The as-prepared Mo5O14-type/C material is compared to α-MoO3 synthesized from the same precursor in air. The morphology and microstructure of the as-prepared samples are characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman scattering (RS) spectroscopy. Thermal gravimetry and elemental analysis indicate the presence of 25.8 ± 0.2% of carbon in the composite. The SEM images show that Mo5O14 is immersed inside a honeycomb-like carbon matrix providing high surface area. The RS spectrum of Mo5O14/C demonstrates an oxygen deficiency in the molybdenum oxide and the presence of a partially graphitized carbon. Outstanding improvement in electrochemical performance is obtained for the Mo5O14 encapsulated by carbon in comparison with the carbon-free MoO3.An amorphous Mo They crystallize in a unique layered structure  and a rutile-like structure (monoclinic P21/c space group), respectively [mO3m−1 phases include Mo4O11, Mo5O14, Mo8O23, Mo9O26, and Mo17O47, which crystallize in the so-called Magnéli phases described in details by Kihlborg [3 by shear mechanism with networks of MoO6 octahedra. These units are connected in three dimensions with empty parallel tunnels involving the presence of potential sites for hosting Li+ ions. They are performant cathode materials in non-aqueous lithium cells [5O14 are available in the literature [5O14 crystal suggest that the unit cell expands in the b direction and contracts in both the a and c directions upon insertion of lithium [−1 for Mo5O14 as the positive electrode of a primary Li cell discharged at 0.5 mA cm−2 and 1.0 V cut-off. [2.8, an oxygen-deficient Mo5O14 prepared by dehydration and annealing treatment of molybdic acid powder (MoO3·1H2O) at 750 °C, exhibited a high electrical conductivity of 10−2 S cm−1. Such a material displayed an excellent reversible capacity (1.45 Li/Mo) in cells with 1 mol L−1 LiClO4 in propylene carbonate (PC) electrolyte in the potential range 3.3–1.3 V vs. Li+/Li.Due to their numerous applications including their vital role as light weight, long life and high-energy density power sources, special attention has been directed towards lithium-ion batteries (LIBs). These power sources find major applications from portable electronic appliances to electric vehicles. As the positive electrode plays a major role in the electrochemical performance of the LIBs, many efforts of research are currently made on active electrode materials with high capability and long cycling life . Among tterature ,11. Prelcut-off.  showed t3 and MomO3m−1 lithiated oxides suffer from a large volume expansion (about 104% for MoO3). During Li+ insertion, pulverization of the cathode occurs at a potential of less than 1.5 V and disconnection from the current collector. As a result, the electrochemical performance is poor [mO3m−1 particles were prepared with low crystallinity [3/C composites: electrospinning method [However, MoO is poor . Differeallinity ,14. Blenallinity . Composiallinity ,17,18,19g method ,16,19,20g method , spray pg method  and ballg method .mO3m−1/carbon is synthesized by a sol-gel method using citric acid as chelating agent. The calcination of dried precursor in argon atmosphere results in the formation of the amorphous-like Mo5O14 (MoO2.8) oxide immersed in a carbon matrix. This new material is characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), surface area analysis (BET) and Raman scattering (RS) spectroscopy. Finally, electrochemical properties of MoO3/C composite for lithium ion batteries were also investigated for comparison.In this paper, a nanocomposite of oxygen-deficient Mo3 (P-MoO3) sample and MomO3m−1/C nanocomposite were prepared by a citrate–gel method using ammonium molybdate tetrahydrate 6Mo7O24∙4H2O). First, a stoichiometric amount of AMT was dissolved in de-ionized water (DI) and stepwise added to the stirring aqueous solution of citric acid (CA) with metal/CA ratio of 1:1 under neutral pH adjusted with ammonium hydroxide; Second, the overall solution was stirred for 3 h to form homogenous mixture through reaction between the metal ion and the chelating agent. Transparent gel was formed after a slow evaporation of the solution heated at 70 °C. Furthermore, continuous heating with stirring led to the transformation of the gel to xerogel, which converted to powder by further drying at ca. 120 °C. Pure MoO3 crystals were obtained by calcining the milled precursor at 500 °C for 5 h in air, while the MomO3m−1/C composite was formed under argon atmosphere. The overall synthesis process is presented in Pure MoO−1. Elemental analysis was performed using a CHNOS analyzer . The structure of the samples was analyzed by X-ray diffraction (XRD) using a X’Pert diffractometer  equipped with a CuKα X-ray source (λ = 1.54056 Å). Data were collected in the 2θ range of 10–80°. The particle morphology was examined by scanning electron microscopy (SEM) . Raman spectra were collected at a spectral resolution of 2 cm−1 with a double monochromator (Jobin-Yvon model U1000) using the 514.5 nm line of an Ar-ion laser (Spectra-Physics 2020) in a backscattering geometry. The laser power was kept below 25 mW to prevent the sample degradation by the laser spot. The specific surface area was measured by nitrogen adsorption/desorption at 77 K using the Brunauer-Emmett-Teller (BET) method (Quantachrome NOVA Automated Gas Sorption).The composition was determined by thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) using an analyzer model TGA-7 series  in the temperature range of 30–1000 °C in air at a heating rate of 10 °C minw/w) of the active material, 10% (w/w) super C65 carbon  and 10% (w/w) polyvinylidenefluoride , in N-methyl-2-pyrrolidone  to get a slurry, which was coated on copper foil at a loading of 2.1 mg cm−2. After drying over night at 80 °C, discs were punched out of this film with a diameter of 1.4 cm. The 2032-type coin cells were assembled in an argon-filled glove box with lithium foil  as the anode, 1 mol L−1 LiPF6 in ethylene carbonate:dimethyl carbonate (EC:DMC) = 50:50 (v/v)  as the electrolyte, and glass microfiber filters (Whatmann®-GF/D 70 mm Ø) as the separator. A multi-channel potentiostat  was used to test the electrochemical performance of the electrodes at 25 °C in the voltage range of 0.6–3.5 V vs. Li+/Li.Electrochemical properties were investigated by cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) techniques. The cathodes were prepared by mixing 80%  reported by Kihlborg [6+3Mo5+2. This valence mixing in turn implies a high concentration of free carriers and thus a large electrical conductivity, which will be beneficial to the electrochemical properties. The crystal structure of Mo5O14 can be depicted as complexes of linked MoO6 octahedra and MoO7 pentagonal bipyramids [θ = 25.7° corresponds to the overlapping (001), (060) and (504) lines of the Mo5O14 lattice, while the less intense peaks at 2θ = 35.1° and 43.5° coincide with the (541) and (002) Bragg lines of the tetragonal structure, respectively [θ = 25.7° is found to be 2.9 nm. The presence of the disordered carbon (low degree of graphitization) is evidenced by the main signal at 2θ ≈ 26° ((002) Bragg line) probably overlapping with the main signals of the Mo5O14 phase. However, the amorphous nature of carbon deposited on the Mo oxide at a low temperature of 500 °C is confirmed by the absence of the (100) and (101) carbon-related peaks in the 40–45° 2θ range [5O14/C composite under argon gas is due to the presence of CO and CO2 gases generated by the combustion reaction of the carbon source using citric acid, which reduces the Mo6+ ions of the ammonium molybdate. The TGA experiments performed during the synthesis (6H8O7 accompanied by the decomposition of (NH4)6Mo7O24  into oxides. At this stage, a weight loss of almost 50% was recorded.Kihlborg . Note thectively . The cry2θ range . The forynthesis a present3 (3 phase that can be indexed in the orthorhombic Pbnm space group (JPCDS card 76-1003). The coarse-grained powder presents a preferred orientation of (0k0) planes, as the (020), (040) and (060) Bragg lines have a large intensity. Rietveld refinement provided lattice constants of a = 0.3965 Å, b = 1.3861 Å, and c = 0.3697 Å.The XRD pattern of P-MoO3 b display5O14/C composite synthesized by sol-gel method was determined using thermal gravimetry (TG) and the differential scanning colorimetric (DSC) experiments. 3 and the Mo5O14/C composite, respectively, recorded in the range 25–1000 °C in air. The thermal behavior of P-MoO3 does not show obvious weight loss until the decomposition of MoO3 (melting point at ca. 795 °C) [2 through reaction between carbon in the composite and the atmospheric oxygen. Thus, the composite contains 26% carbon coming from the pyrolysis of citric acid. Upon further heating, the small exothermic peak appearing at ca. 800 °C is attributed to the melting of Mo5O14 in the composite similarly to the feature observed for pure MoO3 crystal. Further elemental determination of carbon was performed using a CHNOS analyzer that gave the same percentage of carbon (25.8 ± 0.2%) in the composite.The carbon content of the Mo 795 °C) . The sit5O14/C composite was analyzed by scanning electron microscopy and compared with that of pure MoO3 synthesized from the same precursor  of the Mo5O14/C composite display a honeycomb carbon matrix with small pores, and irregular particles of molybdenum oxide are dispersed inside the matrix.The morphology of the Morecursor . While t5O14/C composite using Raman spectroscopy. 3 (a) and the Mo5O14/C composite (b). The Raman spectrum of Mo5O14/C composite prepared at 500 °C in argon atmosphere can be considered as the sum of the Mo5O14 and carbon contribution. In the low-frequency region, the spectrum shows features of the molybdenum oxide phase, while the high-frequency range (greater than 1200 cm−1) displays the fingerprint of the carbon matrix. Note that the weak intensity of Mo5O14 Raman pattern is due to the presence of carbon environment absorbing the scattering light. The five low-frequency Raman bands located at 972, 780, 700, 430 and 225 cm−1 can be identified as the vibrational patterns of tetragonal Mo5O14. The Mo-O stretching mode appears at 972 cm−1, the Mo-O-Mo bridging modes are recorded at 780, 700 and 430 cm−1, while the deformation modes of the Mo-O bonds appear at ca. 225 cm−1. This deformation mode was observed at 229 cm−1 in Mo4O11. It should be noted that this Raman spectrum definitely differs from those of either α-MoO3  = 4.4 ID/IG [5O14 active material.Since XRD experiments are not efficient tools to determine the structure of ill-crystallized materials, we investigated the short-range local structure of Mor α-MoO3 a or Mo4O11 phase ,29. For acancies  showed tistances . The str.4 ID/IG . The lim3 and Mo5O14/C composite. The uptake of nitrogen adsorption of both samples increases gradually with the increase of relative pressure  behavior for pristine MoOnsertion ,38. The t cycles a. Such at cycles . The mesV curves b.3 and Mo5O14/C composite electrodes, respectively, recorded at C/10 rate (70 mA g−1). In a fresh cell, pristine MoO3 and Mo5O14/C composite are electrodes in a “charged state” with all Mo ions with the 6+ oxidation state. Thus, the first process of lithiation occurs during discharge (reduction of Mo to 5+ and 4+ states) providing a capacity Q. During the subsequent charge Q’, Mo is oxidized, but not all Li ions can be extracted from the lithiated phase, thus less electrons are transferred and Q’<Q. Definitely, the electrochemical