In this review, the research progress of Sb2S3-based nanomaterials in the SIB field in recent years is summarized, including Sb2S3, Sb2S3/carbon composites, Sb2S3/graphene composites, and Sb2S3/MxSy composites. Furthermore, the challenges and prospects for the development of Sb2S3-based nanomaterials are also put forward. We hope this review will contribute to the design and manufacture of high-performance SIBs and promote its practical application.Recently, sodium-ion batteries (SIBs) have attracted extensive attention as potential alternatives to lithium-ion batteries (LIBs) due to the abundance, even distribution, low cost, and environmentally friendly nature of sodium. However, sodium ions are larger than lithium ions so that the anode materials of LIBs are not suitable for SIBs. Therefore, many negative electrode materials have been investigated. Among them, Sb Consequently, electrode materials matched with LIBs are not suitable for SIBs  have developed rapidly and are extensively used in electronic devices such as notebook computers, electric vehicles, and mobile phones . Neverthfor SIBs . Therefo. Metal sulfide anodes have a higher sodium storage capacity, and generally have lower redox potential, better electrochemical reversibility, and longer cycle life than metal oxides in charge/discharge reaction   , and L-Smposites , have be of SIBs . Based omaterial . Xu et amaterial  reviewedmaterial . However2S3-based nanomaterials for SIBs in recent years are summarized   . As showlectrode .2S3 nanosheets by using a facile and scalable Li intercalation assisted stripping method. The 2D-Sb2S3 nanosheets (2D-SS) showed a good layered structure with a mean thickness of 3.8 nm  nanomaterials with large surface area and ultrafine thickness have attracted more and more attention. For instance, f 3.8 nm . The larf 3.8 nm , the 2D-2S3 materials with three-dimensional (3D) hierarchical architecture were designed and synthesized to expand the contact surface area of the electrode and electrolyte and adapt it to volume expansion   . Used ass (MOFs) , the mul2S3 will undergo transformation and alloying reaction, which results in excessive volume expansion/contraction of the material, and hinders the application of Sb2S3 energy storage effect. Therefore, Sb2S3 is usually combined with carbon materials to inhibit the volume change, such as Sb2S3/carbon-rods . The obtained composite materials were denoted as SS/CFC. The flexible carbon fiber cloth was completely covered by spherical Sb2S3 in −1 at 0.5 A g−1, and displayed a reversible capacity of 736 mA h g−1 after 650 cycles in the voltage range of 0.01–2.00 V. After two initial cycles, the corresponding Coulombic efficiency of SS/CFC rapidly increased to ∼100%.For instance, l method . The Sb20 cycles . Liu et 0 cycles  reported2S3 can be combined with carbon doped with heteroatoms , thus improving the conductivity, the storage regions, and the active sites  . This was due to the double control synergy of Sb-shell structure and S-doped carbon structure, which effectively expanded the polysulfide diffusion path, enhanced the reversibility of conversion reaction, and thus improved the Na-storage capacity of SIBs  . The fin carbon) , SS/Sb@C of SIBs . This ki2S3 with graphene can provide excellent Na+ energy storage properties. Therefore, many composites have been designed in recent years, such as Sb2S3/RGO (RGO = reduced graphene oxide) (2S3-RGO (tin assisted Sb2S3 decorated on RGO) (2S3 (sulfur-doped RGO-based composite with Sb2S3) (2S3/N-C/RGO (Sb2S3@nitrogen-doped carbon decorated on RGO)  , Sn@Sb2S on RGO) , S-RGO/Sh Sb2S3) , and Sb2 on RGO) , to impr2S3 on RGO (RGO/Sb2S3) through a solution-based synthesis method and applied it as SIB anode materials. The RGO/Sb2S3 composite with a small particle size of 15–30 nm allows Na+ to move in and out of the particles rapidly during charge and discharge process. In addition, the 2D-layered structure of graphene and Sb2S3 can form oriented layered composites with excellent properties. Compared with traditional synthesis techniques, the ultrasound sonochemical method can create particular reaction conditions, and make it possible to prepare nanostructured materials with special properties by acoustic cavitation effects. 2S3/graphene by an ultrasound sonochemical synthesis technique  via firm chemical binding of nano-Sb2S3 structure on S-doped graphene nanosheets (SGS). Schematic illustration of the preparation process of the Sb2S3/SGS composite is displayed in 2S3 nanoparticles are wrapped by flexible SGS and exhibit a size of 30–80 nm. When tested at 0.05 A g−1 current rate, the Sb2S3/SGS anode reaches a high specific capacity of 792.8 mA h g−1 after 90 cycles  on graphene-based materials by surface chemical modification can effectively improve the properties of SIBs . For exacles see . After 9ee −1 in , the Sb2xSy) have hierarchical structures, and Na+ can easily move in the interlayers of metal sulfides without damaging their hierarchical structures (xSy), including SnS2  with a unique morphology of formicary microspheres was formed to solve the poor cycling stability and rate performance of SIBs  as a superior SIB anode material , capitalizing on full advantages of the zeolitic imidazolate framework (ZIF-8). The structure of ZnS-Sb2S3@C core-double shell composites had enough space to greatly adapt to the volume expansion during the repeated insertion/extraction of Na+, and exhibited a superior reversible capacity of 630 mA h g−1 at a current density of 0.1 A g−1 after 120 cycles with a high Coulombic efficiency of ∼100%  and In of SIBs . As showmaterial . After 6mA h g−1 . AdditiomA h g−1  designedof ∼100% .2S3@FeS2 hollow nanorods used as high-performance SIB electrode materials was reported. 2S3@FeS2 hollow nanorods (SFS) into a nitrogen-doped graphene matrix, and synthesized Sb2S3@FeS2/N-doped graphene composite (denoted as SFS/C) via a simple two-step solvothermal synthesis technique   . The SFS g−1 see . When te0 cycles .2S3-based nanomaterials, polypyrrole (PPy) , n = 0,1,2,3,4. e.g., Ti3C2Tx, Ti3C2)  (2S3 to fabricate better SIB anodes.In addition to the aforementioned Sble (PPy) , MXene  , and met., SnO2)  can also2S3/meso@microporous carbon nanofibers@polypyrrole composites (denoted as Sb2S3/MMCN@PPy) though a novel multi-step method combining polymerization, sulfidation and solvothermal process  via a solvothermal method  During the charge/discharge cycles, Sb spheres , to full2S3 to build dense conductive physical barriers. However, the content of Sb2S3 and the corresponding specific capacity of composite materials are reduced. Therefore, the carbon content should be optimized so that the Sb2S3-based materials achieve better electrochemical performance. In addition, Sb2S3/carbonaceous composites fabricated by traditional synthesis techniques suffer from the poor mechanical adhesion and high interface resistance between Sb2S3 and carbonaceous materials. It is highly desirable to optimize the preparation methods and explore more carbonaceous materials  to establish compact conductive physical barriers to further enhance the electrochemical performance of Sb2S3-based materials.2) Carbonaceous materials are often the main choice to combine with Sb2S3-based materials have been tested at room temperature. In order to satisfy the demands of different applications, it is very urgent to explore Sb2S3-based anode materials that can cycle under either higher temperature (up to 60 °C) or lower (−20°C).3) Until now, the cycle lives of many Sb+ storage in Sb2S3-based nanomaterials and the phase changes during repeated charging/discharging still need to be explored. Operating technologies, such as in situ X-ray technology, in situ scanning probe microscopy, technologies based on synchronized X-rays, as well as in situ electron microscopy, are very helpful in acquiring time-related information and studying the mechanism of Na+ storage of Sb2S3-based nanomaterials. Therefore, more research using operating technology is needed to deeply understand Sb2S3-based electrode nanomaterials used in SIBs.4) The mechanism of Na


