me3 levels, respectively, to test this hypothesis. UNC0638 and GSK126 have reported ICapproach , we firsapproach ; n = 2, ll cells . Similarll cells ; n = 2. ntiation , respectn = 3) but failed to rescue cell cycle withdrawal during differentiation of Q133H and emerin-null progenitors (n = 3). EZH2 inhibition failed to significantly alter cell cycle exit in any of the progenitor lines tested compared (n = 3).Now that we confirmed functionality of UNC0638 and GSK126 throughout differentiation, we wanted to test the impact of G9a or EZH2 inhibition on wildtype and EDMD1 mutant myogenic differentiation. EdU incorporation was measured 24 h after differentiation induction to assess cell cycle withdrawal. G9a inhibition increased cell cycle withdrawal during differentiation of wildtype and S54F progenitors , n = 3 bgenitors ; n = 3. compared ; n = 3.+ multinucleated myotubes + number of nuclei in MyHC+ mono- or di-nuclear cells/total nuclei) and the fusion index ) to assess myoblast commitment and fusion to form myotubes, respectively, in the presence of UNC0638 and GSK126. To monitor myoblast commitment and fusion, expression of MyHC was detected by immunofluorescence microscopy 48 h after differentiation induction, while the fusion index is determined at 72 h post-induction. G9a inhibition significantly decreased the differentiation index in wildtype emerin and S54F mutant cells (n = 3), while no significant difference was detected in emerin-null and Q133H mutant cells (n = 3). The fusion index was significantly decreased in emerin-null and Q133H mutant cells upon treatment with UNC0638 (n = 3), while no effect was seen in emerin-null and S54F mutant cells. Treatment with GSK126 decreased myotube formation in wildtype emerin, emerin-null and Q133H mutant cells (n = 3), while no significant difference was seen in S54F mutant cells.We next measured the differentiation index  to localize correctly in emerin-null or emerin S54F mutant progenitors and its failure to rescue differentiation (The emerin-HDAC3 interaction is important for the coordinated temporal relocalization of fLADs during myogenic differentiation . Howeverze cLADs . In thisze cLADs . The lacze cLADs . This isntiation . In thisntiation . HoweverIt is possible that HMTs could be important for fLAD organization and HDAC3 could be important for its reorganization, as our studies and our proposed model fail to differentiate between these possibilities. For example, emerin-null and mutant progenitors have reduced global H3K27me3 and H3K9me2/3—a potential contributor to their impaired differentiation. Future studies will be necessary to investigate the effect of increasing global H3K9 and H3K27 methylation using specific demethylase inhibitors. More experiments are also necessary to examine how specific myogenic loci are dynamically localized and how this correlates with their specific chromatin post-translational modification states in emerin mutant myogenic progenitors.Although these and our previous studies  support It is unclear from these studies as to whether emerin binds directly to EZH2 or G9a. Emerin may bind EZH2 and G9a through binding to their respective complex components. EZH2 and G9a may also interact with emerin through other known binding proteins. One candidate protein is Msx1. Emerin was shown to bind Msx1 and bridge an interaction between EZH2 and emerin . G9a wasThe functional interaction of emerin with EZH2 and Msx1 is important for myogenic differentiation. Emerin knockdown in proliferating C2C12 myoblasts was shown to displace EZH2 and H3K27me3 from the nuclear periphery , suggestvia Msx1 would be seen in vitro, but this increase would be masked in cells. Expression of exogenous G9a or EZH2 will be used in future experiments to test these possibilities.Although the relative levels of H3K9me2 and H3K27me3 at the nuclear envelope are increased in HDAC3-binding emerin mutants, we do not observe more EZH2 or G9a at the nuclear envelope. This could be due to epitope masking of EZH2 or G9a upon binding to emerin at the nuclear envelope. Alternatively, it is possible that in live cells the interactions between EZH2 or G9a and emerin are more transient, as might be expected for an enzyme. This transient interaction would likely be sufficient to add the H3K9me2/3 or H3K27me3 modification. In this scenario, the conditions of the immunoprecipitation may increase the stability of the interactions between emerin and EZH2 that occurs in cells. Lastly, the interaction between emerin, Msx1, and G9a or EZH2 may mask the epitopes of G9a or EZH2 only when bound to Msx1 in cells. In this scenario more binding of G9a or EZH2 to emerin In summary, it is clear the coordinated temporal reorganization of the genome occurs progressively, as some myogenic differentiation genes become active while cell cycle regulation and mitotic genes are silenced. Thus, this process occurs through multiple steps, all of which are crucial for proper differentiation into myotubes. It is possible that the failure to rescue myotube formation we observed in G9a and EZH2 inhibition experiments is a consequence of enriched H4K5ac from loss of HDAC3 interaction in S54F, Q133H, or emerin-null progenitors. One potential explanation would be that deacetylation of H4K5 is a critical step in the myogenic differentiation program. Further, the binding of HDAC3 to these regions would help limit the amount of more stable chromatin repression by limiting the amount of EZH2 or G9a at the nuclear envelope, thereby allowing precise control of the regions to be methylated with H3K9me2 or H3K27me3. In this way, emerin may also be important for maintaining homeostasis of H3K9me2 and H3K27me3 localization at the nuclear periphery, as well as its dynamic reorganization during differentiation. We posit that the interaction of S54F or Q133H emerin with EZH2 or G9a-containing complexes lack proper regulation due to their inability to bind HDAC3, resulting in defective chromatin organization at the nuclear periphery. Cells lacking emerin also exhibit a decrease in repressive marks, likely due to this inability to properly regulate repressive chromatin organization and are thus unable to appropriately repress cell cycle genes. Previous data from our lab and the experiments in this study strongly suggest emerin plays multiple roles in regulating dynamic genomic organization at the nuclear periphery.