behaviors of these electrodes are different. The difference between the shape of charge/discharge curves of pristine MoO3 and Mo5O14/C is mainly due to the difference in the structure and morphology of the active material. In contrast with the 2D structure of α-MoO3, Mo5O14 has a 3D structure with empty site in the tunnels inducing a different Gibbs energy that results in unlike voltage profile. In addition, the voltage profile of an amorphous material is a S-shape curve due to the random distribution of the available site in the framework.3 shows two typical plateaus , which induces a pronounced expansion of the interlayer spacing (from 0.69 to 1.175 nm) [2O5 host [3 electrode decreases continuously over subsequent discharge-charge cycles from 330 mAh g−1 (1st cycle) to 136 mAh g−1 (25th cycle). This large capacity loss (2% per cycle) reveals the poor electrochemical stability of the α-MoO3 phase prepared by citrate-gel method. In contrast, the mesoporous Mo5O14/carbon composite exhibits better electrochemical performance. GCD curves are featureless (without plateau) showing a steady decay of the cell voltage upon Li insertion. Such discharge-charge profiles are characteristic of an amorphous phase, which implies a wide variation in energy for the Li sites available for insertion [−1, decreasing to 388 mAh g−1 at the 2nd cycle. During next cycles, the discharge capacities slightly decrease to reach 325 mAh g−1 at the 50th cycle. The cyclability of pristine MoO3 and Mo5O14/C composite electrodes was tested at C/10 rate in the cell voltage range of 0.6–3.5 V as shown in 5O14/C composite shows an almost stable electrochemical behavior up to 85 cycles with a rate of capacity fade of 0.8% per cycle. Results of the rate capability are presented in 5O14/C composite is clearly revealed with a specific discharge capacity of 155 mAh g−1 at 10C rate (7 A g−1).In the first discharge process, the crystallized pristine MoOplateaus a, in accplateaus a. The in.175 nm) ,40. Simi2O5 host . The spensertion ,43. For mO3m−1 and carbon have been examined for the first time. MomO3m−1 compounds are particularly interesting, mainly due to the structure with large tunnels accessible for Li+ ions. In addition, they have a high electrical resistivity, i.e., ρ = 1 Ω cm for Mo8O23 and ρ = 78 Ω cm−1 for Mo18O52 that are semiconductors, while η-Mo4O11 is metallic with ρ = 1.7 10−4 Ω cm [3 obtained by the decomposition of citric acid as chelating agent and ammonium molybdate tetrahydrate as raw material has been performed in closed atmosphere. The as-prepared mesoporous Mo5O14/C composite was successfully obtained by a moderate annealing temperature of 500 °C. This new material is composed of a honeycomb carbon matrix, in which amorphous Mo5O14 nanoparticles are introduced. It is assumed that the Mo6+ ions were reduced by the presence of CO, CO2 and NH3 gases coming from the chelate and raw material forming the non-stoichiometric (oxygen-deficient) Mo5O14/C compound. This tunnel-like structure accommodates oxygen deficiency via oxygen vacancies and crystallographic shear planes. The formation of an intermediate compound MomO3m−1 between MoVIO3 and MoIVO2 during the reduction of MoO3 has been reported many times [The structural and electrochemical properties of a composite formed of oxygen-deficient Mo0−4 Ω cm . In the ny times ,45,46, b3, the excellent electrochemical behavior of the Mo5O14/C composite electrode is due to the formation of large mesopores. The unique cycling performance of the composite is attributed to the presence of carbon, which plays a dual role in enwrapping the tunnel-like Mo5O14 nanoparticles and accommodating the volume change. Graphitized carbon provides a large contact surface and acts as an excellent conductive agent facilitating the charge-discharge process. The porosity formed by carbon facilitates the transport of charge carriers and the delocalized insertion site in the amorphous matrix enhances the kinetics of Li+ ions. According to its high reversible capacity and good cycling stability, the Mo5O14/C composite is a promising alternative electrode material for application in lithium-ion batteries.Also, this work illustrates the synergy between a molybdenum oxide and carbon that exhibits excellent electrochemical performance of the composite. Also, it reveals the importance of electronic conductivity and the mesoporous nature for substantial lithium insertion in tunnel-like molybdenum-oxide compounds. In contrast to the large capacity fade due to the interlayer spacing expansion in crystalline α-MoOmO3m−1 compounds compared with stoichiometric MoO3. In an early work, Besenhard and Schöllhorn [18O52 and Mo8O23 electrodes in organic Li+ electrolytes occur via reversible topotactic redox processes. Cignini et al. examined the performance of various non-stoichiometric MomO3m−1 compounds as cathodes in Li cells, i.e., Mo8O23, Mo9O26, Mo17O47 and Mo4O11 and showed that Mo5O14 can deliver a specific capacity of 310 mA g−1 when discharged at 0.5 mA cm−2 in the voltage range 2.5–1.0 V [4O11 (MoO2.75), Mo17O47 (MoO2.765), Mo8O23 (MoO2.889), and Mo9O26 (MoO2.875) the highest reversible capacity (1.5Li/Mo) was obtained for Mo17O47 after several deep discharge-charge cycles in the potential range 2.9–1.4 V. The reversible capacities of other MomO3m−1 compounds are considerably less than that of Mo17O47 except for Mo4O11, which exhibits a reversible capacity of 0.75Li/Mo after 20 deep cycles. These results suggest that both channel/site size and electronic conductivity are the predominant factors influencing the extent of reversible Li insertion by MomO3m−1 oxides with tunneled framework. Distortions and nonequivalence of the available sites should also affect the thermodynamics of the reduction processes. Julien et al. showed that the first discharge profile of a crystalline LixMoO2.8 (lithiated Mo5O14) displays a stepped behavior with a voltage plateau at ca. 2.2 V followed by a potential decline for x > 0.7Li/Mo and a total Li uptake of 1.45Li/Mo for a cut-off voltage of 1.0 V. For all Mo ions reduced to Mo4+, the expected lithium uptake would be 8/5(1.6) Li/Mo; a value close to 258 mAh g−1 measured by electrochemical titration [2.895 prepared by ball milling delivered a specific of ca. 600 mAh g−1 that decreased to 420 mAh g−1 (2nd cycle) in the voltage range 3.5–0.5 V. For comparison, electrochemical properties of composites formed by a Mo oxide and a carbonaceous material are listed in Several studies have demonstrated the improved cycling performance of oxygen-deficient Mohöllhorn  have sho5O14/C composite are 703 mAh g−1 and 55%, respectively. In the same voltage range, a composite electrode made of MoO3 nanocrystals distributed in an amorphous carbon matrix delivered a specific capacity of approximately 630 mAh g−1 and an initial Coulombic efficiency of 75% [−1 with a Coulombic efficiency of 69% was reported for crumpled graphene–MoO3 composite cycled at a constant current density of 2 A g−1 [5O14-type/C composite electrode is attributed to the accommodated volume expansion. Christian et al. [5O14 crystal expands in the b direction and contracts in both the a and c directions upon insertion of lithium. Also, we believe that the presence of 26 wt.% carbon homogeneously distributed and associated with the intrinsic properties of the active particles minimizes the volume expansion.According to the GCD profile presented in y of 75% . An init 2 A g−1 . Further3 and mesoporous Mo5O14-type/C composite were successfully prepared by a citrate–gel method from ammonium molybdate precursor. Crystalline orthorhombic α-MoO3 is observed for xerogel calcined in air, while the Mo5O14/C composite is prepared under argon atmosphere. The latter one is a poorly crystalline material that is composed of Mo5O14 nanograins incorporated into a carbon matrix as noticed from X-ray diffraction and Raman spectroscopy. The carbon content of approximately 26 wt.% was estimated from TGA and elemental analysis. Analysis of the vibrational features of D- and G- Raman bands of the carbon reveals that it is moderately graphitized. BET measurements show that the Mo5O14/C composite exhibits a surface area of 7.1 m2 g−1, average pore size of approximately 4 nm and total pore volume of 0.07 cm3 g−1, which are favorable for enhanced electrochemical insertion of Li+ ions into the mesoporous lattice.Pure α-MoO5O14/C composite appears to be electrochemically stable showing a specific capacity of 325 mAh g−1 and a capacity fade of 0.8% after 50 cycles at 0.1C. Such a good performance is attributed to the mesoporous nature of the electrode material and the presence of carbon, which suppresses the aggregation of molybdenum-oxide particles and thus increases their structural stability during cycling, while keeping the particles electrically connected.The Mo


The scalable graphitic carbon quantum dots (CQDs) are obtained with a high yield of more than 50%.2S4 composite which shows enhanced electrochemical properties.The CQDs are utilized to synthesize novel NS-CQDs/NiCoThe assembled novel alkaline aqueous battery delivers superior electrochemical performances.The online version of this article (10.1007/s40820-019-0355-0) contains supplementary material, which is available to authorized users. 2S4 composite cathode materials, manifesting the enhanced electrochemical properties resulted from the synergistic effect of highly conductive N,S-codoped CQDs offering fast electronic transport and unique micro-/nanostructured NiCo2S4 microspheres with Faradaic redox characteristic contributing large capacity. Moreover, the nitrogen-doped reduced graphene oxide (N-rGO)/Fe2O3 composite anode materials exhibit ultrahigh specific capacity as well as significantly improved rate property and cycle performance originating from the high-capacity prism-like Fe2O3 hexahedrons tightly wrapped by highly conductive N-rGO. A novel alkaline aqueous battery assembled by these materials displays a specific energy (50.2 Wh kg−1), ultrahigh specific power (9.7 kW kg−1) and excellent cycling performance with 91.5% of capacity retention at 3 A g−1 for 5000 cycles. The present research offers a valuable guidance for the exploitation of advanced energy storage devices by the rational design and selection of battery/capacitive composite materials.Carbon quantum dots (CQDs) as a new class of emerging materials have gradually drawn researchers’ concern in recent years. In this work, the graphitic CQDs are prepared through a scalable approach, achieving a high yield with more than 50%. The obtained CQDs are further used as structure-directing and conductive agents to synthesize novel N,S-CQDs/NiCoThe online version of this article (10.1007/s40820-019-0355-0) contains supplementary material, which is available to authorized users. In partpability . Since tpability –14, whicCurrently, the methods for the synthesis of CQDs are usually classified into “top-down” and “bottom-up” types. The former is usually breaking down large-size carbon materials into CQDs, including arc discharge, electrochemical exfoliation, hydrothermal technique and chemical oxidation synthesis –18, wher2S4 is seen as the most prospective candidate for future utilization in such energy storage as supercapacitors and lithium ion batteries. To further enhance its comprehensive electrochemical performances, an effective way is to design various unique micro-/nanostructured NiCo2S4, and the enhanced electrochemical properties have been reported by researchers in the literature [Besides, another