Cell death was analyzed using TUNEL and propidium iodide staining. The correlation between METTL3 and glutathione peroxidase 4 (GPX4) was analyzed using Spearman’s correlation test and further confirmed using the CHIP assay. Western blotting and RT-qPCR were performed to measure the relative expression levels. Mice were injected with 0.2 units collagenase IV to establish an ICH model in vivo. We found that the Fe2+, reactive oxygen species, LPO, and MDA levels were enhanced, and glutathione was depleted in OGD/H-treated BMVECs as well as in ICH mice. Additionally, cell viability and SLC7A11 protein levels decreased, and cell death and TFR1 protein levels increased in OGD/H-treated BMVECs. METTL3 silencing relieves OGD/H-induced injury in BMVECs. In addition, METTL3 was significantly negatively related to GPX4, which was further confirmed by the CHIP assay. Silencing of METTL3 decreased the N6-methyladenosine levels of GPX4 and increased its mRNA levels of GPX4. GPX4 knockdown neutralized the role of METTL3 in OGD/H-treated BMVECs. These results implied that ferroptosis occurred in the ODG/H-treated BMVECs and ICH mouse models. METTL3 silencing effectively suppressed ferroptosis by regulating N6-methyladenosine and mRNA levels of GPX4.This study examined the effects of methyltransferase-like 3 (METTL3) on ferroptosis during intracerebral hemorrhage (ICH) progression. The brain microvascular endothelial cells (BMVECs) were stimulated with oxygen and glucose deprivation (OGD) and hemin to establish an ICH model. Cell viability was tested using a CCK8 assay. The levels of Fe  Ferroptosis was occurred in the ODG/H treated BMVECsMETTL3 was negatively related to GPX4Ferroptosis was occurred in the ICH miceIntracerebral hemorrhage (ICH) refers to hemorrhage caused by the increase and rupture of vascular brittleness in the non-traumatic brain parenchyma . In the Ferroptosis, a new type of cell death, was first proposed by Dixon et al. In 2012 . FerroptN6-methyladenosine (m6A), the most common modification in epigenetic changes, drives a variety of biological functions including promotion of tissue development, stem cell differentiation, and repair of DNA damage responses . MethyltTherefore, this study aimed to investigate whether METTL3 could alleviate ICH by blocking ferroptosis. We hypothesized that METTL3 silencing suppressed ferroptosis by regulating m6A and mRNA levels of GPX4.2, 95% N2) for 10 min. Accordingly to a previous study [2, 95% N2). Finally, normal culture conditions were used to terminate oxygen and glucose deprivation (OGD). Additionally, the ferroptosis inducer RSL3 (5 μM) and inhibitor Fer-1 (1 μM) were used to treat the BMVECs to inhibit ferroptosis.Brain microvascular endothelial cells (BMVECs) were obtained from SAIOS Biology  and were selected to establish a cell model of ICH. Briefly, BMVECs were maintained in Dulbecco’s modified Eagle’s medium without glucose and serum  were seeded in 96-well plates and cultured for 24 h . Next, 10 μl CCK8 solution was used to treat the cells in each well for 4 h. The absorbance at 450 nm was measured using a microplate reader.CCK-8 assay was carried out to test the cell viability . 1 × 1052+ levels in the cells were measured using an iron colorimetric assay kit . Additionally, GSH, ROS, MDA, and LPO levels in the cells were analyzed using the corresponding kits purchased from Nanjing Jiangcheng Bioengineering Institute . All experimental steps were performed in strict accordance with the manufacturer’s instructions [The Feructions .The death of HT22 cells in each group was evaluated using TUNEL and PI staining assays  [−ΔΔCt method [The RNA from cells in each group was collected using TRIzol (Beyotime). Subsequently, a Nanodrop 2000 spectrophotometer was used to determine RNA quality. To obtain cDNA, reverse transcription was performed using a Reverse Transcription Kit . Next, cDNAs were used for the RT-qPCR with HiScript® Q RT SuperMix for the qPCR kit (Vazyme): 95°C, 10 min; 94°C, 15s, 40 cycles; 55°C, 30s; 70°C, 30s. The results were analyzed using the 2t method , and GAPMETTL3: Forward 5’-TTGTCTCCAACCTTCCGTAGT-3’, Reverse 5’-CCAGATCAGAGAGGTGGTGTAG-3’;TFR1: Forward 5’-ATGTTGGATGGGTAGCCAAAG-3’, Reverse 5’-TTCGAGAGCGCAAATCTTCTG-3’;SLC7A11: Forward 5’-TCTCCAAAGGAGGTTACCTGC-3’, Reverse 5’-AGACTCCCCTCAGTAAAGTGAC-3’;GPX4: Forward 5’-GAGGCAAGACCGAAGTAAACTAC-3’, Reverse 5’-CCGAACTGGTTACACGGGAA-3’;Primary antibodies  were obtained from Abcam  and used to test the levels of SLC7A11, TFR1, and GPX4 in the cells. Briefly, proteins were extracted from cells and tested using a BCA kit (Beyotime). Next, 10% SDS-PAGE was performed, and the proteins were transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 2 h and then incubated with primary antibodies for 12 h at 4°C. The membranes were then incubated with the secondary antibody for 1  h. Finally, the chemiluminescence kit was utilized to visualize the proteins, and the bands were photographed and analyzed using ImageJ, with GAPDH as the internal parameter .Cells transfected with sh-METTL3 or sh-nc were used to perform the MeRIP assay using the Magna MeRIP™ m6A Kit (Millipore) . BrieflyAccording to a previous study , the celC57BL/6 mice were purchased from Nanjing Jiangcheng Bioengineering Institute  and divided into sham (n = 8) and ICH (n = 8) groups. All mice were housed in a controlled environment  with free access to food and water. Mice were acclimatized for 1 week before the experiments. According to a previous study , the micAfter successful modeling, mice in each group were weighed and anesthetized. Then the head was cut off, and the left and right brain tissues were collected and weighed Next, the brain tissues were baked at 100°C for 24 h and weighed. The water content of brain tissue = (wet weight – dry weight)/wet weight×100% .SPSS analyses were performed using analysis 25.0. All results are shown as mean ± SD. The difference analysis was performed using the Student’s t-test and analysis of variance (ANOVA). Spearman’s correlation test was used to analyze the relationship between METTL3 and other genes. Statistical significance was set at P < 0.05.This study demonstrated that ferroptosis occurred in the ODG/H-treated BMVECs and ICH mouse models. METTL3 silencing effectively suppressed ferroptosis by regulating m6A and mRNA levels of GPX4.2+ , ROS (F2+ ), LPO (F2+ ), and MD2+ ) levels 2+ ) were de2+, GSH, ROS, LPO, and MDA levels of BMVECs . Additif BMVECs ).FigureSubsequently, through TUNEL and PI staining, we found that ODG/H treatment prominently enhanced the number of TUNEL- and PI-positive cells, and Fer-1 or sh-METTL3 treatment prominently neutralized the ODG/H effects. Meanwhile, Fer-1 prominently enhanced sh-METTL3 transfection effects ). AdditiNext, we analyzed the relationship between METTL3 and GPX4, NQO1, HMOX1, ACDH3A1, and AKR1C1 using Spearman’s correlation test and found that METTL3 was significantly negatively related to GPX4 (r = 0.7227) ). The CH2+, GSH, ROS, LPO, and MDA levels in ODG/H-treated BMVECs . Next, d BMVECs ). Additid BMVECs ), SLC7A1d BMVECs ) in the 2+ . In add2+ ), ROS (F2+ ), LPO (F2+ ), and MD2+ ) levels 2+ ) were de2+ ).FigureStudies have shown that ICH is a subtype of stroke that has a high mortality rate. Accumulating studies have shown that ferroptosis is crucial to the progression of brain injury after ICH ,30. TherIn recent years, m6A modifications have become a popular topic in epigenetic research. M6A modification is involved in many biological processes in mammals, such as RNA splicing, protein translation, and stem cell renewal . Li et a3+ carried by transferrin to the cytoplasm by binding to transferrin, which is the main mechanism by which cells absorb iron [2+, GSH, ROS, LPO, and MDA levels, along with SLC7A11 and TFR1 levels in the ODG/H-treated BMVECs. These results indicate that METTL3 regulates ferroptosis by targeting GPX4.Ferroptosis involves multiple pathways including iron metabolism, lipid metabolism, and oxidative damage ,36. SLC7orb iron . Therefoorb iron . Furtherorb iron ,41. ThroHowever, there are still some limitations in this study. This study was a basic research. It needs to be further verified in clinical treatment. Our future research will conduct clinical trials to test our conclusions.In conclusion, our research demonstrates that ferroptosis occurs during ICH progression. METTL3 silencing suppressed ferroptosis development by regulating m6A levels of GPX4 in ODG/H-treated BMVECs. These findings provide new insights into ICH treatment.Click here for additional data file.


Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (P < 0.05). The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight, ovulation rate, mean litter weight, number of live foetuses, percentage prenatal survival, and sex ratio of the litter was assessed. Maternal 25(OH)D (P = 0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (P < 0.05) concentrations were inversely associated with gilt weight. Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90 (P < 0.05). This study has provided novel insights into temporal changes in maternal vitamin D status throughout gestation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight, percentage prenatal survival and percentage of male foetuses in the litter. Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species. However, the temporal changes in maternal vitamin D status throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated. The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (The online version contains supplementary material available at 10.1186/s40104-022-00760-w. Vitamin D then enters the systemic circulation and is transported to the liver where it is hydroxylated to form the main circulating metabolite of vitamin D . This metabolite is transported to the kidney where it can be further hydroxylated to 1,25-dihydroxyvitamin D  2D) –6. Addit5[OH]2D) .In addition to well-characterised roles postnatally, emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species . VitEvidence from multiple species has proven unequivocally that phosphate, calcium, and vitamin D are essential during pregnancy for the appropriate regulation of foetal growth . YetAll procedures were performed with approval from The Roslin Institute (University of Edinburgh) Animal Welfare and Ethical Review Board and in accordance with the U.K. Animals (Scientific Procedures) Act, 1986.n = 5), 30 (n = 9), 45 (n = 6), 60 (n = 12) and 90 (n = 9). Gilts were euthanised on the GD of interest with sodium pentobarbitone 20% w/v  at a dose of 0.4 mL/kg by intravenous injection via a cannula inserted in the ear vein. Immediately before euthanasia, cardiac puncture was performed using an EDTA-coated syringe to collect maternal blood. Plasma was obtained from this sample by centrifugation (1500 × g for 15 min at 4 °C) and stored at −20 °C until required.Large White × Landrace gilts (age 11-14 months) were observed daily for signs of oestrus and were housed indoors in groups of 6-8 animals per pen. Oestrous cyclicity and ovarian function were controlled in accordance with routine normal practice at The Roslin Institute Large Animal Unit . All gilFollowing confirmation of death, gilts were hysterectomised. On GD18, the uterine tract was rinsed with saline and string was used to tie the end of the right and left uterine horns at the bifurcation. The uterine horns were cut between the two pieces of string and each uterine horn was placed in a floatation device. The device contained a solution to preserve the integrity of the RNA  and 40 mL of 0.5 mol/L ethylenediaminetetraacetic acid (EDTA) were added. The solution was adjusted to pH 5.2 using concentrated sulphuric acid and stored at room temperature until required. Using dissection scissors, the uterine horn was opened along the mesometrial side, and the conceptuses floated in the solution. Individual conceptuses were removed from the floatation device with forceps and weighed. On GD30, 45, 60, and 90, both uterine horns were dissected from the ovary towards the cervix as described previously . DNA wasThe ovaries were dissected to determine the number of corpora lutea present, representing the ovulation rate of the animal. The percentage prenatal survival was then calculated from the ovulation rate, and the number of live conceptuses (embryo/foetus and associated extra-embryonic membranes) present in the uterine tract at the time of euthanasia.2 and 25(OH)D3 metabolites were determined by HPLC–MS/MS analysis as described previously [The concentration of 25(OH)Deviously .2 stock solution  and 30 µL of 25(OH)D3 stock solution  into 1 mL artificial serum [50 mg bovine serum albumin  were dissolved in 1 mL of phosphate buffered saline], then 1 in 2 serial dilution with artificial serum. The concentrations of calibration standards were 230.8, 115.4, 57.7, 28.9, 14.4, 7.2, 3.6 and 1.8 nmol/L for 25(OH)D2; and 356.6, 178.3, 89.2, 44.6, 22.3, 11.2, 5.6 and 2.8 nmol/L for 25(OH)D3. These calibration standards were used to generate standard curves for quantification of the concentration of 25(OH)D2 and 25(OH)D3 in pig plasma by HPLC–MS/MS analysis.Eight calibration standards were freshly prepared, by adding 20 µL of 25(OH)D2H3-25(OH)D2  and 2 µL of 23,24,25,26,27-13C5-25(OH)D3 , as internal standards. After adding 20 µL of 1 mol/L NaOH, each plasma sample or calibration standard was then protein precipitated by the addition of 200 µL of acetonitrile. The supernatant of the plasma sample or the calibration standard was purified by solid phase extraction using a Discovery DSC-18 SPE-96 Plate . Briefly, the plate was activated with 3 mL of ethyl acetate, 3 mL of methanol and 3 mL of distilled water. After addition of a mixture of supernatant (approximately 300 µL) from protein precipitation and 1 mL 0.4 mol/L K2HPO4, the plate was washed with 3 mL of distilled water and 2 mL of 40% methanol sequentially and eluted with 1.5 mL of acetonitrile. After evaporating to dryness, samples were derivatized by 2 additions of 25 µL of 0.1 mg/mL DMEQ-TAD ethyl]-3H-1,2,4-triazole-3,5(4H)-dione; Abcam, Cambridge, U.K.) in ethyl acetate. After evaporation to dryness, derivatized extracts were reconstituted in 25 µL of 60:40 (vol:vol) methanol and 0.1% formic acid:water for HPLC–MS/MS analysis.After plasma samples (0.5 mL) were thawed at room temperature, 100 µL of each sample, or calibration standard, was spiked in a 1.5-mL micro-tube with 2 µL of 6,19,19-The HPLC–MS/MS analyses were conducted using an UltiMate 3000 HPLC system interfaced to an amaZon ETD tandem mass spectrometer . Chromatographic separations were achieved using an ACE UltraCore 2.5 SuperC18 column , maintained at 40 °C. Gradient elution was performed, with the mobile phase consisting of 10 mmol/L ammonium formate  with 0.15% formic acid (buffer A) and methanol with 0.1% formic acid (buffer B). The elution was detected using multiple reaction monitoring with positive electrospray ionisation, with a total runtime of 12 min per sample. Quantitation was carried out using QuantAnalysis 2.0 software . The standard curve was generated based on the ratio of the peak area of the standard to that of the corresponding internal standard.P value of ≤ 0.05 was obtained, then the data were not considered to have a normal distribution. Log10 transformations were carried out where required to achieve a normal data distribution. ANOVA was performed for GD, with a post-hoc Tukey analysis. Linear regressions were performed for total 25(OH)D with ovulation rate, mean litter weight, gilt weight, litter size, and percentage prenatal survival. These regressions were performed both within GD and with all GD combined. Results were considered significant when P ≤ 0.05, trending towards significant when 0.05 > P < 0.1, and not significant when P > 0.1.All statistical analyses were performed using GenStat 13.1  and Minitab. Mean values were calculated for each individual sample for each parameter investigated and the normality of the distribution of the data was assessed by an Anderson–Darling test. If a 2 was present in small amounts in maternal plasma (2.63 ± 0.19 ng/mL) when compared with 25(OH)D3 (58.30 ± 7.41 ng/mL) representing 7.28 ± 0.83% and 92.72 ± 0.83%, respectively, of total 25(OH)D. Total 25(OH)D metabolite concentration is presented in this study and reflects the sum of 25(OH)D2 and 25(OH)D3 metabolites.As anticipated, 25(OH)DP < 0.05).Maternal plasma total 25(OH)D Fig.  concentrP = 0.059). On GD90, maternal plasma total 25(OH)D concentrations were inversely associated with gilt weight (Table P < 0.05). An inverse association between maternal plasma total 25(OH)D concentrations and the percentage of male foetuses in the litter was observed on GD90 (Table P < 0.05). Maternal plasma total 25(OH)D concentrations tended to be inversely associated with ovulation rate when analysing data with all GD of interest combined (Table P = 0.085). Litter size and mean litter weight were not associated with maternal plasma total 25(OH)D concentrations on any GD investigated D concentrations tended to be positively associated with percentage prenatal survival on GD60 D was greater on GD30 compared to GD18. GD30 corresponds to several important events in pregnancy in the pig , includi2 and/or 25(OH)D3. Additionally, it has been suggested that wild ewes from St. Kilda with higher vitamin D status pre-mating had more lambs surviving to one year of age [Maternal plasma total 25(OH)D concentrations tended to be positively associated with percentage prenatal survival at GD60. It has been suggested that maternal vitamin D status is not associated with the number of lambs born in sheep . Howeverr of age . CollectVDR mRNA peaks on day 90 of gestation, and that the endometrial expression of CYP2R1 (25-hydroxylase) and CYP27B1  mRNAs increase with advancing gestational day [This study demonstrated novel associations between the concentration of 25(OH)D in maternal plasma and the sex ratio of the litter. On GD90, maternal plasma total 25(OH)D was inversely associated with percentage of males in the litter. Across mammalian species it has been demonstrated that sex influences birth weight, with males being heavier than females; a characteristic that can be observed from an early stage of gestation –38. Maleonal day . Collectonal day –44. Addional day –52. CollEmerging evidence suggests a critical role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of conceptus growth across mammalian species. This study suggests in pigs that maternal vitamin D status may be altered from as early as GD30 to ensure appropriate transport of minerals to the conceptus. Further, this has provided novel insights into the relationship between maternal vitamin D status and several economically important litter characteristics across gestation.Additional file 1. Table S1: Regressions between litter size and mean litter weight, and total 25(OH)D in maternal plasma on days 18, 30, 45, 60, and 90 of pregnancy.


In addition, due to the presence of silicon, the Si3N4-doped materials showed excellent angiogenesis properties, including the promotion of cell migration and tubule formation, which play essential roles in early recovery after implantation.Titanium (Ti) implants have been widely used for the treatment of tooth loss due to their excellent biocompatibility and mechanical properties. However, modifying the biological properties of these implants to increase osteointegration remains a research challenge. Additionally, the continuous release of various metal ions in the oral microenvironment due to fluid corrosion can also lead to implant failure. Therefore, simultaneously improving the bioactivity and corrosion resistance of Ti-based materials is an urgent need. In recent decades, micro-arc oxidation (MAO) has been proposed as a surface modification technology to form a surface protective oxide layer and improve the comprehensive properties of Ti. The present study doped nano silicon nitride (Si With increasing success rates, dental implants have become the treatment of choice to replace missing teeth. Due to its good mechanical properties and biocompatibility, titanium (Ti) has been widely used in dental implants . Howevere.g., roughness, wettability, chemical/crystalline phase composition, etc.) of Ti. Ti-based implants with micron/submicron hierarchical structures produced by sandblasting/acid etching (SLA) treatment are representative of commercial implants used in the clinical setting for many years. Since SLA implants are exposed to a body fluid microenvironment rich in chloride ions and proteins after implantation, they also show certain defects caused by corrosion , even those with excellent osteointegration  has been increasingly favored by researchers for its excellent physiochemical properties, biocompatibility, and osteogenic inductivity  further