The synthesis of BaTiO3 ceramics with weak temperature dependence and a high dielectric constant at room temperature (εRT′) is an urgent problem to meet the miniaturization and large capacity of dielectric capacitors. Doping rare earth elements into BaTiO3 can solve this problem, but it is still challenging. In this work, we adopt a synergistic strategy of increasing εRT′ and improving the temperature stability by codoping Gd3+ and Ho3+, respectively, to address this challenge. By carefully adjusting the synthesis conditions in the solid-state reaction, codoping 7% Gd3+ and 7% Ho3+ in BaTiO3 (BGTH7) ceramics were synthesized. The temperature-dependent dielectric constant reveals that the obtained optimal BGTH7 ceramic satisfies the X7U specification and displays a stable ε′ in the temperature range of −55~125 °C. The optimal BGTH7 ceramic after sintering at 1400 °C for 6 h exhibits a high dielectric constant of 5475 and low dielectric loss (tan δ) of 0.0176, hitherto exhibiting the best performance in X7U ceramics. The findings in this work are conducive to the miniaturization and stabilization of dielectric energy storage devices.BaTiO The colerance ,7.C) of dielectric capacitors is governed by the dielectric constant (ε′), capacitance plate area (S), and distance between two pole plates (d), as shown in the formulas below:9 N·m2/C2). BaTiO3 dielectric ceramics are a key component of dielectric energy storage capacitors. However, the unstable and low capacity of BaTiO3 dielectric capacitors cannot meet the demand for miniaturization and the high performance of dielectric capacitors [ε′RT) for BaTiO3 ceramics can meet the requirements of miniaturization and high performance of dielectric capacitors, which are more widely used in miniaturized and integrated circuit systems [The capacitance  to room temperature contributes to high ε′RT. Gd-doped BaTiO3 can significantly reduce the Tm and increase εRT′ [3-0.3Sr0.2Bi0.7TiO3 ceramics possess high ε′RT (ε′RT ≈ 4000) and Tm near −19 °C, exhibiting excellent energy storage. The difference in the valence of ions at the A-site induces a decreased tolerance factor, resulting in a decrease in Tm [During the tetragonal to the cubic transition of BaTiOric peak . Shiftinase εRT′ ,16. Gd-dse in Tm .3 can broaden the dielectric peak and obtain stable dielectric constant [2O3 and Ho2O3 codoped BaTiO3-based ceramics by a solid-phase method, with a stable ε′ between 2400 and 2600 and satisfactory temperature stability of capacitance with an X8R dielectric specification. La-, Pr-, Nd-, Sm-, and Ho- codoped BaTiO3 all had wide dielectric peaks, satisfying the dielectric specifications of X5T, X6T, X7T, and X8S, respectively [Moreover, the incorporation of Ho in BaTiOconstant . In receconstant  successfectively .3, doping Gd or Ho alone is not sufficient to increase ε′RT and stable ε′ in the temperature range of operation. The dielectric performance of doped ceramics is also dependent on sintering temperature (Ts) and dwell time (td). Therefore, we propose a synergistic strategy codoping 7% Gd3+ and 7% Ho3+ in BaTiO3 (BGTH7), carefully adjusting the synthesis conditions by the solid-phase method to obtain high ε′RT and stable ε′, achieving stable and high capacities for BaTiO3 dielectric capacitors. The specific roadmap was shown in Although the dielectric performance was enhanced using the method reported above for BaTiOTm was caused by the charge-coupled substitution of Gd3+ and Ho3+ at both Ba and Ti sites, which was confirmed by X-ray photoelectron spectroscopy (XPS), electron paramagnetic resonance (EPR), and Raman spectrum. The scanning electric microscope (SEM) images show that optimizing Ts can improve the density and average grain size of the ceramic samples. Eventually, high ε′RT and stable ε′ at work temperature can be obtained, which is promising for miniaturization and high performance of devices.In this work, the phase structure of BGTH7 ceramics was studied by X-ray diffraction and Raman spectroscopy. The improved performance with dielectric diffuse phase transition behavior can be obtained from dielectric measurement. The shift of 3+ and Ho3+ codoped BaTiO3 ceramics were synthesized by a solid-state method to achieve a high ε′RT and stable ε′ in the operating temperature range. Usually, the sintering temperature (Ts) and dwell time (td) greatly affect the phase composition in solid-phase reactions. Thus, we systematically adjusted Ts and td to optimize the sintering conditions (Ts = 1200 and 1300 °C contained impurity phases Ba12Ho4.67Ti8O35 (PDF # 43−0420) and Ho2O3 (PDF # 43−1018) (marked with * and # in 3+ into the perovskite structure owing to the large radius of Ho3+ (0.901 Å) in octahedral coordination compared to that of Ti4+ (0.605 Å) [Ts can change the boundary of the phase diagram in multiple composition compounds but also increase the solution limit of doped ions in perovskite [Ts = 1400 °C, BGTH7 ceramics exhibited a pure phase of perovskite.Gdnditions a. Two BG0.605 Å) . And as rovskite . Thus, atd was examined by XRD. BGTH7 ceramics sintered for different td  are pure phases, as shown in td increased, the XRD peaks gradually narrowed, and their intensities were enhanced, demonstrating an improvement in crystallinity. To accurately determine the shift of the peak, the internal standard KCl was added to the BGTH7 ceramic powder. The enlarged peak at approximately 38° slightly shifted toward a higher 2θ angle as td increased, confirming a slight contraction in unit cell volume (V0). Notably, the diffraction peak (200) at approximately 45° for all ceramics did not show any splitting, which may be attributed to the cubic structure, similar to cubic BaTiO3 (PDF # 31−0174). Certainly, a slight distortion in the tetragonal phase structure or broadening of the XRD peaks can also obscure such a splitting, and this obscuring effect is difficult to detect