major challenge for CQDs is that they are apt to aggregate and stack together, which results in a rapid decrease in their specific surface areas and kinetic behaviors, thus worsening the electrochemical properties of CQDs . These oterature –33. Anotterature , 35. The2S4 microspheres for the first time. In comparison with the pure NiCo2S4 electrode, the N,S-CQDs/NiCo2S4 composite electrode, when for the first time used as cathode for alkaline aqueous batteries, exhibits enhanced electrochemical properties with a high specific capacity of 124.4 mAh g−1 at 2 A g−1, exceptional capacity retention of 77.3% at 50 A g−1 in comparison with 2 A g−1 and excellent cycling property with 97.9% of initial capacity retention at 3 A g−1 for 5000 cycles. Meanwhile, the N-rGO-wrapped prism-like Fe2O3 hexahedrons composite anode materials are obtained, which manifest substantially enhanced electrochemical performances with an ultrahigh specific capacity (249.6 mAh g−1 at 1 A g−1) as well as greatly enhanced rate property and cycling life compared with those of the pristine Fe2O3 electrode. A novel N,S-CQDs/NiCo2S4//N-rGO/Fe2O3 alkaline aqueous battery fabricated by these materials achieves a specific energy (50.2 Wh kg−1) and ultrahigh specific power  with excellent cycle property (91.5% of capacity retention for 5000 cycles). The encouraging results display the enormous potential of the new alkaline aqueous battery as highly promising energy storage system for potentially practical applications.As discussed above, the scalable graphitic CQDs were first synthesized with a high yield of more than 50% utilizing highly conductive Super P powders as precursors. Then, the CQDs were employed as both structure-directing and conductive agents to prepare new N,S-codoped CQDs-decorated urchin-like NiCo2SO4/HNO3 mixed solution  and then sonicated for 1 h. The resulting mixture was refluxed for 24 h. Subsequently, the mixture was further neutralized with Na2CO3·10H2O, and the pH was controlled at 7.0. After further dialysis and vacuum-drying, the final CQDs powders were obtained.CQDs were obtained by a scalable chemical oxidation approach. Super P powders  were first added into concentrated H2S4 composite materials were synthesized by a one-step hydrothermal approach. Firstly, CQDs powders (22.3 mg) were dissolved in H2O/ethanol mixed solution  and agitated for 10 min. Subsequently, NiCl2·6H2O (0.06952 g) and CoCl2·6H2O (0.13919 g) were added into the obtained mixture and stirred for 20 min. Then, thioacetamide (0.13185 g) was further introduced into the above mixture. After continual agitating for another 20 min, the as-resulted mixed solution was transferred into a stainless steel autoclave lined with PTFE (100 mL) and hydrothermally treated at 180 °C for 24 h. The products were obtained by alternate centrifuging with H2O and ethanol for three times and further vacuum-dried overnight at 70 °C. For comparison, the pure NiCo2S4 was synthesized by the similar method only without adding CQDs powders in this preparation process.The N,S-CQDs/NiCo2O3 composite materials were prepared by a facile hydrothermal approach. Firstly, graphite oxide  was dissolved in H2O solution (30 mL) and sonicated for 1 h. Then, FeCl3·6H2O (0.5406 g) was dissolved in H2O solution (15 mL) and stirred for 10 min. The as-resulted FeCl3 aqueous solution was added into the GO solution and sonicated for 30 min. Meanwhile, urea (0.6006 g) was dissolved in H2O solution (15 mL) and magnetically stirred for 10 min. Furthermore, the resulting urea solution was slowly introduced into the above mixture under strong agitation. After stirring for 30 min, the as-obtained mixture was transferred into a stainless steel autoclave lined with PTFE (100 mL) and subjected to hydrothermal reaction at 180 °C for 10 h. Lastly, the products were obtained by alternately centrifuging with H2O and ethanol for three times and further vacuum-dried overnight at 70 °C. For comparison, the pure Fe2O3 was synthesized by the similar method only in the absence of GO in this preparation process.The N-rGO/Fe+). Raman spectra were executed on a Raman spectrometer (Labram-010). X-ray photoelectron spectroscope (XPS) analysis was conducted on a photoelectron spectrometer (K-Alpha). FESEM images, EDS and elemental mappings were captured on a field emission scanning electron microscopy . Transmission electron microscope (TEM) images, HRTEM images and SAED patterns were obtained on a transmission electron microscopy (JEM-2100F). TG curves were recorded from a thermal analysis instrument (NETZSCH STA449C). N2 adsorption/desorption isotherms were acquired on a BET specific surface area analyzer (BELSORP-miniII).X-ray diffraction (XRD) analysis was implemented on an X-ray diffractometer .To fabricate the working electrodes for electrochemical tests, 70 wt% of electroactive material, 20 wt% of Super P and 10 wt% of PVDF with NMP as solvent were adequately mixed to obtain an uniform slurry. Afterward, the resulting slurry was dipped into Ni foam current collector, which was pretreated based on our previous work [2S4 composite cathode and N-rGO/Fe2O3 composite anode are demonstrated in Fig. 2S4 microspheres composite cathode materials via a one-step hydrothermal approach, in which thioacetamide is used as precipitant to generate the NiCo2S4 and codoped agent to achieve nitrogen and sulfur codoping into the CQDs. In this process, the CQDs are not only utilized as structure-directing agents to induce the formation of nanorods-assembled urchin-like composite microsphere structure, but also served as conductive agents to improve the electrical conductivity of the composite cathode materials. Meanwhile, the N-doped rGO-wrapped prism-like Fe2O3 hexahedrons composite anode materials are prepared by a facile hydrothermal synthetic route, where urea serves as precipitant to generate the Fe2O3 and acts as doped agent to achieve nitrogen doping into the GO. The Fe2O3 hexahedrons are tightly wrapped by the N-rGO sheets, and the close contact between the Fe2O3 hexahedrons and the N-rGO sheets can improve the electrochemical kinetic behavior and structural stability of the composite anode materials.The synthetic processes of the graphitic CQDs, the N,S-CQDs/NiCo2SO4/HNO3 mixed solution after the treatment of sonication. However, the digital photograph  reveals that the obtained CQDs are highly soluble in H2O solution, and also the resulted CQDs aqueous solution does not show any change after 6 months at ambient temperature as manifested in Fig. Figure −1 are well fitted to the C–OH, C = O, C = C, C–H and C–O–C bonds [−1 belong to the D-band and G-band of CQDs, which are attributed to the disordered carbon and ordered graphitic structure, respectively [Figure –C bonds , 39, resectively . XPS wasectively . Figure 2S4 composite were examined by FESEM, TEM, HRTEM, EDS and elemental mappings. As demonstrated in Fig. 2S4 where only the microspheres with irregular and larger sizes are obtained as exhibited in Fig. S3, which is ascribed to the induced role of CQDs during the formation process of the composite. The micro-/nanostructured morphology of the composite was further surveyed by TEM and HRTEM. The TEM images  and (400) planes of NiCo2S4. In addition, the EDS analysis , suggesting that the structure of the composite is not changed in the combination of N,S-CQDs and NiCo2S4. However, it is hard to notice the peak of N,S-CQDs due to the relatively low content and weak diffraction peak intensity. Raman spectra were used to further characterize the structure of the pristine NiCo2S4 and N,S-CQDs/NiCo2S4 composite , one Eg (472 cm−1) and one Ag1 (675 cm−1) Raman modes of NiCo2S4 [−1 are characteristic Raman modes of carbon, which are seen from the Raman spectroscopy of the N,S-CQDs/NiCo2S4 composite.XRD was employed to examine the phase and structure of the pristine NiCo NiCo2S4 . Moreove2S4 composite, as presented in Fig. 2+, and another two peaks situated at 854.24 and 876.04 eV are indexed to Ni3+, respectively [2+, and another two peaks situated at 778.74 and 793.79 eV can be attributed to Co3+, respectively [2− in low coordination at the surface and the metal–sulfur bonds [2S4 composite.XPS was further undertaken to study the elemental composition and valence state of the as-synthesized N,S-CQDs/NiCoectively . Similarectively . The S 2ur bonds . For theur bonds . Based o2S4 composite, the thermogravimetric analysis (TGA) was executed in air atmosphere from ambient temperature to 900 °C as demonstrated in Fig. 2S4 and N,S-CQDs/NiCo2S4 composite both show two apparent weight losses and one obvious weight gain. For the N,S-CQDs/NiCo2S4 composite, the first weight loss before 500 °C is more obvious than that of the pristine NiCo2S4, which results from the burning of CQDs and the loss of physically adsorbed and crystalline water. The second apparent weight gain between 500 and 650 °C is attributed to the oxidation of NiCo2S4 to NiSO4 and CoSO4, and the third evident weight loss in the temperature range of 650 and 800 °C is ascribed to the further burning of CQDs as well as the decomposition of NiSO4 and CoSO4 to stable NiO and CoO as reported in the literature [2S4 and N,S-CQDs/NiCo2S4 composite, it is deduced that the content of CQDs in composite is about 15.9 wt%. N2 adsorption/desorption test was employed to research the specific surface area of the pristine NiCo2S4 and N,S-CQDs/NiCo2S4 composite, as presented in Fig. 2S4 composite manifests a greater specific surface area (27.2 m2 g−1) than that of the pristine NiCo2S4 (14.9 m2 g−1), which contributes to the enhancement of electrochemical properties.To further determine the composition of the prepared N,S-CQDs/NiCoterature –45. From2S4 and N,S-CQDs/NiCo2S4 electrodes were investigated in a three-electrode configuration with 2 M KOH solution as electrolyte. Figure 2S4 and N,S-CQDs/NiCo2S4 electrodes at 20 mV s−1 in the potential range of 0 to 0.6 V. The two electrodes both manifest strong and broad redox peaks in the CV curves, implying the combination of Faradaic battery and double-layer capacitive behavior. The Faradaic battery behavior is derived from reversible redox reactions of Ni2+/Ni3+ and Co2+/Co3+/Co4+ according to Eqs. The electrochemical performances of the NiCo2S4 composite during different electrochemical steps, as exhibited in Fig. 2S4 electrode is greater than that of the NiCo2S4 electrode, revealing that the N,S-CQDs/NiCo2S4 electrode shows higher specific capacity. Figure 2S4 and N,S-CQDs/NiCo2S4 electrodes at different scan rates, respectively. The N,S-CQDs/NiCo2S4 electrode manifests a smaller peak position shift than that of the NiCo2S4 electrode as the scanning speed increases from 5 to 100 mV s−1, suggesting that the composite electrode has lower polarization, better conductivity and faster electrochemical kinetic. To further explore the difference of the electrochemical behaviors of the NiCo2S4 and N,S-CQDs/NiCo2S4 electrodes, the quantitative analysis was conducted based on the mechanism of charge storage. The relationship of the peak currents (ip) and scan rates is expressed as I = avb shown in Fig. b-value of 0.5 and 1 suggests the diffusion-controlled and surface capacitive processes of the electrochemical reaction, respectively. The calculated b-value of the NiCo2S4 and N,S-CQDs/NiCo2S4 electrodes is, respectively, 0.80 and 0.89, indicating a mixed behavior of diffusion-controlled and surface capacitive processes. The higher b-value of the N,S-CQDs/NiCo2S4 electrode signifies more surface capacitive behaviors. Furthermore, the