explore the potential of Si3N4-doped coatings in promoting angiogenesis and osteointegration.We successfully constructed a series of Sie by MAO . This st3N4  were added, including 2, 4, and 6 g/L. MAO was applied by DC pulse power for 5 min with a platinum sheet as the cathode. The parameters were an applied voltage of 500 V and fixed duty cycle and frequency of 20% and 1000 Hz, respectively. Depending on the Si3N4 concentration, the samples were named MAO-SN2, MAO-SN4, and MAO-SN6, respectively. MAO referred to the pristine micro-arc oxidized specimen. After MAO treatment, the samples were cleaned in a 100 W ultrasonic device for 10 min to remove unnecessary impurities or adhered Si3N4 particles. All samples were sterilized in 75% ethanol and with ultraviolet radiation for 30 min before cell culture.Commercial pure titanium plates  with dimensions of 1 cm × 1 cm were used as the substrates for MAO treatment. The plates were polished with sandpaper and washed in acetone, ethanol, and deionized water by ultrasound to remove unnecessary pollutants. The electrolyte solution contained 10 g/L trisodium phosphate  and deionized water. Different concentrations of SiThe surface morphology and roughness of samples were observed by scanning electron microscopy  and atomic force microscope . An instrument to measure contact angle  was used to determine the wettability. The chemical compositions of the different coatings were determined by X-ray photoelectron spectroscopy .in vivo. Therefore, in this experiment, the samples were immersed in PBS solution (1.2 cm2/ml) at 37°C. The PBS extract containing Si4+ was collected at five different time points  and the Si4+ concentrations were determined using an inductively coupled plasma optical emission spectrometer .Phosphate buffered (PBS) solution has a similar ion concentration to physiological liquid and is usually used to model the environment An electrochemical workstation  was used to test the corrosion resistance. First, a working area measuring 0.5 cm × 1 cm was marked on different substrates, with the remaining uninspected area encapsulated in epoxy resin. Normal saline was used as the electrolyte, a platinum plate as the counter electrode, the sample as the working electrode, and a saturated calomel electrode as the reference electrode. Before measurement, pure nitrogen was filled into the electrolyte for 30 min to reduce the oxygen concentration. After another 30 min to stabilize the current, the Tafel curves of the different samples were scanned at 1 mV/s. Dynamic potential scanning was performed in the potential range of −1.5 V–1.0 V. All operations were performed at ambient temperature. The corrosion potential (Ecorr) and corrosion current density (Icorr) were finally calculated using ImageJ software.4 cells/cm2 on the surface of each sample in 24 well plates. After 3 d, the cells were fixed with 4% fixative solution at 4°C for 40 min. Subsequently, 1% Triton X-100 solution was added for 15 min to increase the cell membrane permeability. Tetramethylrhodamine phalloidin  and 4′,6-diamidino-2-phenylindole  were then added and the samples were incubated in the dark for 40 and 15 min to dye the cytoskeleton and nucleus respectively. Finally, a confocal laser scanning microscope  was used to observe the cell morphology.The commercial MC3T3-E1 osteoblast cell line (American Type Culture Collection) was cultured at 1 × 104 cells/cm2, the MC3T3-E1 cells were fixed, dyed with DAPI to color the nucleus, and observed by CLSM. The cell numbers were calculated using ImageJ software.After culturing for 3 h at a density of 2 × 104 cells/cm2) were incubated on each sample to evaluate cell viability. After 3 or 7 d, the media was replaced with 200 μL fresh media and 20 μL 3--2,5-diphenyltetrazolium bromide (MTT) solution. The plates were then incubated for 4 h. The solution was then removed and 1 ml dimethyl sulfoxide (DMSO) was added per well. After incubation on a 37°C shaker for approximately 15 min, 200 μL of the solution was transferred to 96-well plates and read on a microplate reader  to determine the OD values at 490 nm. All procedures were performed under dark conditions.Each group of samples was placed into 24-well plates in six replicates. MC3T3-E1 cells (2 × 104 cells/cm2) on various samples for 3 and 7 d, an ALP Assay Kit  was used to determine the level of ALP activity. Briefly, 1% Triton-100 was added to lyse cells for 40 min after washing the samples in PBS. The substrates, deionized water for the blank, standard solution for the control, and corresponding cell lysates for samples were added to 96-well plates, respectively. They were then mixed with buffer solution and Matrix fluid before incubation at 37°C for 15 min. Chromogenic agent was then added and the OD values were measured at 520 nm. The protein content of each sample was also measured using a BCA Protein Assay Kit .After culturing MC3T3-E1 cells (2 × 104 cells/cm2) for 14 d, the samples were washed with PBS and fixed with 4% fixative solution at 4°C for 15 min. Mineralized nodules on the surfaces were observed by microscope after soaking 20 min in Alizarin Red S solution and thorough washing in deionized water. Finally, cetylpyridinium chloride was added to dissolve the nodule. The OD values were measured at 490 nm.After culturing MC3T3-E1 cells (2 × 104 cells/cm2) were cultured on the samples for 7 d. Total RNA was extracted using an RNA simple Total RNA kit . The total RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit . Finally, the expression of target genes  was detected using a SYBR Premix EX Taq Kit . The relevant primers are listed in MC3T3-E1 cells  were cultured at 2 × 104 cells/cm2) were cultured in 24-well plates in high-glucose DMEM for 3 d until the cells reached 80%–90% confluency. A cross scratch was made using a white spear in every well. The peeling cells caused by the scratch were then gently rinsed with sterile PBS. The cells were cultured in an extraction solution with 1% fetal bovine serum. The wound healing in the same area was observed and photographed using an inverted phase contrast microscope after 0, 12, and 24 h. Finally, ImageJ software was used to measure and calculate the migration areas of the HUVECs.HUVECs (2 × 105 cells/cm2) were inoculated onto the plate with different extract solutions containing 15% fetal bovine serum. Tube formation was observed and photographed using an inverted phase contrast microscope after 0, 3, and 10 h. Finally, Angiogenesis Analyzer in ImageJ was used to measure and calculate the number of nodes (points adjacent to three pixels) and junctions (the border of the overlaying of four nodes), as described previously (Frozen Matrigel matrix glue was melted in advance at 4°C and added to a 6-well plate (50 μL/well). The plate was then transferred to a 37°C incubator for 30 min. HUVEC cells (1.5 × 10eviously .4 cells/cm2) were cultured in an extraction solution containing 10% fetal bovine serum. After 3 d of culture, total RNA was collected and reversed-transcribed to cDNA as described above. Finally, the expression of target genes  was detected using a SYBR Premix EX Taq kit . The relevant primers are listed in HUVECs  and Student’s t-test, and expressed as means ± standard deviation. 3N4-doped coatings, we performed a series of tests were performed on the samples. As shown in et al. , MAO , MAO-SN2 , MAO-SN4 , and MAO-SN6 .3N4, the WCA showed a slightly declining tendency from MAO-SN2 to MAO-SN6. The WCA values were: MAO, ∼20.8°; MAO-SN2, ∼25.6°; MAO-SN4, ∼15.6°; and MAO-SN6, ∼13.2°. After MAO treatment, the hydrophilicity of the materials decreased significantly (p < 0.05) compared to pure Ti, but did not differ significantly among the four groups of MAO-treated samples. The possible mechanisms of the hydrophilicity change might be as follows: 1) according to Cassie–Baxter’s solid-liquid-air complex contact model, the droplets were not only in contact with the surface structure but also with the air in the surrounding space (Assessment of the wettability of the different samples illustrated in ng space  and 2) ang space .3N4 doping, Si and N in the MAO-SN2 group reached 12.8 at% and 5.4 at%, respectively. The Si and N content in the MAO-SN4 and MAO-SN6 groups further increased with increasing of Si3N4 doping concentration . In addition, the content of elemental Ti and P in the coating gradually decreased . Further analysis of Si2p , 1.2:1 (MAO-SN4), 0.6:1(MAO-SN6)].The XPS results of the various MAO-treated coatings are shown in  of Si2p  showed tet al. . Although the MAO and Si3N4-doped groups did not differ significantly, the corrosion resistance improved with increasing Si3N4 concentrations. The Icorr values for Ti, MAO, MAO-SN2, MAO-SN4, and MAO-SN6 were approximately 1.506, 0.290, 0.245, 0.081, and 0.075 μA/cm2, respectively. The Ecorr values were approximately -0.174, -0.002, 0.063, 0.038, and 0.052 V, respectively, indicating that the MAO-treated groups had a lower thermodynamic tendency toward electrochemical corrosion . In addition, as shown in To comprehensively evaluate the osteogenic properties of the SiM images  revealed3N4 doping. At 7 d, MAO-SN4 and MAO-SN6 showed excellent performance, with significantly better performance for early osteogenesis compared to Ti (p < 0.05). Regarding mineralization, the results of the quantitative analysis (p < 0.05). The light microscopy images . Similar results were reported by Xu et al.  after 7 d, indicating the good effects of MAO-SN6 on cell viability in HUVECs.To fully consider the vascularization ability of Si vessels . Because vessels  did not 3N4 doping groups were significantly better than those of the Ti and MAO groups. The scratch wounds in the MAO-SN6 group basically healed, showing the best effect, followed by the MAO-SN4 group. Next, cell migration experiments were used to explore wound healing. As shown in in vitro. HUVECs are connected into lines, in which the nodes represent typical early-stage angiogenesis. In the middle and late stages, vascular branches are cross-linked to form a vascular network structure   . Osumi eogenesis .3N4-doped coatings on Ti by MAO. Si3N4 oxidation at local high temperatures finally formed a mixture of Si3N4 and SiO2. The Si3N4-doped samples showed better hydrophilicity compared to pure Ti due to improved surface roughness and promoted cell adhesion without damaging cell viability. The results of the osteogenesis experiments indicated that the Si3N4 modification performed well in the early and late stages of osteogenic differentiation in a dose-dependent manner. These experiments also showed good angiogenesis ability, including cell migration and tubular formation, by releasing silicon ions. The results of the corrosion test verified excellent corrosion resistance of the Si3N4-doped coatings. Therefore, the multifunctional Ti-based implant showed good osteogenesis, angiogenesis, and corrosion resistance, which suggested its potential in clinical applications.In this study, we designed a series of Si