by XRD. Therefore, in addition to XRD data, spectroscopic evidence could be helpful to prove the accurate phase structure of BGTH7 ceramics sintered at 1400 °C.The phase structure of BGTH7 ceramics sintered at 1400 °C with different Pmm does not give any Raman signal due to a centrosymmetric structure belonging to the O1h space group [3 structure deviates from cubic symmetry, such as tetragonal with P4mm, four degenerate 3Fu1 (IR) + Fu2 (inactive) modes for the O1h space group split into eight Raman active 3A1 + B1 + 4E modes [Raman spectroscopy can effectively distinguish the tetragonal or cubic phase of perovskite. Ideal cubic perovskite with ce group . Once th4E modes . Thus, t−1, usually from the B1 and E modes, was seen in the Raman spectra of sintered BGTH7 ceramics , indicating that BGTH7 ceramics sintered at 1400 °C had a tetragonal structure [−1 was not obvious for the BGTH7 ceramic sintered at td = 1 h due to incomplete grain growth. With these observations in mind, we refined the XRD data of BGTH7 ceramics sintered at 1400 °C by the tetragonal phase structure. The obtained lattice parameters in a and c are very close , i.e., the structure of BGTH7 ceramics sintered at 1400 °C slightly deviates from cubic symmetry. On the other hand, the Raman peak at approximately 520 cm−1 in 6)4− octahedra, confirming the presence of a small structure distortion. A similar distortion was also observed in previous reports [ε′, which is desirable for BaTiO3 dielectric energy storage capacitors [As shown in tructure ,26. Diff reports ,28. The pacitors ,30.td = 1, 3, 6, and 12 h, respectively. As illustrated by the SEM images in the inset of td = 1 h exhibited tiny grains and pores, showing incomplete grain growth. The grain size was improved by prolonging the dwell time. These gradually increased grains filled the pores, resulting in a denser microstructure. The relative density (ρr) of the BGTH7 ceramics was 73, 79, 86, and 93%, sintered at 1400 °C for td = 1, 3, 6, and 12 h, respectively, in which ρr was estimated using the following formula:The dwell time greatly affected the morphology of intact disc BGTH7 ceramics sintered at 1400 °C. According to the grain size analysis in ρ0 is the theoretical density obtained from Rietveld refinement, and ρa is the actual bulk density that can be measured using the Archimedes method.3+ and Ho3+, have different ion radii than Ba2+ and Ti4+, which often leads to their preferential occupation once they are doped in BaTiO3. Such a preferential occupation has an important influence on the dielectric property. In addition to the ionic radius, some other factors can also change the occupation of rare earth ions in the perovskite, such as the valence state of Ti and oxygen vacancies, which can be determined by XPS, EPR, and photoluminescence analysis.Rare earth ions, such as GdTs = 1400 °C. Well spin-orbital splitting peaks Ti2p3/2 (at approximately 457.8 eV) and Ti2p1/2 (at approximately 463.6 eV) were observed, and the splitting energy of 2p3/2 and 2p1/2 was 5.8 eV, confirming the presence of Ti4+ ions [3+ 2p3/2 appears at approximately 455 eV [3+, i.e., only one oxidation state Ti4+ in the BGTH7 ceramics sintered at 1400 °C.The valence state of Ti ions was investigated by XPS. i4+ ions ,32. Geney 455 eV . Deconvo3+/Ho3+ and host ions Ba2+ and Ti4+ in BaTiO3, the incorporation of Gd3+/Ho3+ could introduce a positive or negative charge center. These charge mismatches can be compensated by vacancies, described as follows by the Kröger–Vink notation:Due to the charge difference between dopant ions Gd3+ or Ho3+. g = 1.974 and 1.955 [g = 1.990 was observed for BGTH7 ceramics sintered at 1400 °C. This signal is attributed to Gd3+ with an electron configuration of 4f7 (ground state is 8S7/2) [3+ is highly dependent on the symmetry of ion coordination in g = 5.953 and 2.451 at temperatures below 223 K in 3+ symmetry from slightly distorted cubic coordination to intermediate coordination because of the increased structure distortion at a low temperature [Tm (270.36 K). Therefore, the incorporation of Gd3+ and Ho3+ could adopt a charge-coupled substitution on both the Ba and Ti sites:Here, Ln represents Gdnd 1.955 ,35. In Fs 8S7/2) . The EPRperature ,38. The 3+ ions in some perovskites exhibit a strong photoluminescence emission. The Raman spectrum of Ho3+ doped BaTiO3 under 532 nm excitation is often used to probe the site occupations of Ho3+ ions because the concentration of Ho3+ ions on the Ba sites is highly dependent on the transition intensities of 5F4/5S2→5I8 at 545 nm, 5F5→5I8 at 653 nm, and 5F4/5S2→5I7 at 755 nm [3+ at the Ba site can be estimated [td values of 1, 3, 6, and 12 h, the contents of Ho3+ substituted for Ba2+ were 0.0059, 0.0050, 0.0022, and 0.0018, respectively. Increasing the dwell time at a high temperature (1400 °C) could reduce the incorporation of Ho3+ into the Ba site. The cross-site occupancy between Gd3+ and Ho3+ is described in Equation (5) and occurred in BGTH7 ceramics sintered at 1400 °C. Therefore, for BGTH7 ceramics with td values of 1, 3, 6, and 12 h, the contents of Ho3+ substituted for Ti4+ were 0.0641, 0.065, 0.0678, and 0.0682, respectively. From the ionic radius in 3+ ions preferentially incorporated into the Ba site, while Ho3+ preferentially incorporated into the Ti site, forming a 3+ and Gd3+ change their occupation on the Ba site and Ti site to Ts = 1400 °C and td = 6 h, the real molecular formula of the obtained ceramic was (Ba0.93Gd0.0678Ho0.0022)(Ti0.93Ho0.0678Gd0.0022) O3, having more Hot 755 nm ,39. Figustimated . For BGTThe Electronic Industry Association (EIA) classifies class II ceramic capacitors into two categories: capacitance-stable and high-capacitance ceramic capacitors. The most commonly used capacitance-stable capacitors are R-type capacitors. Over the operating temperatures, the capacitance change of R-type capacitors does