result is further verified by the quantitative calculation of the contribution of the diffusion-controlled and surface capacitive charge, which is obtained from the following equation [i(V) = k1v + k2v1/2, in which k1v represents the surface capacitive current and k2v1/2 denotes the diffusion-controlled current. The fitted lines of the i/v1/2 to v1/2 of the two electrodes are presented in Fig. k1 and k2. The charge contributions of the NiCo2S4 and N,S-CQDs/NiCo2S4 electrodes are manifested in Fig. 2S4 electrode displays larger capacitive contribution ratio than that of the NiCo2S4 electrode at all scan rates, which reasonably elucidates why the N,S-CQDs/NiCo2S4 electrode possesses faster electrochemical kinetic than that of the NiCo2S4 electrode.Ex situ XRD measurement was utilized to characterize the phase change of the N,S-CQDs/NiCoequation : i(V) = 2S4 and N,S-CQDs/NiCo2S4 electrodes at different current densities in the potential window of 0–0.5 V, respectively. The GCD curves of the N,S-CQDs/NiCo2S4 electrode present a better symmetry than that of the pristine NiCo2S4 electrode, signifying that the composite electrode has lower resistance and higher rate performance. The specific capacities of the two electrodes are calculated according to the GCD curves, as exhibited in Fig. 2S4 electrode is, respectively, 124.4, 122.4, 118.6, 112.8, 106.4 and 96.1 mAh g−1 at 2, 5, 10, 20, 30 and 50 A g−1, which is better than that of the NiCo2S4 electrode (from 92.8 mAh g−1 at 2 A g−1 to 59.1 mAh g−1 at 50 A g−1). Correspondingly, the N,S-CQDs/NiCo2S4 electrode manifests a greater capacity retention of 77.3% at 50 A g−1 than that of the NiCo2S4 electrode (63.7% of capacity retention at 50 A g−1). The improved specific capacity and rate performance of the N,S-CQDs/NiCo2S4 electrode originate from the positive synergistic effect between highly conductive N,S-CQDs with the capacitive feature and high-capacity NiCo2S4 with Faradaic characteristic. Note that the specific capacity and rate performance of the as-obtained N,S-CQDs/NiCo2S4 composite are comparable or even higher than those of nickel cobalt sulfide-based composite as reported in the literature [2S4 and N,S-CQDs/NiCo2S4 electrodes was surveyed by the repeated GCD tests at 5 A g−1 in the potential range of 0–0.5 V over 5000 cycles, as exhibited in Fig. 2S4 electrode manifests a better cycle property with 97.9% of the initial capacity retention than that of the NiCo2S4 electrode (90.3%), revealing the excellent cycling stability of the as-resulted composite electrode. Additionally, the EIS test was utilized to illustrate the superior electrochemical behaviors, which was implemented in the frequency range of 0.01 Hz to 100 kHz at the open-circuit potential . Note that the characteristic peaks of rGO cannot be observed in the XRD pattern of the composite, which results from the weak and broad peak and low rGO content in composite. Raman spectra were used to further examine the structure of the pristine Fe2O3 and N-rGO/Fe2O3 composite  and three Eg  Raman modes of Fe2O3 [−1 are assigned to the D-band and G-band, respectively, further suggesting that the Fe2O3 is successfully combined with the N-rGO to form the composite.XRD was utilized to further characterize the structure and phase of the pristine Feite Fig. f. The XRof Fe2O3 . Besides2O3 composite, as presented in Fig. 2O3 composite. For the Fe 2p spectrum  than that of the pristine Fe2O3 (13.8 m2 g−1), and thus, it is expected that the N-rGO/Fe2O3 composite can present excellent electrochemical performances.The content of GO in N-rGO/Fe2O3 and N-rGO/Fe2O3 composite were also studied in a three-electrode cell with 2 M KOH solution as electrolyte. Figure 2O3 and N-rGO/Fe2O3 electrodes at 20 mV s−1 within the potential range of − 1.2 to− 0.2 V. Apparently, the composite electrode manifests a distinctly greater area of CV curve than that of the Fe2O3 electrode, implying that the composite electrode shows much larger specific capacity than that of the pristine Fe2O3 electrode. The substantially improved specific capacity of the composite electrode is ascribed to the positive synergistic effect of the double-layer capacitive behavior of N-rGO and Faradaic redox reaction of Fe2O3. Ex situ XRD test , implying the lower resistance and better kinetic behavior. Figure 2O3 and N-rGO/Fe2O3 electrodes, respectively, and no distinct distortion can be noticed with the increasing scanning speed from 5 to 50 mV s−1. Figure 2O3 and N-rGO/Fe2O3 electrodes at various current densities in the potential range between− 1 and − 0.2 V, respectively. The calculated specific capacity of the N-rGO/Fe2O3 electrode . Moreover, the specific capacity of the N-rGO/Fe2O3 electrode is not only far higher than that of carbon-based anode materials, but also comparable to or even larger than that of most Fe2O3-based anode materials reported in the literature [2O3 and N-rGO/Fe2O3 electrodes was further compared by the repeated GCD tests at 3 A g−1 over 5000 cycles, as manifested in Fig. 2O3 electrode manifests a better cycle stability with 92.1% capacity retention than that of the Fe2O3 electrode (71.8%) after 5000 cycles, suggesting that the unique N-rGO-tightly-wrapped Fe2O3 composite structure can prevent the agglomeration of Fe2O3 and N-rGO and alleviate the volume change in the repeated charging-discharging processes, leading to the improvement of cycle performance. Moreover, the EIS measurement was taken, and the Nyquist plots are demonstrated in Fig. 2O3 electrode shows smaller intercept and semicircle diameter in the high-frequency region as well as higher slope value in the low-frequency region than those of the Fe2O3 electrode. Therefore, the results well demonstrate why the N-rGO/Fe2O3 electrode presents improved electrochemical performances in comparison with the Fe2O3 electrode.The electrochemical properties of the pristine Feest Fig. d was alsode Fig. g is, resterature , 60–64. lectrode 1.8% afte2S4 and N-rGO/Fe2O3 composites, a novel alkaline aqueous battery was assembled by, respectively, employing the two composites as the cathode and anode, and the electrochemical performances, including CV, GCD and EIS, were tested in a voltage window of 0–1.5 V with 2 M KOH solution as electrolyte. Figure −1, and the shape of CV curves indicates that the capacity originates from the combined contribution of both Faradaic redox reaction and capacitive EDLC at all scan rates. The GCD curves of the alkaline aqueous battery at current densities of 1-20 A g−1 are presented in Fig. E) and specific power (P) of the alkaline aqueous battery according to the following two formulas [E = 0.5C(∆V)2/3.6 and P = E/t, where the C, ∆V and t are, respectively, the specific capacity, voltage difference and discharge time of the alkaline aqueous battery. As demonstrated in Ragone plot of Fig. −1 at a specific power of 485 W kg−1 and still maintains a decent specific energy of 16.1 Wh kg−1 even at an ultrahigh specific power of 9.7 kW kg−1, implying the excellent power capability. Moreover, the highest specific energy of the assembled alkaline aqueous battery in our work is comparable to or even higher than that of alkaline aqueous devices reported in the literature, including NiCo2O4/CQDs//AC [2S4/NCF//OMC/NCF [2O4/CuO//Fe2O3/rGO [2/FGS//Fe2O3/FGS [2//AC [2S4@PPy//CF/MoS2 [2S4@G//PC [2O3/NCA [−1 over 5000 cycles, and 91.5% of capacity retention is maintained, suggesting the superior cycle stability.To further clarify the superior electrochemical properties of the N,S-CQDs/NiCoformulas : E = 0.5CQDs//AC , NiCo2S4/OMC/NCF , Ni–Co–S/OMC/NCF , CuCo2O4e2O3/rGO , MnO2/FGe2O3/FGS , Ni(OH)2S [2//AC , CF/NiCo/CF/MoS2 , NiCo2S4S4@G//PC , Ni-Co-SS4@G//PC  and MnO/[2O3/NCA . Additio2S4//N-rGO/Fe2O3 device are attributed to the following factors: (1) The utilization of CQDs as structure-directing agents induces the formation of unique N,S-codoped CQDs-decorated urchin-like nanorods-assembled NiCo2S4 microspheres composite. The nanosized building blocks of the composite with a large surface area afford more efficient electroactive sites for charge storage, contributing to the enhanced specific capacity, and the micrometer-sized assemblies of the composite possess desirable and mechanical properties and superior structure stability, leading to the improved cycling life. Meanwhile, the utilization of CQDs as conductive agents, together with the N,S-codoped modification, greatly enhances the electronic conductivity of the composite, leading to the improvement of rate property; (2) the unique prism-like nanostructured Fe2O3 hexahedrons can provide sufficient surface active sites for redox reaction, giving rise to the enhanced specific capacity, and the conductive N-rGO-decorated Fe2O3 can significantly boost the electrical conductivity of the composite anode materials, facilitating the fast electron transport kinetic and resulting in the enhanced rate property. Moreover, the Fe2O3 hexahedrons are tightly wrapped by the N-rGO layers, which effectively prevents the agglomeration and buffer against the volume change during the charging/discharging cycle process, substantially improving the cycling performance; (3) the ultrahigh-capacity N-rGO/Fe2O3 composite anode materials are utilized to couple with the high-performance N,S-CQDs/NiCo2S4 composite cathode materials to fabricate the alkaline aqueous battery, and the rational design and selection of cathode and anode materials render the resulting device achieve the enhanced specific energy, ultrahigh specific power and superior cycling life.The superior electrochemical properties of the N,S-CQDs/NiCo2S4 composite microsphere cathode materials for the first time, which display evidently improved electrochemical properties including specific capacity, rate performance and cycle life benefited from the synergistic effect of highly conductive N, S-codoped CQDs and unique micro-/nanostructured NiCo2S4 with Faradaic redox feature. Meanwhile, the N-rGO-wrapped prism-like Fe2O3 hexahedrons composite anode materials are prepared, which exhibit an ultrahigh specific capacity, substantially enhanced rate performance and cycle property due to the synergistic effect between highly conductive N-rGO and high-capacity nanostructured Fe2O3 hexahedrons. In view of their superior electrochemical properties and rational coupling of cathode and anode materials, a novel alkaline aqueous battery is assembled by employing N,S-CQDs/NiCo2S4 as cathode and N-rGO/Fe2O3 as anode in 2 M KOH aqueous electrolyte. The fabricated N,S-CQDs/NiCo2S4//N-rGO/Fe2O3 device delivers a high specific energy of 50.2 Wh kg−1 at a specific power of 485 W kg−1 or an ultrahigh specific power of 9.7 kW kg−1 at a decent specific energy of 16.1 Wh kg−1 and good cycling life with 91.5% of capacity retention at 3 A g−1 over 5,000 cycles. The present research gives a valuable reference for the design and exploitation of superior energy storage devices using battery/capacitive composite materials simultaneously as both cathode and anode in aqueous electrolyte.In a word, the graphitic CQDs are prepared via a scalable mixed-acid chemical oxidation method employing highly conductive and low-cost Super P powders as precursors, and the high yield of more than 50% is successfully acquired. Then, the resulting CQDs are further used as both structure-directing and conductive agents to controllably synthesize the N,S-codoped CQDs-decorated urchin-like NiCoSupplementary material 1 (PDF 644 kb)Below is the link to the electronic supplementary material.