The nanocomposite has ZnO, MnO2, and ferrites with a surface area of 21.03 m2 g−1. The adsorbent was effective in mineralizing acidic sulfurous gases better in wet conditions. The material exhibited a maximum H2S and SO2 removal capacity of 1.31 and 0.49 mmol g−1, respectively, in the optimized experimental conditions. The spectroscopic analyses confirmed the formation of sulfide, sulfur, and sulfite as the mineralized products of H2S. Additionally, the nanocomposite could convert SO2 to sulfate as the sole oxidation by-product. The oxidation of these toxic gases was driven by the dissolution and dissociation of gas molecules in surface adsorbed water, followed by the redox behaviour of transition metal ions in the presence of molecular oxygen and water. Thus, the study presented a potential nanocomposite adsorbent for deep desulfurization applications.A ternary Mn–Zn–Fe oxide nanocomposite was fabricated by a one-step coprecipitation method for the remotion of H H2S is a toxic pungent-smelling gas released from decayed organic matter, oil industry, coal and natural gas-based thermal power plants, and sewage treatment facilities2. Acute exposure to H2S at levels of 200–500 ppm could paralyze the olfactory nerve and beyond 500 ppm could lead to sudden death3. Furthermore, H2S conversion to SO2 and its hydrolysis to form acid rain could acidify soil and water bodies, which could be disastrous to plants and marine life, respectively5. SO2 is a colourless toxic gas with a sharp odour, which could cause various respiratory ailments such as chronic bronchitis and infections of the respiratory tract6. While a low SO2 concentration of 1–5 ppm is enough for human discomfort, exposure above 100 ppm could be life-threatening7. The main sources of atmospheric SO2 are thermal power plants and vehicular emissions8. Thus, H2S and SO2 removal from point of origin should be prioritized to limit air contamination and prevent catastrophic events like smog formation and acid rain.Air contamination is a global issue which has been amplified by various anthropogenic activities in the last many decades. Among numerous air contaminants toxifying the air, hydrogen sulfide  and basic OH– groups, which could react with acidic gases like H2S and SO2 (acting as electron donors)12. The surface reactivity of metal oxides for these gases could be amplified in the presence of water molecules. Firstly, the water layer on the metal oxide surface dissociatively reacts and improves the hydroxyl density. Secondly, the surface water film dissolves the gas molecules, which lowers the energy barrier for reactive interaction with the metal oxide surface and thus favours the overall chemisorption process17. Thus, it is worth exploring the positive effect of water during the adsorption of acidic gases over metal oxides, which is the focus of this research work. Also, it is equally important to explore adsorbent materials for the remediation of low H2S/SO2 concentrates to confirm the applicability of adsorbents in deep desulfurization and gas purification applications.Chemical adsorption of these toxic gases over an adsorbent surface is one of the most simplistic and affordable methods to adsorb and mineralize H2S and SO2 gases in wet conditions. The gas concentration of 500 and 100 ppm for H2S and SO2 was adopted for their industrial application and suitability in capturing these pollutants in the toxicity range for humans. The oxide showed better adsorption performance in wet conditions with complete mineralization to non-toxic by-products. Besides studying the factors affecting the adsorption process, the adsorption mechanism was studied in detail using various microscopic and spectroscopic techniques. The study confirmed that the oxide nanocomposite has the potential to eliminate and mineralize low concentrations of gaseous H2S and SO2 in dry–wet conditions.In this study, we have fabricated an affordable Mn-Zn-Fe metal oxide nanocomposite by a one-step coprecipitation method for room-temperature adsorptive removal of H3)2·4H2O), zinc(II) nitrate hexahydrate (Zn(NO3)2·6H2O), iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O), and 2.0 mol L−1 NaOH solution were procured from Samchum Pure Chemicals, Korea. H2S gas (0.05 Vol.%) and SO2 gas (0.01 Vol.%) balanced with pure N2 gas were procured from Union gas, Korea. All solutions were prepared in double-distilled water.Manganese(II) nitrate tetrahydrate (Mn(NO3)2·4H2O, 4.45 g of Zn(NO3)2·6H2O, and 4.04 g of Fe(NO3)3·9H2O were dissolved to give a final Mn2+:Zn2+:Fe3+ ratio of 3:3:2. A higher ratio of divalent to trivalent cations was adopted for the formation of MnO2 and ZnO in the nanocomposite (for a higher acidic gas adsorption capacity). Under vigorous stirring, 2.0 mol L−1 NaOH solution was added dropwise until the solution pH reached 12.5. This pH was sufficient for the formation of ternary oxide nanocomposites as reported earlier18. After stirring for 2 h, the precipitate was phase separated and dried at 393 K overnight in a hot air oven. The use of an excess of water for washing was avoided to reduce the overall impact on the environment during the material fabrication process.In 50 mL of deionized water, 3.76 g of Mn instrument after degassing at 423 K for 6 h with a mass of 0.324 g. The powder X-ray diffraction (PXRD) patterns were obtained at room temperature (2θ = 5–50°) on an Ultima IV  X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å) and a Ni filter. Fourier-transform infrared (FTIR) spectra of samples were recorded using KBr pellets over a Cary670 FTIR spectrometer . For X-ray photoelectron spectroscopy (XPS) analysis, a K-alpha XPS instrument  was used with a monochromatic Al Kα X-ray source. The pressure was fixed to 4.8 × 10−9 mbar. Spectra were charge corrected to the main line of the C 1 s (aromatic carbon) set to 284.7 eV. Spectra were analysed using CasaXPS software (version 2.3.14).The morphology in the surface and transmission mode was probed over field emission scanning electron microscopy  and field emission TEM , respectively. SEM analysis was done on finely grounded dried samples after coating them with a gold-platinum alloy by ion-sputtering (E-1048 Hitachi ion sputter). The elemental analysis was done using energy-dispersive X-ray spectroscopy . The specific surface area and porosity were determined by analysing the standard N19. The H2S gas (0.05 Vol.%) or SO2 gas (0.01 Vol.%) was passed through it at a fixed flow rate. The outgoing gas was analyzed using an H2S gas analyzer  or an SO2 gas analyzer . The analyzer recorded the effluent gas concentration every minute in real-time until the breakthrough points of 20% (100 ppm for H2S and 20 ppm for SO2) were reached and after that the experiment was completed. The wet samples were prepared by passing water vapours (80% relative humidity) directly through the adsorbent bed before passing the gas through it. The gas adsorption capacity was measured using the following equation:C0-initial concentration (mg L−1), C-concentration at time ‘t’ (mg L−1), Q-flowrate (L min−1), m-the mass of adsorbent (g), and tb-breakthrough time (s).The gas adsorption experiments were performed by taking 0.5 g of the adsorbent in a Pyrex tube (height: 50 cm and diameter: 1 cm) at 298 K. The sample was fixed between the glass wool and supported on silica beadsd) values from the XRD pattern. The fringe width of 0.308, 0.530, and 0.261 nm were assigned to the MnO2 (110)20, ZnO (0002)21, and MFe2O4 (311)22, respectively. The EDAX elemental analysis confirmed peaks for Mn, Zn, Fe, and O at respective energies having the atomic contribution of 13.80, 7.14, 3.57, and 75.48%, respectively 23, ZnO (pink circle)24, ferrites (green square)25, and NaNO3 (blue square)26, which confirmed a poly-oxide nature of the composite 27, CeO2/Mn2O3/Fe2O3 28, and Fe2O3/Na2SO4 29 used for the same applications. The spectrum has a broad band centred at 621 cm−1 for the metal–oxygen stretching vibrations31. The bands at 835 and 1385 cm−1 were attributed to the asymmetric stretching vibration (ν3-NO3−) and out-of-plane bending vibration (ν2-NO3−), respectively, of nitrate32. The bands at 3433 and 1635 cm−1 were assigned to the stretching and bending vibration modes of adsorbed water molecules, respectively , Mn3+ (43.9%), and Mn4+ (32.9%) oxidation states of Mn ions, respectively34. The analyses showed that multivalent Mn ions were related to the formation of MnO2 and Mn-based ferrites  and Fe3+ ions (29.6%), respectively , surface hydroxyl groups/nitrate ions (24.6%), and water molecules (18.4%), respectively , which had a poor adsorbate-adsorbent interaction. The maximum adsorption capacity of 1.31 mmol g−1 achieved for the synthesized nanocomposite is similar to or higher than those reported for commercial ZnO (1.16 mmol g−1)39, CeO2-Mn2O3-Fe2O3 (0.83 mmol g−1)28, Mn2O3-Fe2O3 (0.35 mmol g−1)27, α-Fe2O3-Na2SO4 (1.06 mmol g−1)29, and ZnFe2O4 (0.05 mmol g−1)40 in similar experimental conditions.The synthesized nanocomposite was tested for Hons Fig. a. The adate Fig. b and adsate Fig. c on the 2 adsorption in dry and wet conditions 41, MnO2 (0.48–1.23 mmol g−1)42, and NaMxOy (0.73 mmol g−1)7 in similar experimental conditions. The nanocomposite possessed a higher H2S adsorption capacity compared to the SO2 gas. The superior H2S adsorption was related to an easy dissociation of H2S molecules due to the much lower energy barrier and higher adsorption energy compared to SO2, which has been previously demonstrated for Zn–MoSe2 structure through computational calculations43.The nanocomposite was also studied for SOons Fig. a. The adion Fig. b. The SOion Fig. c. Thus, 2S and SO2 adsorption showed no significant variation in the surface morphology . These two peaks were assigned to the presence of ZnSO344. The absence of additional new peaks in these samples could be related to the formation of oxidized sulfur species on the surface  than Fe2O3 (Fe3+ sites)46. Even in our previously reported work on the adsorption of H2S over Mn2O3/Fe2O3, bulk Fe2O3 phase did not take part in the oxidation process27. The slight variation in the peak position could be linked to the involvement of Fe2+ sites in the H2S adsorption process. For the SO2-adsorbed sample, the Fe2+ and Fe3+ peak positions red-shifted by 0.1 and 0.4 eV, respectively, with a significant drop in the Fe2+ proportion (70.4–62.3%). It has been proven that the SO2 molecules are much more reactive to the Fe2+ sites than the Fe3+ sites. Thus, a drop in the Fe2+ contribution suggested that the divalent Fe sites catered the oxidation of SO2 molecules  species, respectively. For the SO2-adsorbed sample, the 531.5 eV peak improved even further due to the consumption of lattice oxygen and the formation of sulfate species , elemental sulfur (25.1%), and sulfite (38.8%)48. While the formation of metal-bound sulfide is initiated by the dissociated adsorption of H2S (into H+ and HS−) in the presence of water molecules. The formation of elemental sulfur and sulfide is mediated by the redox behaviour of transition metal ions, surface-adsorbed molecular oxygen, and water molecules31. Wang et al.have reported the dissociation of H2S over In2O3 thin film in the presence of moisture, where the reactive dissociation of H2S molecules with adsorbed water produced HS− and H+ species. The formed HS− and H+ ions reacted with the surface-chemisorbed oxygen species to yield sulfide and sulfite species49. The HRXPS S 2p spectrum of SO2-adsorbed MFZO has a set of doublets with a 2p3/2 peak at 168.4 eV, which was assigned to the sulfate species (Fig. 48. The adsorption of SO2 over the oxide surface is generally driven by the reactive interaction of SO2 molecules with the lattice oxygen or surface hydroxyl groups to form sulfite/bisulfite, which further oxidized to sulfate via redox behaviour of transition metal oxide and gaseous oxygen molecules17. Moreover, just like H2S dissolution in the surface water, SO2 could be readily adsorbed and hydrolysed by surface water molecules, which makes the oxidation of SO2 molecules, energetically favourable17.The HRXPS S 2p spectrum of Hsulfite 3.8%48. Whies Fig. 48. The a2, ZnO, and ferrites with a surface area and pore volume of 21.03 m2 g−1 and 0.07 cm3 g−1, respectively. The nanocomposite was tested for room-temperature adsorptive removal of H2S and SO2 in dry and wet conditions. The oxide exhibited better gas adsorption capacity in wet conditions owing to the dissolution and dissociation of gaseous molecules in the surface water film. The adsorbent showed a better adsorption capacity at a lower flow rate and adsorbent loading. In the optimized conditions, a maximum of 1.31 and 0.49 mmol g−1 of H2S and SO2 was removed by the nanocomposite, respectively. The in-depth spectroscopic analysis confirmed the mineralization of H2S gas into sulfide, sulfur, and sulfite, which was mediated by the Fe and Mn redox cycles in the presence of adsorbed water and molecular oxygen. Though Zn ions did not participate in the oxidation process, Zn2+ probably interacted with the sulfides and sulfites. The SO2 mineralization was associated with the formation of sulfates, driven by the redox behaviour of Fe and Mn in an oxidative environment. Thus, we have presented a novel adsorbent material for the successful mineralization of toxic sulfurous gases, which could be suitable for deep desulfurization applications.In conclusion, we have fabricated an Mn-Zn-Fe metal oxide nanocomposite via a one-step coprecipitation reaction. The fabricated nanocomposite has MnOSupplementary Information.