not exceed 15%. However, these capacitors have relatively low dielectric constant, which are not suitable for miniaturized applications in electronic products. Another capacitor is the Y-type capacitor. Even though the Y-type capacitor has a high capacitance compared to the R-type capacitor, it is difficult to apply because of its unstable dielectric constant. A U-type capacitor combines the advantages of temperature stability and high capacitance, eventually having a stable and high dielectric constant over the entire operating temperature range ,41,42,43ε′ and dielectric loss (tan δ) measured at 1 kHz for BGTH7 ceramics sintered at 1400 °C. According to td = 1, 3, 6, 12 h) satisfy the X8R, X7U, X7U, and X6U specifications, respectively. The dielectric properties for all BGTH7 ceramics are shown in ε′RT (ε′RT > 5000) and ultralow tan δ (tan δ < 0.02), with the location of the dielectric peak near the room temperature. This finding is very important for solving the problem of the location of the dielectric peak for conventional BaTiO3 ceramics being too high (close to 125 °C) to satisfy the requirement of the development and application of small electronics at room temperature.ε′ and tan δ of BGTH7 ceramics sintered at 1400 °C with different td exhibited the same changes with frequency  and low tan δ (tan δ = 0.0176) at 1 kHz, as well as enhanced frequency stability from 1 to 107 Hz. In addition, this BGTH7 ceramic satisfied the X7U specification according to ε′ in the operating temperature range (−55~125 °C). As shown 3 dielectric energy storage capacitors.Ceramics sintered at 1200 and 1300 °C exhibited low dielectric constant and high tan ε′RT gradually increases from 1762 to 6580 as the average grain size changes from 0.71 to 3.17 μm , as shown in ε′RT can be understood from the following two points: (1) Air acts as a stress snubber and reduces the entrapment forces between grains in the BaTiO3 ceramic, resulting in the formation of pores. Polarization is a critical factor in improving the dielectric constant. Normally, polarization is weakened by the depolarization field that occurs near the pores, resulting in decreasing dielectric constant [ρr shields the depolarization field, resulting in a high ε′ of ceramics. Among the four examined BGTH7 ceramics sintered at 1400 °C, the one sintered at td = 12 h, having ρr as high as 93%, gives the highest ε′RT of 6580. (2) An easier and more regular motion of the domain wall can also increase ε′RT [ε′. As the grain sizes increased, the dielectric peak was gradually enhanced, resulting in an increased dielectric constant (ε′RT) at room temperature. Therefore, a denser structure and large grain size are beneficial to obtaining high ε′RT in BGTH7 ceramics sintered at 1400 °C.constant . High ρrase ε′RT ,58. Genet) can predict the symmetry of ABO3 perovskites that significantly affect their dielectric performance and is defined as the ratios of the constituent ionic radii of A, B, and O:The Goldschmidt tolerance factor , 0.605 , and 1.4 Å; thus, t ≈ 1.06. A previous investigation has shown that the dielectric peak temperature (Tm) of BaTiO3 is highly dependent on the tolerance factor (t). Reducing t can shift the Tm to a lower temperature [3+ ions are incorporated into Ba sites, the decrease in t shifts Tm to a low temperature. Instead, the substitution of Ho3+ for Ti4+ ions leads to Tm moving toward higher temperatures, similar to the incorporation of Ca2+ [3+ and Ho3+ at both Ba and Ti sites (Equation (5)) has different effects on Tm, which was caused by the different effects of substitution for Gd3+ and Ho3+ on Ti-O octahedral distortion [3+ content in the Ti site increases. Consequently, Tm first decreased and then increased, as shown in For pure BaTiOperature . When so of Ca2+ . In othe3+ and Ho3+ codoped BaTiO3 ceramics can also broaden the dielectric peak, similar to La-, Pr-, Nd-, Sm-, and Ho- codoped BaTiO3 ceramics [3+ with Gd3+ has an important effect on the stability of ε′ over a wide temperature range.All studied BGTH7 ceramics sintered at 1400 °C exhibit dielectric diffusion phase transition (DPT) behavior. The broadening dielectric peak was related to the grain size, which is consistent with numerous past reports ,62. In aceramics . Codopin3+ and Ho3+ at both Ba and Ti sites, decrease of tolerance factor, distortion of octahedral, as well as increased density can be optimized by adjusting sintering temperature, and time. Eventually, the optimal BGTH7 ceramic not only exhibited high ε′RT but also dielectric DPT, which can ensure stable operation for BaTiO3 dielectric energy storage capacitors.The detailed mechanisms for improving dielectric properties is illustrated in 3 (99.5%), TiO2 (99.5%), Ho2O3 (99.95%), and Gd2O3 (99.90%) were used as raw materials, weighed according to 7% Gd3+ and 7% Ho3+ codoping in BaTiO3 (BGTH7), and milled. After drying and calcining at 1100 °C for 5 h for decarburization, the obtained powder was reground and pressed into discs with a diameter of 12 mm and a thickness of 2 mm at 150 MPa using an aqueous solution of PVA (12% by mass) as a binder. The final sintering conditions to form the BGTH7 ceramics were chosen as heating up directly from room temperature to different Ts  and different td  in the air at a heating rate of 100 °C/h, a cooling rate of −200 °C/h to 700 °C, and then furnace cooling to room temperature.BaCO1 radiation (λ = 1.540562 Å). The microstructure of the sintered samples was observed using an EVOMA 10 scanning electric microscope  operated at 15 kV. The conducting Au atoms were sputtered on the specimen surface for SEM observations. XRD Rietveld refinements were conducted with the General Structure Analysis System (GSAS) program. The 532 nm and 785 nm lasers were used for excitation to obtain the Raman spectra (RS) of the ceramics and photoluminescence (PL) of Ho3+ using a LabRAM XploRA Raman spectrometer . X-ray photoelectron spectroscopy  measurements were performed to study the valence states of cations. The measurement curves of XPS data were fitted by a mixed Gaussian–Lorentzian function, and Shirley-type background subtraction was used. EPR measurements were carried out with an X-band (≈9.4 GHz) spectrometer  at 90–298 K. The gyromagnetic constant (g) was calculated using hv = gμ0H, where h is the Planck constant (h = 6.626 × 10−34 J·s), v is the microwave frequency, μ0 is the Bohr magnetron (μ0 = 9.262 × 10−24 J/T), and H is the magnetic field strength. The dielectric properties of the ceramic samples were measured on a broadband dielectric spectrometer  in a temperature range of 198–473 K and a frequency range of 1 Hz–10 MHz.The crystallographic structures were studied by using X-ray diffraction  with Cu Kα3 dielectric capacitors are critical energy storage devices due to their ultrafast charge/discharge ability, exceptional cycle life and high-temperature tolerance. However, the unstable and low dielectric constant (ε′RT = 1600) near the room temperature of BaTiO3 ceramics cannot meet the needs of miniaturization and the high capacity of dielectric capacitors. In this work, Gd3+ and Ho3+ codoped tetragonal BaTiO3 ceramics were prepared by carefully adjusting the synthesis conditions to address this issue. The optimal ceramic sintered at 1400 °C for 6 h had an ultrahigh room-temperature dielectric constant (ε′RT = 5475) with low dielectric loss (tan δ = 0.0176). In addition, the BGTH7 ceramic exhibits good X7U dielectric properties in the temperature range of −55~125 °C, which is superior to other X7U BaTiO3-based ceramics reported previously. The mechanisms for improving dielectric properties can be attributed to the tetragonal perovskite structure, large grain size, and the formation of the Converting renewable energy to electricity and efficient electricity storage are keys to addressing the energy crisis. BaTiO


We have investigated the thermally and photoinduced structural and chemical changes of a polyvinylpyrrolidone (PVP)-covered silver nanocube (AgNC) array on Au(111). The Langmuir–Blodgett (LB) method was utilized to fabricate the highly ordered array of the AgNC monolayer on Au(111). In the Raman spectra obtained at room temperature, sharp vibrational peaks were observed owing to the surface-enhanced Raman scattering (SERS) effect of AgNCs. When AgNCs were annealed, their corners became rounded, followed by their height decrease and lateral expansion. Simultaneously, PVP decomposed into nanocarbons, which were eliminated from the gap between AgNCs. Further annealing AgNCs/Au(111) resulted in obvious decreases in Raman signal intensity and AgNC height due to the sintering of AgNCs. We also confirmed the photochemical transformation of PVP to nanocarbons without the deformation of AgNCs when an intense laser was irradiated on the AgNC surface. We have investigated the thermally and photoinduced structural and chemical changes of a polyvinylpyrrolidone (PVP)-covered silver nanocube (AgNC) array on Au(111). For example, NP arrays made of gold (AuNP)4–7 and silver (AgNP)8–13 are representative and have been extensively studied. The key factors in the determination of the physical and chemical properties of metal NP arrays are their shape, size and arrangement. Microscopic structures of NP arrays obviously affect the surface-enhanced Raman scattering (SERS) effect, localized surface plasmon resonance (LSPR) and reaction activity.14–17Metal nanoparticles (NPs) forming an array on a substrate have been attracting much attention in relation to technological applications such as sensing, optoelectonics and catalyses.et al. showed that isolated AgNPs sublimate uniformly upon heating without transforming to the liquid phase.18 Generally, the sublimation temperature increases as the size of NPs increases. For example, 5 nm AgNPs sublimate at around 500 °C, whereas 30 nm AgNPs sublimate at around 675 °C.18 On the other hand, it is known that thermal changes in the morphology and arrangement of NP arrays occur at a temperature lower than that in the case of isolated NPs.4,5,7,19 For example, Zhang et al. demonstrated that heating treatment causes the aggregation of AuNPs at 100 °C and the coalescence of AuNPs at 140 °C.4 Laser-induced shape conversions have been also reported for Au nanorods, Ag nanoplates and AgNPs in colloidal suspensions.20,21 Thermal and photoinduced structural changes of NPs are often accompanied by a shift in LSPR wavelength and are proposed to control the plasmon properties of NPs.4,5,7,19–24Many studies have been conducted to control the shape, size of individual NPs and arrangement of NP arrays through thermal treatment. Asoro 25–31 than and a superior catalytic activity to AgNP of spherical shape.32,33 Unique nanoscale properties, such as single-molecule detection by the SERS effect,25 superior catalytic activity on (100) facets32,33 and plasmonic chemical reactions,34–36 and brightening of an organic light-emitting device,37 have been realized by utilizing AgNCs. Similarly to the spherical NP system, the shape and size of individual AgNCs can be controlled by heating and light irradiation. Vijayan and Aindow heated isolated AgNCs and examined the temporal change of the AgNC structure.38 They revealed that AgNCs remained intact below 400 °C, whereas they were sublimated and became spherical at around 760 °C. Several research groups demonstrated that AgNCs behave as quasiliquids above 500 °C and (110) facets are preferentially formed by surface relaxation during sublimation.39,40 As described above, although shape changes of isolated AgNCs have been well studied, those of AgNCs in an array have not been reported. Several research groups pointed out that unique characteristics, such as the enhanced SERS effect26,29 and selective excitation of localized surface plasmon (LSP) modes,27 appear in the AgNC array. Thus, it is highly expected that the optical properties of AgNC arrays can be tuned by changing the size and alignment of AgNCs. To the best of our knowledge, there has been no report on the structural changes of the AgNC array upon heating or laser irradiation.Recently, a silver nanocube (AgNC) has drawn much attention since it has a higher electric field enhancementIn this study, we have investigated the structural change of an AgNC array upon heating and laser irradiation, and also characterized the simultaneous changes in the chemical states of polyvinylpyrrolidone (PVP) covering the AgNCs. About 100nm-sized AgNCs covered with PVP were synthesized by the polyol method. The Langmuir–Blodgett (LB) method was utilized to fabricate a monolayer array of AgNCs on Au(111). The AgNC array on Au(111) was heated in an ultrahigh vacuum (UHV) chamber and irradiated with a laser in atmosphere. Then, the AgNC shapes and arrangements were evaluated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Furthermore, we used Raman spectroscopy to elucidate the chemical state of PVP before and after heating and laser irradiation.2.3 After 1 mL of 3 mM HCl was added in 5 mL of ethylene glycol (EG) heated to 130 °C, 3 mL each of 94 mM AgNO3/EG solution and 147 mM PVP /EG was dropped into with a flow rate of 0.75 mL min−1. The AgNCs were synthesized by keeping the temperature at 130 °C for 20 hours. The synthesized solution was purified by centrifuging at 2000 × g for 20 minutes. It is known that PVP adsorbed selectively to the (100) faces, leading to the growth of cubically shaped Ag nanoparticle. Therefore, each AgNC facet was fully covered with PVP immediately after the AgNC was synthesized. It is important to use AgNCs with a uniform size since the size of AgNC affect the SERS activity. For this reason, we used 100 nm-sized AgNCs because we succeeded in producing AgNC of this size with good reproducibility. In addition, we chose the size of AgNC in terms of the resonance wavelength of AgNC's LSP mode. The Au(111) substrate was obtained by evaporating Au  on a freshly cleaved mica surface under vacuum (3 × 10−4 Pa), followed by annealing at 500 °C.41–43 The 2D array of AgNCs was fabricated on Au(111) by the LB method.27 We evaluated the shape and array structures of AgNCs by SEM  and AFM . Raman spectroscopy  was performed at room temperature under an atmosphersic condition using a laser of 532 nm wavelength , a 600 lines per mm diffraction grating, and a ×100 objective (N.A. = 0.90). AgNCs/Au(111) was subsequently heated from room temperature to 111, 179, 222, 265, 395 and 482 °C in an UHV chamber (1 × 10−7 Pa). The sample was heated by resistive heating. After reaching the set temperatures, AgNCs/Au(111) was retained at the temperature for 20 min, followed by cooling to room temperature. After the annealing treatment, AgNCs/Au(111) was removed into the atmosphere, followed by SEM, AFM and Raman measurements. Photochemical reactions were induced at room temperature in atmosphere by laser irradiation to AgNCs/Au(111) using the laser equipped in the Raman spectroscope. The weakness of silver is low resistance to oxidation and sulfidation, which causes instability in the various physical and chemical properties.44 However, it has been reported that strongly adsorbed ligand molecules on metal are known to provide long-term stability to the particles, protecting them from oxidation.45 In this study, the surfaces of AgNC are always covered with PVP and/or decomposed products. Thus, we presume that contaminations of AgNC in air is sufficiently suppressed.100 nm-sized AgNCs were synthesized by the standard technique of the polyol method with reference to previous studies.3.27−1 are assigned to stretching modes of CH and CH2. A peak at 1607 cm−1 is assigned to the stretching mode of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O. Peaks between 800 and 1000 cm−1 are assigned to stretching modes of C–C and C–N. The details of the assignments of the vibrational peaks have been reported in the previous paper.27 Although the thickness of PVP is small (4–6 nm), the SERS effect of AgNCs enables us to observe vibrational peaks of PVP.27−1. This peak consists of two peaks at 2157 and 2214 cm−1, which are assigned to the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N stretching modes.46 This is strong evidence of the ring opening of pyrrolidone. Therefore, we consider that PVP starts to decompose at 111 °C. We presume that such a chemical change of PVP accompanies its structural change, leading to the increase of roughness.Next, this sample was annealed in UHV to induce the structural change of AgNCs. 38 This structural change is more clearly seen in the AFM image  of a pure PVP film in nitrogen atmosphere revealed a large reduction in mass due to decomposition and desorption of PVP at around 400 °C.2 stretching peak intensity as a function of annealing temperature. The intensity of the ν(CH2) peak decreased from room temperature to 111 °C because of the opening of pyrrolidone rings. At 179 °C, the G band appeared and PVP started to be transformed to nanocarbons. Further decrease and vanishment of ν(CH2) peak intensity at 179 and 222 °C, respectively, are associated with the decomposition of PVP. In contrast, the G band peak intensity increased owing to the formation of nanocarbons up to 265 °C. However, when the sample was annealed at 395 °C, the G band intensity significantly diminished by two orders of magnitude. One can note that the decrease in G band intensity stems from the decrease in the amount of nanocarbons. According to the previous TGA studies,49–52 nanocarbons start to desorb from the sample at around 400 °C. However, it was reported that almost 33% of nanocarbons remains on the metal nanoparticles after annealing at 500 °C.54 Thus, it is difficult to explain the drastic