Progressive fibrosis of the dermal tissues is a challenging complication of radiotherapy whose underlying mechanism is not fully understood, and there are few available treatments. The canonical Wnt/β-catenin signaling pathway plays an important role in fibrosis as well as in the epithelial-to-mesenchymal transition (EMT). We investigated whether inhibition of Wnt/β-catenin signaling with sLRP6E1E2, a molecule that binds to extracellular Wnt ligands, ameliorated radiation-induced fibrosis both in vitro and in vivo. Radiation with a single dose of 2 Gy not only facilitated fibrosis in cultured human dermal fibroblasts via activation of the Wnt/β-catenin pathway but also initiated EMT in cultured keratinocytes, developing collagen-producing mesenchymal cells. sLRP6E1E2-expressing adenovirus treatment exerted anti-fibrotic activity in irradiated cultured dermal fibroblasts and keratinocytes. In a mouse model, a single fraction of 15 Gy was delivered to the dorsal skins of 36 mice randomized into three groups: those receiving PBS, those receiving control adenovirus, and those receiving decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2). The mice were observed for 16 weeks, and excessive deposition of type I collagen was suppressed by sLRP6E1E2-expressing adenovirus treatment. These results demonstrate that the modulation of the Wnt/β-catenin pathway has the potential to decrease the severity of radiation-induced dermal fibrosis. Since the skin is usually the first site of entry of radiation, various skin reactions can occur. In the acute phase of radiation injury, erythema may be evident within hours of irradiation, and desquamation and ulceration appear at higher doses. These acute events can be addressed symptomatically and generally resolve without exacerbation2. However, progressive fibrosis of the dermal tissues is a late adverse effect of radiation that is not always resolved. Pain, limited range of motion, and poor cosmesis are well-described adverse events attributed to radiation-induced fibrosis of the skin3. Unfortunately, there are few anti-fibrotic therapies available.Radiotherapy is an effective treatment for controlling solid tumors such as head and neck cancer, breast cancer, and soft tissue sarcoma. However, collateral injury to the surrounding healthy tissues may result in toxicity and frequently limits the therapeutic dose of radiation that can be delivered4. Several mediators are involved and act on fibroblasts to cause tissue fibrosis. In addition to the TGF-β signaling pathway, which is a key mediator of fibroblast activation, the Wnt/β-catenin signaling pathway plays an important role in fibrotic reactions5. In the setting of radiation, β-catenin mediates the effects of radiation in fibroblasts, and its modulation has the potential to decrease the severity of radiation-induced complications4.Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components following a breakdown in the normal balance of extracellular matrix synthesis and degradation6. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt ligand, is required for activation of the canonical Wnt/β-catenin pathway7. A novel soluble Wnt receptor, sLRP6E1E2, which is composed of the LRP6 E1 and E2 regions, was used to inhibit the pathway8. Therefore, we explored the biological effects of the binding of sLRP6E1E2 to extracellular Wnt ligands after irradiation.In this study, we tested the hypothesis that inhibition of the Wnt/β-catenin signaling pathway would ameliorate radiation-induced fibrosis. Inhibition of the pathway was accomplished using adenovirus gene therapy expressing a Wnt antagonist that binds Wnt ligands to block interactions with receptorsDermal fibroblasts and keratinocytes, representative cells that compose the skin, were selected to determine the effect of irradiation on skin tissues. We began with the premise that the Wnt/β-catenin signaling pathway would be activated in the setting of radiation. To verify that radiation contributed to the activation of the Wnt/β-catenin pathway, we examined Wnt-3a and β-catenin expression level in human dermal fibroblasts (HDF) and HaCaT keratinocytes at 48 h after a single dose of 2-Gy irradiation. Both Wnt3a and β-catenin are upregulated at the protein level Fig. a,b. This6. Therefore, we examined the extent of cytosolic and nuclear localization of β-catenin by immunofluorescence staining of irradiated HDFs and HaCaT keratinocytes after transduction with dE1-k35/sLRP6E1E2 (sLRP6E1E2-expressing virus) or dE1-k35 (control virus). In absence of radiation, β-catenin was rarely observed in the cytosol and nucleus. Upon radiation, control groups (radiation + PBS and radiation + sLRP6E1E2 (−) virus) showed increased β-catenin localization on the cytosol and nucleus, while dE1-k35/sLRP6E1E2-transduced cells showed lower levels of nuclear localization of β-catenin than control groups  virus) Fig. b, indicaWe next evaluated changes in the Wnt/β-catenin pathway in the setting of radiation in a mouse model treated with PBS, control adenovirus (dE1-k35), or decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2) using western blot analysis of Wnt-3a and β-catenin. In the PBS control, levels of Wnt-3a began increasing after irradiation and peaked at 12 weeks, while β-catenin increased rapidly at 4 weeks, peaked at 8 weeks, and decreased 12–16 weeks after irradiation . As we sought to focus on radiation-induced fibrosis in this study, fibrosis caused by wound healing should be excluded. After 15 Gy radiation was delivered to the dorsal skin, tiny dry desquamations appeared in approximately 1/3 of the mice in each group within 4 weeks, and fields receiving radiation healed without ulceration in all mouse. Upon examination by planimetric analysis and laser Doppler flowmetry, there were no significant differences between groups in almost values , and sLRP6E1E2 (+) groups) compared to those in normal dermal tissue. Thus, radiation seems to be responsible for increasing collagenous fibrosis. Among the irradiated groups, a relatively thin dermis and many miniaturized follicles were observed in the sLRP6E1E2-treated group, while mice in the control groups ) had thicker dermal tissues and fewer follicles. In addition, adnexa were replaced by collagenous fibers in both control groups Fig. a.Figure To test the potential effect of sLRP6E1E2 on in vivo fibrosis, mRNA expression of collagen type I α1, collagen type III α1 and TGF-β1 was examined at 12 weeks post-radiation, and immunohistochemistry for type I collagen was performed at each time point. The mRNA expression of collagen type I α1, collagen type III α1 and TGF-β1 was significantly reduced in dE1-k35/sLRP6E1E2-transfected mouse compared to controls In conclusion, radiation facilitates the fibrotic reaction in cultured keratinocytes as well as cultured human dermal fibroblasts, while sLRP6E1E2 exerts anti-fibrotic activity in irradiated cultured cells (in vitro study). In a mouse model, excessive collagen deposition was controlled in the sLRP6E1E2-treated group (in vivo study). Taken together, these results support the idea that modulation of the Wnt/β-catenin pathway has the potential to decrease the severity of radiation-induced dermal fibrosis.2 until forming a confluent monolayer. An X-RAD320 irradiator  was used to irradiate cell cultures with a single dose of 2-Gy radiation.HDF and HaCaT keratinocytes  were seeded at passage 4 in 6-well plates and grown in a complete cell culture medium consisting of Dulbecco’s modified Eagle medium (DMEM) with 100 U/ml penicillin, 100 g/ml streptomycin sulfate, and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO6. The replication-incompetent dE1-k35 was used as a negative control8. Titers of generated adenoviruses were determined using the optical density of the viral genome. For both dE1-k35/sLRP6E1E2 (sLRP6E1E2-expressing virus) and dE1-k35 (control virus), a multiplicity of infection (MOI) of 100% was transfected into cultured dermal fibroblasts and HaCaT keratinocytes. Irradiation of cell cultures was followed immediately by viral transfection.The virus dE1-k35/sLRP6E1E2 was obtained as previously describedCOL1A1), collagen type 3 α1 (COL3A1), TGF-β1 (TGFB1) in cultured HDF cells and those of E-cadherin (CDH1), vimentin (VIM), and twist (TWIST1) in cultured HaCaT keratinocytes, qRT-PCR was performed 48 h after irradiation. Total RNA was prepared with the RNeasy Mini Kit , and complementary DNA was prepared from 0.5 µg of total RNA by random priming using a first-strand cDNA synthesis kit  under the following conditions: 95 °C for 5 min, 37 °C for 2 h, and 75 °C for 15 min. Applied Biosystems TaqMan primer/probe kits were used to analyze mRNA expression levels with an ABI Prism 7,500 HT Sequence Detection System . For cDNA amplification, AmpliTaq Gold DNA polymerase was activated by 10-min incubation at 95 °C; this was followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C for each cycle. The mRNA expression levels were normalized to the levels of the GAPDH housekeeping gene, and then relative quantities were expressed as fold-inductions compared with the control gene after determining the threshold cycle and drawing standard curves. The following primers were used: Hs00164004_m1 (COL1A1), Hs00164103_m1 (COL3A1), Hs00998133_m1 (TGFB1), Hs 01023894_m1 (CDH1), Hs 00185584_m1 (VIM), Hs 00361186_m1 (TWIST1), and Hs99999905_m1 .To evaluate mRNA levels of collagen type 1 α1 , 1% Nonidet P-40, 150 mM sodium chloride, and 0.1 mM zinc acetate in the presence of protease inhibitors. Protein concentration was determined by the Lowry method , and 20 µg of each sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were then transferred electrophoretically onto a polyvinylidene difluoride membrane . The membrane was blocked with blocking buffer for 1 h and then incubated overnight at 4 °C with primary antibodies against β-catenin , Wnt , and actin . After a 2-h incubation with the secondary antibodies horseradish peroxidase-conjugated rabbit antibody (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated mouse antibody (Santa Cruz Biotechnology), protein bands were visualized using an ECL detection kit  according to the manufacturer’s instructions. Protein expression was analyzed using ImageJ software .After irradiation, HaCaT keratinocytes were cultured for 48 h, and phenotypic changes were examined using phase-contrast microscopy .After 48 h post-radiation, cultured cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 15 min at room temperature, and then permeabilized by incubation for 15 min with 0.01% Tween 20 in PBS. Samples were blocked with 5% bovine serum albumin followed by incubation with anti-collagen I, anti-phospho-Smad 2/3 and anti-β-catenin  overnight at 4 °C. The next day, cells were washed with PBS and incubated with bovine anti-rabbit IgG-FITC  secondary antibody for 2 h at room temperature. Cells were mounted on slides with mounting solution