The study of the molecular mechanisms of the pathogenesis of Alzheimer’s disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of β-amyloid (Aβ) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aβ) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aβ based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aβ was estimated in the total Aβ-pool with a detection limit of 1 fmol for pSer8-Aβ (1–16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples. Alzheimer’s disease (AD) is a neurodegenerative disorder responsible for 60–80%  of all cStudies devoted to quantitative assessment of pSer8-Aβ content mainly concern brain tissues ,7, whileThe main difficulty in the development of analytical methods for early diagnostics is the achievement of high sensitivity, since the concentrations of specific Aβ proteoforms in the biological fluids of patients are low. In addition, the most popular methods for studying the phosphoproteome involve the enrichment of phosphopeptides using metal oxide affinity chromatography or immunoassays and do not allow to measure the different proteoforms simultaneously.MALDI-TOF MS-based methods have high sensitivity, high throughput, and speed due to the automation of the mass spectrometric analysis process, and also allow simultaneous detection of different forms of Aβ ,11,12. TPhosphorylation is one of the ubiquitous PTMs involved in many regulatory cellular pathways; however, correct identification of modified forms is difficult due to the lability of the phosphoryl group. Mass spectrometric analysis of pSer8-Aβ is complicated by the relatively low ionization efficiency compared with the unmodified Aβ. Thus, the study of phosphorylated peptides requires the selection of appropriate experimental conditions.m/z values can overlap with the target peak giving false positive or negative results. This limitation can be bypassed by the reflectron mode, which has a higher resolution [The analysis was performed in linear and reflectron modes for binary mixtures of pSer8-Aβ:Aβ (1:1) with a total concentration of 25 fmol/μL for the hydrolytic amyloid fragment 1–16 and 54 pmol/μL for the full-length peptide 1–42. In negative ion mode, the pSer8-Aβ form had a higher relative intensity than the unmodified Aβ, but the overall intensity was worse in contrast to the positive ion mode. The linear mode is more sensitive than the reflectron mode, which allows us to use the method with lower sample quantities. However, the linear mode has a low resolution, thus analytes with close solution . For thesolution ,15,16  was obtained by using the DHB matrix solutions with addition of 0.1% TFA, 0.2% PA, or 0.5% PA . Matrix The objective of the study was to develop a highly sensitive method to estimate p-Aβ/Aβ ratio at concentrations observed in blood and CSF samples from AD patients. Practically for this purpose, calibration curves that would allow to quantify the relative amount of phosphorylated form by measuring the intensities of pSer8-Aβ and Aβ peaks were obtained.Measurements were carried out for a series of Aβ binary solutions containing pSer8-Aβ form in molar ratios from 0% to 100%. The solutions were applied to the target in an amount of 2.5 fmol to 5 pmol of total Aβ16 or from 4 pmol to 44 pmol of total Aβ42. Bruker MTP 384 polished steel target plate with a matrix solution of 20 mg/mL DHB, 0.5% PA in 30% ACN (DHB-PA), and Bruker MTP 384 AnchorChip 400 target plate with DHB 10 mg/mL, 0.1% TFA, and 30% ACN were used. To determine the intensity of the peaks corresponding to different Aβ proteoforms, the area under the isotopic cluster of the corresponding proteoform was determined. For the calibration curve, the input of the pSer8-Aβ isotope cluster to the sum of pSer8-Aβ and Aβ areas was calculated and plotted versus the fraction of pSer8-Aβ in the content of the binary mixture. The obtained curves  have a fη). Each compound has its own ionization coefficient:To obtain the theoretical equation of the curve, a statistical ionization model was used . In thisI—intensity; α—ionization coefficient; η is the amount of substance on the target; indices n—for unmodified Aβ-peptides; and p—for phosphorylated Aβ.fr.pI) and the relative amount of pSer8-Aβ in the analyzed sample (x):The coefficient α is considered constant for a particular analyte and independent of its total amount. For further transformations, the following variables were chosen: the relative intensity of pSer8-Aβ , beta-amyloid 1–42 (Aβ42), as well as their phosphorylated at serine 8 forms (pSer-8-Aβ16 and pSer8-Aβ42) were obtained from BioPeptide Co  . PeptideThe following reagents were used for matrix preparation: 2,5-dihydroxybenzoic acid (DHB), α-Cyano-4-hydroxycinnamic acid (HCCA), and 2,5-Dihydroxyacetophenone (DHAP). Matrix solution additives: phosphoric acid (PA), trifluoroacetic acid (TFA), and diammonium citrate (DAHC). Solvents: acetonitrile (ACN), deionized water, and ethanol. All reagents were of HPLC/MS grade or higher and obtained from Merck KGaA .v/v) ACN: five samples with PA addition (ranging from 0.2–2% (v/v)) and one with 0.1% TFA. Solutions with 10 mg/mL HCCA in 30% ACN: one with 1% PA and another with 0.1% TFA.Several matrix solutions were tested. Solutions of 10 mg/mL DHB in 30%  DAHC were used to prepare 12 solutions with various combinations of matrix concentrations. DHAP: 0.2 mg/mL in 1% ethanol, 0.5 mg/mL in 2.5% ethanol, 1 mg/mL in 5% ethanol, 2 mg/mL in 10% ethanol—in combination with DAHC: 20 mM, 50 mM, and 100 mM  [An amount of 20 mg/mL DHB solution  was chosen as a standard for peptide ionization and the following solutions were used: DHB 20 mg/mL, 0.1% TFA, 30% ACN; HCCA saturated, 0.1% TFA, 30% ACN and DHAP 15.2 mg/mL, DAHC 20 mM (4.524 mg/mL), and 75% ethanol.For matrix optimization experiments, a solution of p-Ser8-Aβ:Aβ (1:1) was used with a total concentration of 5 pmol/µL for the 1-16 peptide fragment and 54 pmol/µL for the 1–42 one. Solutions of pSer8-Aβ:Aβ (1:1) were applied to the Polished Steel target in 0.5 µL at three points of the target for each matrix, which was further applied in 0.5 µL after the peptide solution had dried.Binary mixtures of phosphorylated and non-phosphorylated forms of beta-amyloid were used to build calibration curves. Total concentrations of 16-amino-acid fragments were 0.025 pmol/µL, 0.05 pmol/µL, 0.125 pmol/µL, 0.5 pmol/µL, 2.5 pmol/µL, and 5 pmol/µL, and in mixtures with 42-amino-acid fragments they were 9 pmol/µL, 13 pmol/µL, 17 pmol/µL, 21 pmol/µL, 27 pmol/µL, and 44 pmol/µL. The percentage of phosphorylated fraction in every solution varied from 0% to 100%. An amount of 1 µL of each mixture was applied to MTP 384 Polished Steel and MTP 384 AnchorChip 400 targets using the “Dried Droplet” method. Matrix formulations: 20 mg/mL DHB, 0.5% PA in 30% ACN for Polished Steel and 10 mg/mL DHB, and 0.1% TFA in 30% ACN for AnchorChip. Each binary mixture was applied to the target three times at different points. All preparations were carried out on air at room temperature.To test the method performance, model samples containing p-Aβ peptides were prepared. Human serum obtained from a healthy volunteer and diluted 1:50 with PBS (pH 7.4), comparable in total protein amount with CSF, was chosen as the “matrix” solution. pSer8-Aβ solutions were prepared from a 1 mg/mL pSer8-Aβ stock dissolved in 10% acetonitrile, which was stored at −80 °C until the experiment. pSer8-Aβ42 was spiked into 1 mL of matrix solution and incubated for 1 h at room temperature. The final sample concentration was 100 ng/mL. Then, pSer8-Aβ42 was immunoprecipitated from the model samples using 1E4E11 monoclonal antibodies  immobilized on Dynabeads  according to the manufacturer’s protocols. Briefly, the model sample was spiked with 1E4E11 beads (up to ~1 μg of antibodies per sample) and mixed with a RIPA (radioimmunoprecipitation assay) buffer , and incubated for 1 h at room temperature. After removal of the supernatant using a magnetic rack, the beads were washed with 1 mL of 0.1% BSA/PBS solution (pH 7.4) 3 times and 1 mL of 10 mM Tris (pH 7.4) once. The target fraction was eluted using 70% ACN solution. The final samples (40 μL) were dried on a vacuum concentrator  to the volume of 20 μL.For the MALDI-TOF analysis, samples were hydrolyzed by the LysC protease  to obtain p-Aβ16 fragments. Samples after immunoprecipitation (20 μL) were mixed 1:1 with 0.5 μg/μL LysC prepared in 100 mM ammonium bicarbonate  and then incubated at 37 °C for 4 h. An amount of 1 μL of a digested sample was spotted on the AnchorChip target plate and analyzed as described above. The completeness of digestion was evaluated by controlling the absence of peaks related to 1–42 peptides.Experiments were conducted on an UltrafleXtreme  MALDI-TOF/TOF mass spectrometer using linear and reflectron modes in positive and negative ion modes.m/z for Aβ16 and 4500–4700 m/z for Aβ42. The spectra were obtained automatically by summing the signal from 5 random rasters of the target point, satisfying the condition for the presence of a peak with S/N >9.Measurements for calibration curve construction were carried out in the ranges of 1900–2100 The resulting spectra were processed using scripts and functions written in MATLAB R2021b with the Bioinformatics Toolbox application. The proportion of p-Ser8-Aβ intensity was calculated from the obtained intensity values.