decrease in G band intensity at 395 °C solely by the decrease in the amount of nanocarbons.From SEM observations, we found that the AgNC gaps are difficult to identify when the annealing temperature exceeds 179 °C. 55 It has been reported that 10–100 nm-sized aggregates of AgNPs, i.e., Ag ink paste, coalesced at 200–250 °C.48,56–60 This coalescence of AgNPs occurs at a temperature significantly lower than the melting points of corresponding sizes of AgNPs61,62 and is widely known to be a result of sintering. According to the previous photoabsorption spectroscopy study, the irradiation of a 532 nm laser to AgNCs induces the excitation of the single-particle mode of AgNCs' LSP.27 This indicates that hot spots for SERS are preserved as long as the individual AgNC structures are maintained. However, when the sintering of AgNCs occurs, the G band intensity is expected to be suppressed because of the absence of the hot spots of AgNCs.63 As seen in the temperature dependence of the AgNC height  to the sample surface for 4 s (2 s × 2 times). We found that individual AgNCs are intact after the irradiation of the intense laser. This indicates that the intense laser used in this study would not have sufficient power to induce the structural changes of AgNCs. However, obvious changes were confirmed in the Raman spectrum after the irradiation of the intense laser, as represented by the appearance of D and G bands, as shown in i.e., PVP/Au(111), indicating that LSP-induced electric field enhancement at AgNCs is involved in this reaction process. D and G band intensities obtained after photoreaction . reaction  are apprreaction . This is4.Thermally and photoinduced structural changes of a PVP-covered AgNC array on Au(111) were investigated. Owing to the LB method, AgNCs are aligned in the face-to-face configuration, forming a two-dimensional monolayer. In the Raman spectrum obtained at room temperature, sharp peaks assigned to vibrational modes of PVP were observed as a result of the SERS effect of the AgNCs. When annealing AgNCs up to 179 °C, the alignment of AgNCs remained unchanged while the corners of individual AgNCs were rounded. Simultaneously, PVP was thermally converted to nanocarbons. The decreases in G band intensity and AgNC height indicated that the sintering of AgNCs occurred between 265 °C and 395 °C. Structural changes of AgNCs and the thermal desorption of nanocarbons lead to the elimination of nanocarbons from the AgNC nanogap, followed by the exposure of the clean Ag surface and finally the sintering of AgNCs. By irradiating an intense laser on the AgNC surface, we also confirmed the transformation of PVP to nanocarbons on AgNCs without the deformation of AgNCs. The strong peaks of both D and G bands appearing after the photoreaction are due to the SERS effect of AgNCs.There are no conflicts of interest to declare.


VP7/VP4 gene sequences of RVAs. G9P[8] was the most predominant strain in all four years but the prevalence of G2P[4] genotype increased rapidly since 2014. We reconstructed the evolutionary time scale of RVAs in Hubei, and found that the evolutionary rates of the G9, G2, P[8], and P[4] genotypes of RVA were 1.069 × 10−3, 1.029 × 10−3, 1.283 × 10−3 and 1.172 × 10−3 nucleotide substitutions/site/year, respectively. Importantly, using a molecular clock model, we showed that most G9, G2, P[8], and P[4] genotype strains dated from the recent ancestor in 2005, 2005, 1993, and 2013, respectively. The finding of the distribution of RVAs in infants and young children in Hubei Province will contribute to the understanding of the epidemiological characteristics and genetic evolution of RVAs in China.Group A human rotaviruses (RVAs) annually cause the deaths of 215,000 infants and young children. To understand the epidemiological characteristics and genetic evolution of RVAs, we performed sentinel surveillance on RVA prevalence in a rotavirus-surveillance network in Hubei, China. From 2013 to 2016, a total of 2007 fecal samples from hospital outpatients with acute gastroenteritis were collected from four cities of Hubei Province. Of the 2007 samples, 153 (7.62%) were identified positive for RVA by real-time RT-PCR. RVA infection in Hubei mainly occurred in autumn and winter. The highest detection rate of RVA infection was in 1–2 years old of outpatients (16.97%). No significant difference of RVA positive rate was observed between females and males. We performed a phylogenetic analysis of the G/P genotypes based on the partial  •A four-year study of sentinel surveillance program of RVAs was performed in Hubei, China.•The key population of rotavirus infection is 1–2 years old of outpatients with acute gastroenteritis.•G9P[8] was the most predominant strain between 2013 and 2016.•VP7 gene was 46 years.The estimating time to the most recent common ancestor for the G9 genotype based on partial •RVA distribution in Hubei Province contributes to the understanding of the epidemiological characteristics of RVAs in China. The viruses are packaged into a triple-layer capsid and divided into seven major groups based on VP6 gene, from groups A to G. Two proteins in the outermost layer of the viruses, VP4 and VP7, are essential for virulence, host specificity, and neutralizing antibody responses . Five genotype combinations, G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8], are prevalent in humans  are the main cause of acute gastroenteritis among children and annually cause 215,000 deaths of infants and young children worldwide . Even inHere, we performed a four-year study of sentinel surveillance program of RVAs in Hubei, China. We assessed the prevalence of G and P genotypes from RVA strains identified in this program and collected baseline data about circulating genotypes before the implementation of RVA vaccination program. We found that the key population of rotavirus infection was 1–2 years