containing DAPI , and cells were viewed under a confocal microscope system .Animals were handled according to national and international guidelines in an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The number of animals used was minimized, and all necessary precautions were fulfilled to mitigate pain and suffering. Protocols were approved by the Institutional Animal Care and Use Committee in Yonsei Biomedical Research Institute.10 viral particles (VP)/ml was administrated. The mice were housed and observed for 16 weeks to allow toxic effects of radiation to develop. At 2, 4, 6, 8, 10, and 12 weeks after irradiation, digital photographs and laser Doppler flowmetry  measurements were taken. At 4, 8, 12, and 16 weeks, mice were euthanized (n = 3 each week), and tissues were harvested for histological analysis.A total of 36 four- to five-week-old mice were randomized into three groups according to the injection materials: PBS, control adenovirus (dE1-k35), and decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2). A single fraction of 15 Gy was delivered to the dorsal skin (dimension: 1.5 × 2 cm) of each mouse using an X-RAD320 irradiator, ensuring that > 90% of the prescribed dose would be limited to skin depth. Post-irradiation, PBS or virus was directly injected into each irradiated dorsal skin at 1, 3, 5, 7, 9, and 11 weeks. For each mouse, 5 × 10Gross changes of the skin after a single fraction irradiation of 15 Gy were observed, and complications related to irradiation such as erythema, dry desquamation, moist desquamation, ulceration, hair loss, and necrosis were recorded. Irradiated areas were marked on the dorsum of each mouse before radiation, and the marked areas were later assessed by digital photographs. The Scion  image program was used by two blinded observers to digitally measure each affected area. Using the program, the length of each image was converted to the actual length, and the surface area was calculated. Sequential changes in the irradiated area were analyzed.To assess changes in blood flow within the irradiated area, the Periflux system 5,000 was used. Skin vascularity was assessed from the center of the irradiated area using laser Doppler flowmetry. Data were measured three times at 1-min intervals, and the mean values were obtained. Data are presented as perfusion units (PU).To analyze the histology of collagen fibers, Masson’s trichrome staining was performed. The staining solution was prepared with a standard protocol.Immunohistochemistry was performed to measure the amount of type I collagen. Slides were incubated overnight at 4 °C with rabbit anti-collagen I antibody (Abcam) and rabbit anti-fibronectin (Santa Cruz Biotechnology) diluted in blocking serum. After several stages of washing with PBS, the tissue sections were incubated with peroxidase-conjugated goat anti-rabbit polyclonal antibody  at a dilution of 1:200 in PBS for 30 min at 37 °C. After several washes in PBS, the signals on the tissues were revealed by incubating the tissues with diaminobenzidine in PBS for 10 min.Semi-quantitative analysis of the synthesis of type I collagen was executed using MetaMorph image analysis software . Results are expressed as the average optical density (OD) of five different digital images.t-test for unpaired observations or Bonferroni’s correction for multiple comparisons. P < 0.05 was considered significant.Each experiment was performed in triplicate. Results are expressed as mean ± standard error of the mean (SEM). Group results were compared by one-way analysis of variance, followed by post hoc Student’s Supplementary file1 (PDF 648 kb)


Low-cost sensors based on WO3 nanoparticles are promising due to their distinct selectivity to detect NO2 at the ppb level. Here, we revealed that controlling the thickness of highly porous (97%) WO3 films between 0.5 and 12.3 μm altered the NO2 sensitivity by more than an order of magnitude. Therefore, films of WO3 nanoparticles (20 nm in diameter by N2 adsorption) with mixed γ- and ε-phase were deposited by single-step flame spray pyrolysis without affecting crystal size, phase composition, and film porosity. That way, sensitivity and selectivity effects were associated unambiguously to thickness, which was not possible yet with other sensor fabrication methods. At the optimum thickness (3.1 μm) and 125 °C, NO2 concentrations were detected down to 3 ppb at 50% relative humidity (RH), and outstanding NO2 selectivity to CO, methanol, ethanol, NH3 , H2, CH4, acetone , formaldehyde (>103), and H2S (835) was achieved. Such thickness-optimized and porous WO3 films have strong potential for integration into low-power devices for distributed NO2 air quality monitoring.Nitrogen dioxide (NO To prevmbustion  or the Umbustion , respectmbustion . Neverthxpensive  detector2 below 20 ppb in ambient air  [Sensors based on reduced graphene oxide , carbon ient air . On the t air  and are3O4 [2O5 , high mi3O4 [2O5 . Despite3O4 [2O5 . Such SM3O4 [2O5 . The WO3o 10 ppb ), optimuo 10 ppb ), and ouethanol) .3 films, the optimum NO2 response has been reported at ~85 nm thickness in the range of 40 to 200 nm [2 sensitivity of WO3 lamella stacks prepared by precipitation increase with porosity by adding hydrothermally-made SnO2 nanoparticles as spacers [2 might also play a role. In addition, layer-by-layer inkjet printing has been used to obtain different thicknesses of Cu2O and CuO films for NO2 sensing; however, the effect of film thickness on sensor sensitivity and selectivity was not investigated [Film morphology optimization is an effective route to improve sensing performance . Consequo 200 nm . Besides spacers , though,stigated . Changesstigated ) or addistigated . As a restigated  or doctostigated ) that instigated  and can 3 sensors featuring outstanding acetone selectivity for breath analysis  have responded to NO at 280–310 °C [2 as with CO [2 for other compounds  and deposition is an attractive method to generate highly porous films  of nanoexercise  and dietexercise ) and in exercise  for humaexercise . Besides 20 days , while fetection . Further0–310 °C . Such fi with CO  and provt 90% RH  or 5 ppbt 90% RH ).3 film thickness for selectively sensing NO2 at realistic 50% RH. Nanoparticles were produced by FSP with crystal size and composition analyzed by X-ray diffraction and Raman spectroscopy. Highly porous films were obtained by direct deposition onto low-power μ-hotplate substrates. Film thickness was controlled through deposition time and characterized by cross-sectional focused ion beam scanning electron microscopy (FIB-SEM). The sensor performance at different thicknesses was evaluated at 125 °C [2 among common confounders—CO, H2, CH4, NH3, H2S, formaldehyde, acetone, methanol, and ethanol—at much higher concentrations and benchmarked to state-of-the-art solid-state NO2 detectors. These confounders were selected as they are among the critical ones outdoors [Here, we investigated the effect of porous WOt 125 °C  for sensoutdoors .3 nanoparticles [3 basis, Sigma Aldrich, Buchs, Switzerland) dissolved in an equivolumetric solution of ethanol  and diethylene glycol monobutyl ether  to obtain final tungsten molarity of 0.2 M [−1, through the FSP nozzle, and dispersed by 5 L min−1 oxygen  at a pressure drop of 1.6 bar into a fine spray. Additionally, sheath oxygen was supplied at 5 L min−1 through an annulus surrounding the nozzle. A ring-shaped pilot flame of premixed methane  and oxygen (3.2 L min−1) ignited and sustained the spray-flame. The as-prepared nanoparticles were collected at 60 cm above the burner on a water-cooled glass-fiber  filtered by a vacuum pump and removed with a spatula. The sieved (250 μm mesh) powders were subsequently annealed at 500 °C for 5 h.A flame spray pyrolysis (FSP) reactor was used to prepare WOarticles . The preof 0.2 M . The pre2) featuring a suspended membrane with an integrated heater and interdigitated electrodes (MSGS 5000i [−1) at 14.5 cm above the burner was applied to enhance adhesion and cohesion [For sensor assembly, nanoparticles were flame-deposited for 1 to 18 min directly onto micromachined μ-hotplate sensor substrates  operated at 40 kV and 30 mA at 2θ (Cu Kα) = 20 to 60°. The step size and scanning speed were 0.0026° and 0.62° min−1, respectively. Crystal phases were identified with reference structural parameters of monoclinic γ-WO3 (ICSD 80056), triclinic δ-WO3 (80053), and monoclinic ε-WO3 (84163), respectively, using the software Diffrac.eva V3.1 . Sample displacement was corrected by aligning the pattern to the peaks of crystalline NiO  that was added as an internal standard [3 peaks at 2θ = 20–36°. Additionally, Raman scattering was recorded with a 785 nm laser at 500 mW (operated at 1% intensity) in the range of 300 to 900 cm−1 using an exposure time of 1 min . The specific surface area (SSA) was measured by nitrogen adsorption . The Brunauer–Emmett–Teller (BET) equivalent particle diameter was calculated with the density of WO3 (ρs = 7.16 g cm−3).Particle morphology was obtained by transmission electron microscopy  at 100 kV. Crystal phases and sizes of the annealed WOstandard . Corresp+ ions at 30 kV. Rough milling to open the cross-section and subsequent polishing were performed at 2.5 and 0.77 nA, respectively. For the determination of film thickness, 40 measurements were taken across one sensing layer of the same sample using the application software of the SEM.Film morphology was obtained by FIB-SEM  operated at 2 kV. For cross-sectional images, a slice was cut into the sensing film by a focused beam of Ga3 films on Al2O3 substrates (20 × 20 mm2) was assessed by X-ray signal attenuation of the Al2O3 peaks by the WO3 film, as done with similar flame-deposited SnO2 films [s and bulk density ρs, resulting in an emerging intensity The porosity of flame-deposited WOO2 films . Thereintion law :(1)IemIi3 at 8.04 keV (Cu Kα) was calculated as 13.59 m2/kg using the XCOM Photon Cross Sections Database [2O3 substrates were split using a wedge to obtain cross-sectional SEM images, and the thickness was evaluated at least 40 points across the cross-section. Finally, the average porosity was calculated from the main Al2O3 peak reflections at 2θ = 25.6, 35.1, 43.3, and 57.5°.The Database . Dense fnt angle  and comp3) arranged between a tubular gas inlet and outlet  were mixed by high-resolution mass flow controllers , using a setup described in detail elsewhere [2, H2S, CH4, NH3, acetone, methanol, ethanol ; H2 (50 ppm in synthetic air); formaldehyde (10 ppm in N2); CO (500 ppm in synthetic air). Note that the O2 concentration in the gas mixture was reduced from 20 to 18 vol% for 1 ppm formaldehyde in N2; however, this should not affect the sensor performance [Up to four sensors were glued  onto leadless chip carriers . Electrical connections were established by wire bonding  before installing the chip in a stainless steel sensor chamber . In specd outlet . Sensorslsewhere , and