Taken together, our findings provide evidence that reduced expression of KDM5B has a critical role in promoting ESCC radio-sensitivity by upregulating PIK3C3, suggesting KDM5B may function as an oncogene in ESCC.Many patients with esophageal squamous cell carcinoma (ESCC) are inoperable because of old age or the advanced stage of the disease; thus radio- and chemotherapy are believed as the standard treatments for these patients. However, due to the radio-resistance of tumor cells that may develop during radiotherapy, results remain unsatisfactory. In this article, the possible relationship between the expression of lysine demethylase 5B (KDM5B) and ESCC radio-resistance is clarified, and the underlying mechanism is evaluated. Using the GSE75241 microarray, we identified KDM5B as a potential oncogene in ESCC. KDM5B was overexpressed in ESCC patients and cells. Inhibition of KDM5B enhanced the H3K4me3 methylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) promoter and induced the expression of PIK3C3. Knockdown of KDM5B or overexpression of PIK3C3 in KYSE-150 and TE-10 cells promoted apoptosis, cell cycle arrest, autophagy, and increased sensitivity to radiotherapy. Silencing of PIK3C3 attenuated the promoting effect of sh-KDM5B on the sensitivity of ESCC cells to radiotherapy. The inhibition of sh-KDM5B in radio-resistance of ESCC cells was also reproduced  Esophageal cancer ranks seventh in terms of incidence (572 000 new diagnoses) and sixth in overall mortality (509 000 deaths), signifying that it will be responsible for an estimated one in every 20 cancer deaths in 2018 worldwide . RadiothThe phosphoinositide 3-kinase (PI3K) family is vital to almost all aspects of cell and tissue biologies and essential to human cancers and aging, and there is a recognition of the significance of distinct roles for each of the three PI3K classes , as well as for the different isoforms within each class . PIK3 caDeclaration of Helsinki. Thirty ESCC tissues and corresponding paracancerous esophageal epithelial tissue specimens were obtained from patients who underwent surgical resection at the First Affiliated Hospital of Soochow University from January 2019 to September 2020. The patients were aged between 58 to 76 years and included 19 male and 11 female cases. The normal paracancerous esophageal epithelial tissues were at least >3 cm from the tumor margin. The 30 samples used in this study were confirmed by experienced clinicopathologists. ESCC tissues from patients were placed in liquid nitrogen and then stored at −80°C until analysis. Patients who had received radiotherapy or chemotherapy, patients with infections, patients with a history of malignancy other than ESCC, and patients with primary immunodeficiency and acquired immunodeficiency syndrome were excluded from this study.Prior to sample collection, informed consent was acquired from all patients in accordance with the protocols approved by the institutional review boards of the First Affiliated Hospital of Soochow University. All procedures involving patients were performed following the 2, and the medium was refreshed at 1 d intervals.ESCC cell lines KYSE-150 and TE-10 were from National Infrastructure of Cell Line Resource . ESCC cell lines ECA-109, EC-9706 and normal esophageal epithelial cells HET-1A were acquired from Shanghai EK-Bioscience Biotechnology Co., Ltd. . The cells were preserved in RPMI-1640 medium  with 10% FBS (Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin . The cultures were incubated in an incubator at 37°C with 5% COShort hairpin RNA (sh)-KDM5B, sh-PIK3C3, overexpression (oe)-PIK3C3 and their corresponding negative controls were synthesized by GenePharma . Prior to transfection, the KYSE-150 and TE-10 cell were plated in 6-well plates to achieve an approximately 80% confluence. Transfection was performed using the lipofectamine 3000 transfection kit (Invitrogen) as per the manufacturer’s protocol, and the cells were screened with 2 μg/mL puromycin  to obtain stably transfected cell lines.-ΔΔCt method.Total RNA was isolated from ESCC tissues, normal adjacent tissues, and cells using TRIzol (Invitrogen) as per the manufacturer’s protocol. The purity and concentration of RNA were assessed by NanoDrop 2000 . Then, the RNA was reverse transcribed into cDNA using a reverse transcription kit . The primer sequences of KDM5B and PIK3C3  were synKYSE-150, TE-10 cells and ESCC cells-formed xenograft tumor tissues were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were assessed using the bicinchoninic acid protein quantification kit (Beyotime). Equal amounts of protein (50 μg) from each sample were separated using 10% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes . The membranes were closed with 5% BSA at 37°C for 60 min and treated with primary antibodies to GAPDH , KDM5B , PIK3C3 , H3K4me3 , Cyclin B1 , CDC2 , pCDC2 , Beclin-1 , LC-3I/II , and Bcl-2  overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-coupled secondary antibody  for 60 min at room temperature. Protein bands were analyzed using Pierce Western Blotting ECL substrate kit (Thermo Fisher Scientific) and BandScan 5.0 system .3 cells/well and cultured overnight. The cells were radiated at 0, 2, 4, 6, 8 Gy for 24 h [The sensitivity of cells to x-rays was detected using the colony formation assay. Briefly, the KYSE-150 and TE-10 cells were plated in 96-well plates at 6 × 10for 24 h . The cel5 per well) were plated in 6-well plates for 24 h and exposed to 0 Gy or 8 Gy X-rays for 24 h. The apoptotic cells were distinguished by dual staining with Annexin-V fluorescein isothiocyanate (FITC)-PI and analyzed by flow cytometry  using an apoptosis detection kit .The transfected KYSE-150 and TE-10 cells  was used to assess DNA content and cell cycle distribution.Chromatin immunoprecipitation (ChIP) assays were performed using a commercial kit (Beyotime) as per the manufacturer’s protocol. KYSE-150 and TE-10 cells were fixed with 1% formaldehyde for 10 min at room temperature. Glycine (0.1 M) was added for a 5 min incubation to quench the cross-linking reaction. The cell lysates were prepared using an ice-cold cell lysis buffer at 4°C for 60 min and sonicated to break chromatin to an average length of 500 to 800 bp. The samples were cleaned with protein A agarose  by gently spinning at 4°C for 60 min and incubated with rabbit antibodies to KDM5B  and H3K4me3  overnight at 4°C. Antibodies were used for immunoprecipitation of cell lysates, and phenol/chloroform was used for extraction, purification and recovery of DNA fragments. Finally, the DNA released from the precipitated complexes was analyzed by qPCR.6/mL with 1× Wash buffer. The cell suspension (90 μL) was stained with 10 μL monodansylcadaverine (MDC) staining for 0.5 h at room temperature in the darkness. The slides were covered with 100 μL collection buffer, observed, and photographed under a fluorescent microscope .Cellular autophagy staining assay kit  was used to detect autophagy in ESCC cells. KYSE-150 and TE-10 cells were exposed to 0 Gy or 8 Gy x-rays for 24 h, and the cell concentration was adjusted to 106) transfected with sh-NC or sh-KDM5B were injected subcutaneously into the right dorsal flank of nude mice. Two weeks after inoculation, the mice were subjected to x-ray irradiation (0 or 8 Gy) 2 times a week. The tumor volume was measured once a week with calipers. The formula for calculating the tumor volume was as follows: Tumor volume = (length × width2)/2. The nude mice were euthanized by intraperitoneal injection of 1% pentobarbital sodium (150 mg/kg) after 4 weeks, and the tumors were weighed.Animal studies were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Soochow University. Twelve BALB/c female nude mice (4 weeks old) were purchased from the Shanghai Lab Animal Research Center . To assess the effect of KDM5B on modulating radiosensitivity in tumor models, KYSE-150 cells  and Ki-67  at 4°C and re-probed with goat anti-rabbit secondary antibody  coupled with HRP for 60 min at room temperature. Next, after being cultured with diaminobenzidine , counter-stained with hematoxylin (Sigma-Aldrich) for 1 min and sealed with neutral gum, the sections were photographed under a microscope .Apoptosis was assessed in xenograft tumor tissue sections (5 μm) by Terminal deoxynucleotidyl transferase-mediated 2′-Deoxyuridine 5′-Triphosphate nick end labeling (TUNEL) using the In-Situ Cell Death Detection Kit-POD (Roche). The sections were treated with proteinase K (10 mmol/L) for 15 min and then treated with TUNEL reaction mixture for 60 min, followed by incubation in converter-peroxidase for 30 min. TUNEL-positive cells were considered apoptotic. Fluorescence images were obtained using a fluorescence microscope (Nikon Corporation). Under the microscope, the percentage of apoptotic cells was calculated as the rate of TUNEL-positive cells to total cells.t test was applied for comparisons between two groups, while one-way or two-way ANOVA followed by Tukey’s post hoc test was adopted for comparison among multiple groups. Differences that produced P values less than 0.05 were accepted as significant.All statistical analyses from at least three separate experiments were performed using SPSS 23.0 software  and GraphPad Prism 8.0.2 . Measurement data were presented as mean ± standard deviation. The paired https://www.ncbi.nlm.nih.gov/geo/) with a data series consisting of 4 ESCC tissues and normal paracancerous tissues. A total of 47 genes with differential expression were screened out with P-values <0.01 and log2 fold change >2, and the heatmap was plotted  demonstrated the presence of the H3K4me3 activation in the PIK3C3 promoter  [et al. determined that broad spectrum KDM inhibitors resulted in decreased cell survival and prolonged DNA damage repair kinetics [In this study, we conducted mRNA-based microarray analysis to screen differentially expressed genes in ESCC. KDM5B was identified as one of the most significantly overexpressed one, which was consistent with a previous study . In addi< 0.001) . The sup< 0.001) , melanom< 0.001) , as well< 0.001) . In addi< 0.001) . Moreove< 0.001) . Bak pro< 0.001) . Using ckinetics . At the kinetics .high cells showed increased PI3K pathway activation in oral cancers [et al. displayed that KDM5B favored cell survival by transcriptional modulation of genes related to cell cycle, DNA repair and cell death [Interestingly, KDM5B was found to promote the radio-resistance of non-small cell lung cancer through the decline of PTEN expression . As a co cancers . In ESCC cancers . Also, w cancers . Beclin- cancers . This stll death . Our obsThe findings of our study support the posit that the KDM5B/PIK3C3 axis is a promising novel regulatory network in the radiotherapy of ESCC. We have provided evidence suggesting that KDM5B reduces the expression of the tumor suppressor PIK3C3 via H3K4me3-mediated demethylation, which ultimately reduces ESCC cell radiosensitivity. However, our research is still a preliminary one, indicating more experiments in this field are required in the future.