old of outpatients. Our findings could be exploited to understand the epidemiological characteristics and genetic evolution of RVAs in China.22.1This active surveillance was conducted in the gastrointestinal clinics of sentinel hospitals in four cities across Hubei Province . All outpatients visiting the clinics with symptoms of diarrhea (≥ 3 loose or watery stools within 24 h), vomiting or fever were included. The epidemiologic settings of this study for RVA infection included people of all ages that meet the case definition. From April 2013 to December 2016, a total of 2,007 stool specimens from outpatients with diarrhea were collected. All the specimens were kept frozen until testing.2.2g for 30 min after shaking. Viral RNA was then extracted from the supernatant using a magnetic bead prefilled DNA/RNA extraction kit  according to the manufacturer's instructions. The extracted viral RNA was immediately stored at −80 °C for further use. The viral RNA was detected for rotavirus using the AgPath-ID one-step RT-PCR kit  on an ABI 7500 real-time PCR platform . The previously published primers and probes were used (Stool specimens were diluted 1:10 with 1 × PBS (pH 7.4). The suspension was centrifuged at 3000×ere used . The rea2.3VP7 and VP4-coding genes of RVA were amplified using the primers, as previously described . The complete or partial escribed . The PCRectively .http://www.dnastar.com) . The G and P genotypes of RVA were determined from partial VP7 and VP4 sequences (452 and 572 bp) by phylogenetic analysis with MEGA 6.0 (http://tree.bio.ed.ac.uk/software/figtree/). G and P genotype reference strains with established genotypes were selected from GeneBank  model, a Bayesian skyline model for population growth was selected using the BEAST software. MCMC analyses were performed for 100 million generations, sampling each tree every 10,000 steps; the first 10% were discarded as chain burn-in. MCMC convergence and the effective sample size estimates (>200) were checked with the Tracer program Version 1.5. After a 10% burn-in, the maximum clade credibility (MCC) tree was generated by Tree Annotator Version 1.8.1 (http://beast.bio.ed.ac.uk/TreeAnnotator). The phylogenetic tree was viewed with FigTree 1.4.3. VP7 and VP4 sequences of G2P[4] RVA strains and G9P[8] RVA strains with clear collection date and genotype information were downloaded. Effective selection is made from the earliest time node (April 2013) to the latest time node (December 2016). The selected reference strains were showed in Using the Akaike information criterion, the best-fit models of nucleotide substitutions were selected by the jModelTest program Version 0.1.1 . The tim2.5χ2) test. A value of P < 0.05 indicates statistically significant differences. The data were analyzed by using SPSS Statistics for Windows, Version 19.0 .Clinical and epidemiological information, including gender, age, seasonality and date of sampling, was collected for all patients. The statistical analyses of different age groups were performed using the chi-square . Seasonal variation of RVAs prevalence was observed  A. In theobserved B. RVA in3.2χ2 = 30.123, P < 0.001) (The RVA-positive patients were comprised of 78 (10.58%) (78/737) children under one year of age, 28 (16.97%) (28/165) children in the 1–2 year age group, 5 (3.94%) (5/127) children in the 2–5 year age group, 3 (2.52%) (3/119) children in the 5–10 year age group, 7 (4.86%) (7/144) youth in the 10–25 year age group, 11 (4.06%) (11/271) persons in the 25–50 year age group, and 21 (4.73%) (21/444) people above 50 years of age . The hig 0.001) . Of the  0.001) .Table 1R3.3VP7 and VP4 genes for genotyping. Among the 153 RVA-positive samples, a total of 148 specimens were successfully amplified and assigned to phylogenetic analysis. Based on the phylogenetic analysis, the G genotype RVAs identified in this study were clustered into five genotypes: G9, G2, G1, G3, and G4  and 107 years , respectively, which dated the most recent ancestor back to 1975 and 1913  recommended two licensed rotavirus vaccines, RotaTeq®  and Rotarix®  in 2003, showing high efficacy against homotypic and heterotypic RVA strains in developed countries . Introdu5In this study, we performed a four-year study of sentinel surveillance program of RVAs in Hubei Province and found that the key population of rotavirus infection is 1–2 years old of outpatients with acute gastroenteritis. Our data showed that G9P[8] was the most predominant strain in all four years and all G9 genotypes of RVA strains have different evolution trend. This rotavirus surveillance can be used as an entry point to further determine the molecular epidemiological characteristics, major circulating genotypes, evolutionary histories, and evolutionary divergence times of RVAs in Hubei Province.All the data generated or analyzed during the current study are included in this published article or available from the corresponding author upon request.All institutional and National guidelines about the use of clinical samples were followed. The informed consent has been obtained from all participants and the studies have been approved by the Ethics Committee of the College of Life Sciences, Wuhan University.Ting Zhang: Conceptualization, Investigation, Methodology, Validation, Writing − Original draft preparation, Visualization. Jing Li: Investigation, Methodology, Validation, Formal analysis. Yong-Zhong Jiang: Investigation, Methodology. Jun-Qiang Xu: Investigation, Methodology. Xu-Hua Guan: Investigation, Methodology. Li-Qiang Wang: Formal analysis, Data curation, Validation. Jie Chen: Project administration, Resources. Yi Liang: Supervision, Project administration, Funding acquisition, Conceptualization, Investigation, Methodology, Validation, Writing- Original draft preparation, Writing − Reviewing and Editing.The authors declare that they have no conflict of interest.