mealsewhere ). Analytformance . All tubair/Ranalyte − 1, where Rair and Ranalyte are the film resistances in the absence and presence of analyte [analyte/Rair − 1. Response and recovery times were defined as the times needed to reach and recover 90% of the resistance change, respectively. Sensor sensitivity was defined as the derivative of the response with respect to the analyte concentration, according to DIN 1319-1:1995-01 5.4.The ohmic film resistances were continuously measured with a multimeter  between the substrate’s interdigitated Pt electrodes. A picoammeter  was used to read-out resistances > 100 MOhm. The analyte response for reducing gases was defined as S = R analyte , respect3 nanoparticles, as shown by TEM (2 g−1) obtained by nitrogen adsorption.The filter-collected and annealed powder consisted of agglomerated WOn by TEM a. The prn by TEM a, inset,3. Between 23 and 25°, three peaks characteristic for monoclinic γ-WO3 (triangles) were visible. Note that also triclinic δ-WO3 (3 (circles), featuring a peak at 2θ = 24.10°, in agreement with the literature [3 (while they should be similar for γ-WO3). The peak at 23.60° might be affected as well due to the overlapping peaks—a characteristic for small crystal sizes. The ε-phase  was obt40 °C [3 , and wast 700 °C . NeitherXRD) of 23 and 16 nm, respectively, in agreement with TEM (3 (2.017 Å) and the (−1 2 2) plane in ε-WO3 (1.994 Å). The crystal sizes compared rather well to reported dXRD of 29 and 21 nm for γ- and ε-WO3, respectively, of flame-made WO3 in the literature [3 , which t 125 °C  and, esp3 films  was most likely due to the small ε-phase crystal size (as estimated by XRD), which leads to peak broadening [The Raman spectrum supported the coexistence of γ- and ε-phases in the WO3 films c. The ma both γ- ,63 , exhibi−1. For thermophoretic deposition, such a linear trend could be obtained when the temperature difference between aerosol and film is constant [The measured film thickness as a function of deposition time is shown in constant , indicatconstant . Therefo3 sensors operated at 125 °C and realistic 50% RH. This sensing temperature was chosen, as identified previously for optimal NO2 sensitivity with hydrothermally prepared films [9 to 2.6∙107 Ohm when the film thickness increased from 0.5 to 7.7 μm and leveled off thereafter. Such a reduction should be attributed to the additional conduction pathways  with increasing film thickness, reducing the overall film resistance, in line with the literature [−1  [2 (~7 nm for H2) [2  . For the, 85 nm)  that inf, 85 nm) . Optima  for H2)  or atomi (~10 nm  or 2.6 n (~10 nm  for CO).10 nm [2 . This wa10 nm [2  rather t10 nm [2  that cou2 limits are below 100 ppb [2 at 50% RH. The sensor response increased continuously with a sensitivity of about 2 ppb−1 up to 25 ppb and thereafter with 1 ppb−1, suggesting the onset of saturation. This was orders of magnitude higher than graphene-based sensors operated at room temperature (~0.1 ppb−1) [3 nanowires at 250 °C (~0.002 ppb−1) [The legal NO 100 ppb , definin1 ppb−1)  or WO3 n2 ppb−1) . Such be2 ppb−1) . Most im2 concentrations of 3 ppb in 50% RH. The film resistance increased from about 104 to 108 MOhm when introducing 3 ppb NO2, as expected for oxidizing gases like NO2 that fill oxygen vacancies located on the surface of WO3 [2 has already been measured only in dry air [2 sensitivity of WO3-based sensors decreased with increasing RH, both at low  [2 concentrations with flame-deposited WO3 films was associated with their highly porous and fine film morphology . Import., 25 °C ) and hig., 25 °C ). Therei readout . In factncrease) . This concrease) . The detrphology . In factnnealing , and higser ones . The corser ones .2 in the presence of interfering gases. Therefore, the responses of the above sensor (3.1 μm thickness) were tested for 100 ppb NO2 and its major confounders at much higher concentration (1000 ppb): H2S, formaldehyde, H2, CH4, CO , H2, CH4, acetone , and formaldehyde (>103), indicating negligible cross-interference. Even to H2S, the selectivity was still >800. Please note that WO3 also showed high NO2 selectivity to other confounders, e.g., 150 for NO at 100 °C and 1 ppm [2/ethanol and H2S, decreasing and increasing trends, respectively, were observed with the thickness . The selectivity could be further increased with filters preceding the sensor, e.g., size-selective membranes [2O3 to retain hydrophilic [In environmental monitoring, a key challenge is the selective detection of NOd 50% RH . Excellend 1 ppm . Intereshickness . These s traffic . In specembranes  or adsorrophilic  or Tenaxrophilic ).2 sensors at 10–80% RH and relevant sub-ppm NO2 concentrations. The present flame-made porous WO3 sensor showed superior selectivities over those prepared by sputtering (for NH3 and CO), drop coating (for acetone), and other coating methods (for ethanol), which could be attributed to their more compact film morphology and/or to the presence of ε-phase WO3 in the flame-made films (3 sensors. Only brush-coated In2O3 [2/ZnO (UV excited) sensors [5 for CO).de films b,c. Furted In2O3  and SnO2 sensors  feature  sensors ,27 opera3 sensing films with controlled thickness were created by FSP. The films consisted of mixed-phase γ- and ε-WO3, as revealed by XRD and Raman spectroscopy. Their thickness was controlled through the deposition time without affecting the crystal size or phase content of WO3. Such flame-deposited films at an optimal thickness  exhibited an order of magnitude higher NO2 response than the thickest films . This was probably related to a competition of increased amount of reaction sites, hindered analyte penetration, and weakened electrode effects for thicker films. The thickness-optimized WO3 films showed excellent NO2 selectivity over confounding gases. Furthermore, NO2 concentrations were detected down to 3 ppb at 50% RH with a high signal-to-noise ratio (>17), superior to state-of-the-art SMOx-based and commercial NO2 sensors. As a result, such morphology-optimized WO3 films have a high potential for integration into low power devices for distributed and remote NO2 air quality monitoring. In a broader sense, such film morphology optimization could be applied also for other metal-oxide sensors and analytes to improve their sensitivity in environmental monitoring or medical breath analysis [Highly porous, crack-free, and homogeneous WOanalysis , where s


Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases ( The pig is a useful animal model because of its physiological and anatomical similarities to humans ,2. Transsuv39h1 and suv39h2 are highly expressed in heterochromatin regions of mammalian cells  and Bcl-2 homologous antagonist killer [Bak]) and increased the expression levels of anti-apoptosis genes (B-cell lymphoma [Bcl]-xl and Bcl2) compared to the control (Chaetocin markedly increased the inner cell mass (ICM) and trophectoderm (TE) cell numbers of SCNT embryos compared to the control but did not affect the ICM to TE cell ratio . The exphaetocin D. Moreov control . Therefosuv39h1 and suv39h2 were also considerably decreased by chaetocin  were significantly decreased by chaetocin treatment at the pronuclear, two-, and four-cell stages of SCNT embryos  were significantly higher in control than in IVF embryos. The expression levels of most of these genes in pronuclear- and two-cell stage SCNT embryos were decreased by chaetocin (We investigated the number of microtubule-associated protein 1A/1B-light chain 3 (LC3) dots, a marker of autophagy induction, and the expression levels of autophagy-related genes in porcine SCNT embryos by immunofluorescence and qRT-PCR. At the pronuclear and two-cell stages, both were significantly increased by chaetocin A–F. In ahaetocin G. Therefsuv39h1, and suv39h2 transcripts compared to IVF embryos, resulting in aberrant reprogramming and defective embryonic development [suv39h1 and suv39h2 than IVF embryos. Therefore, we attempted to enhance the developmental competence of porcine SCNT embryos using chaetocin, an inhibitor of suv39h1 and suv39h2.Transgenic pigs are used in biomedical and regenerative medicine research as disease and xenotransplantation models ,27. To eelopment ,12,32. Ioct4, nanog, and sox2 were markedly increased. This is consistent with previous reports that H3K9me3 marks heterochromatin foci in the ICM and that reducing the H3K9me3 level upregulates the expression of pluripotency-related genes [Microinjection of the mRNAs of Kdm4d, Kdm4a, and Kdm4e H3K9-specific demethylases, into mouse, human, and bovine SCNT embryos significantly decreases the H3K9me3 level and increases developmental efficiency ,9,33. Ined genes ,41,42,43suv39h1, suv39h2, and G9a [suv39h1, suv39h2, and G9a mRNA or their protein in ovine cells and human cancer cells [suv39h1 and suv39h2 in the early stage of porcine SCNT embryos, indicating that chaetocin could efficiently downregulate the H3K9me3 levels possibly via reduction of the expression or protein stability of H3K9 methyltransferases. However, the exact mechanism of chaetocin governing the reduction of H3K9me3 has not yet been fully elucidated or was largely unknown until recently. Thus, the understanding of chaetocin function as a transcriptional modifier of suv39h1 and suv39h2 genes requires further detailed investigation, such as ChiP-seq analysis between modification types of histone H3 and the promoter region of both genes.Chaetocin is the first inhibitor of histone lysine methyltransferase, which was found to be a specific inhibitor of the SUV39 family, such as  and G9a ,44. Prever cells ,32,45,46DNMT1 is the most abundant one and acts to maintain methylation, whereas DNMT3a and DNMT3b are responsible for de novo methylation [suv39h1- and suv39h2-knockout embryonic stem cells show decreased DNA methylation [suv39h1 and suv39h2 directly interact with HP1 to methylate heterochromatin regions [suv39h1 and suv39h2, suggesting that H3K9me3 and DNA methylation act in concert to maintain a repressed chromatin state. In this study, the DNMTs’ expression levels in SCNT embryos were higher than in IVF embryos and were significantly restored by chaetocin. Moreover, chaetocin treatment noticeably downregulated the 5-mc levels, an indicator of DNA methylation, at the early stage of SCNT embryos. These results demonstrate that chaetocin enhances epigenetic reprogramming in porcine SCNT embryos by reducing the levels of H3K9me3 and global DNA methylation.Global DNA methylation, which occurs on cytosine-guanine dinucleotides, is important for epigenetic reprogramming and normal embryonic development in mammals . DNA methylation . SCNT emhylation ,51. A prhylation , and suv regions , indicatAutophagy is a crucial cellular mechanism that degrades unnecessary or dysfunctional cellular components to maintain intracellular homeostasis . During In conclusion, an aberrant H3K9me3 level is a major epigenetic barrier during the development of porcine SCNT embryos. We found that chaetocin treatment of early stage SCNT embryos increased developmental competence by enhancing epigenetic reprogramming and autophagic activity. Therefore, chaetocin could be applied to enhance our understanding of the role of H3K9me3 in the regulation of autophagy in early stage SCNT embryos. Our findings will enable the production of SCNT embryos with high developmental competence and the generation of transgenic pigs for biomedical research.This study was carried out in strict accordance with the recommendations of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee .All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co.  unless otherwise indicated.2 in air. The IVM medium consisted of tissue culture medium 199 supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 10 ng/mL β-mercaptoethanol, 10 ng/mL epidermal growth factor, 10 IU/mL pregnant mare serum gonadotropin, and 10 IU/mL human chorionic gonadotropin. After 22 h of IVM, COCs were further cultured in IVM medium without hormones for another 22 h. After 44 h of IVM, expanded cumulus cells with oocytes were treated with 0.1% hyaluronidase and then removed after vortexing for 1 min. Matured MII oocytes with a visible polar body, regular morphology, and homogenous cytoplasm were used for experiments.To obtain porcine oocytes, ovaries were collected from prepubertal gilts at a nearby local slaughterhouse and transported to the laboratory in 0.9% saline containing 75 µg/mL potassium penicillin G and 50 µg/mL streptomycin sulfate at 38.5 °C within 2 h. Cumulus–oocyte complexes (COCs) were aspirated from follicles (3–6 mm in diameter) using an 18-gauge needle into a disposable 10 mL syringe. Collected COCs were washed three times in Tyrode’s Albumin Lactate Pyruvate-HEPES medium and then approximately 50 COCs were sequentially matured in 500 µL of IVM medium in a 4-well multi-dish  for 44 h at 38.5 °C in 5% CO2·2H2O, 20 mM Tris , 11 mM glucose, and 5 mM sodium pyruvate, and no antibiotics. MII oocytes were washed three times in mTBM containing 2.5 mM caffeine sodium benzoate and 1 mg/mL bovine serum albumin (BSA), and 10–15 oocytes were placed into a 48 µL droplet of IVF medium under mineral oil pre-equilibrated at 38.5 °C in 5% CO2 in air. For preparation of the spermatozoa using the swim-up method prior to fertilization, freshly ejaculated semen was washed three times with sperm washing medium  supplemented with 1 mg/mL BSA, 100 µg/mL penicillin G, and 75 µg/mL streptomycin sulfate). After washing, 2 mL of sperm washing medium were gently added to the spermatozoa pellet and incubated for 15 min at 38.5 °C in 5% CO2 in air. After incubation, supernatant was washed with mTBM, and resuspended with 1 mL mTBM. Then, 2 µL of diluted spermatozoa were added to 48 µL of mTBM containing 10–15 oocytes to a final concentration of 1.5 × 105 spermatozoa/mL. Oocytes were co-incubated with the spermatozoa for 6 h at 38.5 °C in 5% CO2 in air. After 6 h, oocytes were stripped by gentle pipetting and transferred to IVC medium, consisting of porcine zygote medium-3 (PZM-3) containing 4 mg/mL BSA, for culture at 38.5 °C in 5% CO2 in air. PZM-3 consisted of 108 mM NaCl, 10 mM KCl, 0.4 mM MgSO4·7H2O, 0.35 mM KH2PO4, 25.07 mM NaHCO3, 0.2 mM sodium pyruvate, 2 mM Ca-lactate·5H2O, and 50 μg/mL gentamicin sulfate.IVF was performed in a modified Tris-buffered medium (mTBM), consisting of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2 in air until confluent. Donor cells were used at passages 4 to 6 for SCNT. To synchronize the cell cycle at the G0–G1 phase, kidney cells were cultured after reaching confluency and then further cultured in culture medium containing 0.5% FBS for 3 days. Donor cells for SCNT were washed with DPBS and digested with 0.25% trypsin-EDTA for 3 min, then trypsin activity was blocked with DMEM containing 10% FBS. The cells were spun down at a low speed (150× g) for 2 min and resuspended with DPBS.Porcine kidney was obtained from a neonatal pig by surgical operation. Harvested kidney tissues were stored in DPBS washing buffer containing 10% (v/v) penicillin/streptomycin  on ice until isolation. Kidney biopsies (2 × 1 × 1 cm) were washed three times in washing buffer, chopped (0.3 × 0.3 × 0.3 cm), and washed with Dulbecco’s modified eagle’s medium . Small pieces of kidney tissue were placed in 60 mm culture dishes and cultured in DMEM containing 10% fetal bovine serum , 10 ng/mL basic fibroblast growth factors , and 1% (v/v) penicillin/streptomycin at 38.5 °C in 5% CO2·2H2O, 0.2 mM MgSO4·7H2O, and 0.01% polyvinyl alcohol (PVA), and incubated at 38.5 °C in 5% CO2 in air. After 2 h, oocyte–cell couplets that were completely fused as observed under an inverted microscope were selected and activated by one direct current pulse of 1.2 kV/cm for 50 µs in activation medium consisting of 280 mM mannitol containing 0.1 mM CaCl2·2H2O, 0.2 mM MgSO4·7H2O, 0.01% PVA, and 0.5 mM HEPES, and then cultured in post-activation medium, which consisted of IVC medium supplemented with 5 µg/mL cytochalasin B and 2mM 6-dimethylaminopurine, for 4 h at 38.5 °C in 5% CO2 in air. After activation, the activated embryos were transferred to IVC medium at 38.5 °C in 5% CO2 in air.SCNT were performed as previously described . MII oocTo confirm the optimal conditions for chaetocin treatment during porcine SCNT embryo development, activated embryos were cultured in activation medium with various concentrations of chaetocin  for 24 h after activation. The concentration that caused the highest percentage of embryos to develop (0.5 nM) was used for various durations of treatment  after activation. The cleavage and blastocyst rates were determined at 48 and 144 h, respectively.Embryos of every stage derived from IVF and SCNT were washed in DPBS supplemented with 0.1% PVA (DPBS-PVA) for 10 min each. For membrane permeabilization, the fixed embryos were incubated in DPBS containing 0.5% Triton X-100 for 40 min at room temperature (RT). For the staining of global methylation, permeabilized embryos were additionally stored in 1 M HCl for 30 min at 38.5 °C. Subsequently, embryos were washed three times in DPBS-PVA and stored in blocking medium, which consisted of DPBS containing 4mg/mL BSA for 1 h at RT. The embryos were incubated with primary antibodies H3K9me3 , 5-mc , or LC3  overnight at 4 °C. After washing three times with DPBS containing 0.05% Tween 20, the embryos were incubated with the secondary antibody, Alexa Fluor 488 goat anti rabbit or mouse IgG (1:1000 for H3K9me3 or 1:200 for 5-mc and LC3) for 1 h at RT. After washing three times with DPBS containing 0.05% Tween 20, embryos were mounted on clean glass slides with 4′,6′-diamidino-2-phenylindole (DAPI) and observed under a fluorescence microscope . Approximately five to eight embryos were used in the immunocytochemistry in each independent experiment.25 were added to each sample. The beads were hybridized for 5 min and then separated from the binding buffer using a Dynal magnetic bar (Invitrogen). Bound poly(A) mRNAs and beads were washed with buffers A and B and then separated by adding 10 µL of Tris buffer. The resulting poly(A) mRNAs were reverse transcribed in 20-µL reactions containing oligo(dT)20, 5× RT buffer (containing 25 mM Mg2+), 10 U of the RNase inhibitor ReverTra Ace , and a 10 mM mixture of dNTPs. The secondary RNA structure was denatured by incubating at 42 °C for 60 min to facilitate cDNA production. The reaction was terminated by incubation at 99 °C for 5 min. The resulting cDNA was used as a template for PCR amplification. The following PCR conditions were used: 95 °C for 30 s, 60°C for 30 s, and 72 °C for 30 s, followed by extension at 72 °C for 5 min. The Mx3000P QPCR system  and SYBR premix Ex Taq  were used for qRT-PCR. The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence passes a fixed threshold above baseline. For the comparative analyses, mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and are expressed as the fold change. The sample delta Ct (SΔCT) value was calculated from the difference between the Ct values of GAPDH and the target genes. The relative gene expression levels between the samples and the controls were determined using the formula 2−(SΔCT−CΔCT). The primers used in the current study are listed in Poly(A) mRNAs were extracted from embryos using the Dynabeads mRNA Direct kit (Invitrogen) according to the manufacturer’s protocol. Briefly, after thawing, samples were lysed in 200 µL of lysis/binding buffer at RT for 10 min, and 20 µL of Dynabeads oligo(dT)Blastocysts were fixed in 4% paraformaldehyde overnight at 4 °C and washed three times in DPBS-PVA for 10 min each. For membrane permeabilization, the fixed blastocysts were incubated in PBS containing 0.5% Triton X-100 for 40 min at RT. Subsequently, blastocysts were washed three times in DPBS-PVA and stored in DPBS-PVA supplemented with 1 mg/mL BSA (DPBS-PVA-BSA) at 4°C overnight. The blastocysts were blocked with 10% normal goat serum for 45 min and then incubated overnight at 4 °C with primary antibody, mouse monoclonal anti-Cdx2 . Subsequently, the blastocysts were washed three times in DPBS-PVA-BSA for 10 min each and incubated for 40 min at RT with conjugated secondary antibodies, Alexa-Fluor-488-labeled goat anti-mouse IgG (1:200 in DPBS-PVA-BSA). After the blastocysts were washed three times in DPBS-PVA-BSA for 10 min each, the DNA was stained with 2 µg/mL DAPI. DAPI-labeled or Cdx2-positive nuclei were observed using a fluorescence microscope (Olympus). Approximately seven to eight blastocysts per treatment group were used in the immunocytochemistry in each independent experiment.To evaluate apoptotic blastomeres in blastocysts, a TUNEL assay was performed using an in situ cell death detection kit . Blastocysts were washed three times in DPBS-PVA and fixed in 4% paraformaldehyde overnight at 4 °C. Fixed blastocysts were permeabilized in DPBS containing 0.5% Triton X-100 at RT for 60 min. Nonspecific binding sites were blocked by incubation with DPBS containing 10 mg/mL BSA for 1 h. Subsequently, blastocysts were washed three times with DPBS-PVA and stained with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5 °C. Subsequently, the blastocysts were washed three times with DPBS-PVA and mounted on clean glass slides with DAPI. DAPI-labeled or TUNEL-positive nuclei were observed under a fluorescence microscope (Olympus). Total and apoptotic cell numbers per blastocyst were judged by counting the nuclei with blue (DAPI) and green (TUNEL) signals. Approximately seven to eight blastocysts per treatment group were used in the TUNEL assays in each independent experiment.p-values less than 0.05 were considered to indicate statistical significance.All experiments were repeated at least three times. Data are expressed as the means ± standard error of the mean (SEM). Data were analyzed by analysis of variance (ANOVA), followed by Tukey’s multiple range test (