X-ray diffraction and X-ray photoelectron spectroscopy confirm the existence of MoOx3−. The MoOx3−/TiO2 electrode prepared at 550 °C exhibits a high specific capacitance of 23.69 mF cm−2 at a scan rate of 10 mV s−1 and good cycling stability with capacitance retention of 86.6% after 1000 cycles in 1 M Na2SO4 aqueous solution. Our study reveals a feasible method for the fabrication of TiO2 nanotubes modified with electroactive MoOx3− as high-performance electrode materials for supercapacitors.Here we report the supercapacitive properties of a novel MoO x3−/TiO2 nanotube composite prepared by a facile galvanostatic deposition technique and subsequently thermal treatment in an argon atmosphere between 350 °C and 550 °C.Here we report the supercapacitive properties of a novel MoO In particular, TiO2 nanotubes (TNTs) with a well-defined open-top and uninterrupted nanotube structure have received tremendous attention in various applications including photocatalytic degradation of environmental pollutants,8 solar cells,9 energy storage devices,10 drug delivery11 and sensors.12 Supercapacitors are one of the most promising electrochemical energy-storage systems and have attracted tremendous attention.13–17 Recently, self-assembled titania nanotube arrays have received immense interest for use in supercapacitors because of their capability to offer large surface area for electrochemical reactions and unique electron transport pathways compared with non-oriented structures. Additionally, the electroactive materials can directly grow on the surface of TNTs, avoiding the use of a polymer binder and conductive additives.In the past decade, various nanostructured materials  and 3.1 eV (for rutile phase).18 Pristine TNTs only contribute a very low capacitance (0.047–0.97 mF cm−2).19–22 Therefore, TiO2 nanotubes need to be modified urgently in order to meet the demands for supercapacitors with high electrochemical performance. A promising method to further enhance the capacitance of TNTs is to incorporate with some transition metal oxides . The composite electrode can utilize the positive features of individual materials and may provide a combined effect, such as large capacitance and good cycling performance.27However, TiO,23 MnO2  and MoO3 MnO2 (3  etc.). T28 catalysts,29 electrochromic devices1 and energy storage30,31 due to their low cost, resource abundance and nontoxicity. In particular, a layered molybdenum trioxide (α-MoO3) has been studied as an attractive electrode material for supercapacitor due to its high theoretical specific capacitance of 1111 mA h g−1.32 However, the poor electrical conductivity and large capacitance loss in initial cycles have limited its application as an electrode material.26,33 Annealing in inert or hydrogen atmosphere could effectively improve electrical conductivity of some transition metal oxides.20,34 Meduri et al. discovered that oxygen deficient MoOx3− had fast intercalation and excellent electrochemical performance in lithium ion batteries.6 Law et al. reported that oxygen vacancies in MoOx3− led to enhanced electrical conductivity.35 Dunn et al. showed that incorporation of oxygen vacancies in MoOx3− could enhance pseudocapacitive charge storage properties.36 To the best of our knowledge, there have been a few studies on incorporating MoOx3− with TiO2 nanotubes and thus exploring its supercapacitive behavior. Compared with other hybrid electrodes,37–39 the prepared electrodes can make full use of TiO2 nanotubes and MoOx3− layer, thus achieving high electrochemical performance. Therefore, we fabricated MoOx3−/TiO2 nanotubes composite and extensively studied its application for supercapacitor.Molybdenum oxides have enabled a wide range of applications in gas sensing,x3−/TiO2 nanotube hybrid by galvanostatic deposition in molybdenum salt solutions and subsequent thermal treatment in argon atmosphere. Various microstructure characterizations and electrochemical measurements were conducted to investigate the crystal phase, surface morphology, chemical composition and electrochemical properties of prepared samples. The electrochemical measurements reveal improved electrochemical performance of MoOx3−/TiO2 nanotubes composite with high specific capacitance as well as stable capacitance behavior.In this work, a feasible approach was proposed to prepare MoO2.2.1.3) was purchased from LuoKe titanium Ltd. Ethylene glycerol , ammonium fluoride , hexaammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O, purity > 99.0%) and sodium sulfate anhydrous  were purchased from KeLong Chemical Ltd. Other chemical reagents used in these experiments were of analytical grade and were used directly without further purification. All aqueous solutions were prepared using deionized water.The titanium sheet 6Mo7O24·4H2O aqueous solution. Afterwards, the as-deposited samples were annealed at 350 °C, 450 °C, and 550 °C in argon atmosphere for 1 h. In consideration of tubular structure collapses at higher annealing temperatures (e.g. 650 °C), we just studied capacitive behavior of deposited samples annealed at 350–550 °C. The sample annealed at 450 °C is designated as M-TNT. For comparison, TNTs just annealed at 450 °C for 1 h in ambient atmosphere are referred as TNT.The highly ordered TNTs were prepared as described elsewhere.2.3.2SO4 aqueous solution with an electrochemical workstation  in a 3-electrode setup consisting of the prepared sample as the working electrode, platinum foil as the counter-electrode, and saturated Ag/AgCl electrode as the reference electrode. Cyclic voltammetry (CV) tests were conducted over a voltage range from −0.9 to −0.2 V (vs. Ag/AgCl) at different scan rates (from 10 to 200 mV s−1). The galvanostatic charge–discharge (CD) testing was performed under different current densities (from 0.4 to 1 mA cm−2). Mott–Schottky plots were measured at a frequency of 1 kHz. Electrochemical impedance spectroscopy (EIS) measurements were conducted over a frequency range of 0.01 Hz to 100 kHz at an AC voltage amplitude of 5 mV. The cycling stability of the samples was tested by galvanostatic CD measurement performed up to 1000 cycles at a current density of 0.6 mA cm−2.A Hitachi S-4800 scanning electron microscopy (SEM) and DX-2700 X-ray diffraction (XRD) were used to investigate the surface morphology and microstructure of the fabricated samples, respectively. Chemical composition and chemical states of deposited samples were characterized by X-ray photoelectron spectroscope . Electrochemical measurements were performed in a 1 M Na3.3.1.2 (JCPDF 21-1272) while peaks at ∼38.4° and ∼40.1° are assigned to Ti (from Ti substrate). Notably, the peaks with 2θ of ∼28.5°, ∼29.4°, ∼30.9° and ∼32.6° are indexed to an oxygen deficient Mo17O47 phase (JCPDF 71-0566).6 Due to the strong sharp diffraction peaks of titanium substrate, the diffraction peaks corresponding to MoOx3− are suppressed.The crystalline phase of prepared M-TNT was characterized by XRD. As shown in 5+, and the Mo 3d3/2 peak at 232.4 eV corresponds to Mo4+, respectively.40 Our early research discovered that coating layer after electrochemical deposition in molybdenum salt solutions was just amorphous Mo5+ oxides and Mo6+ oxides.26 So, the formation of Mo4+ from M-TNT indicates that thermal treatment in argon atmosphere could lead to oxygen loss on the surface of deposition coating, resulting in the generation of oxygen deficiency and low oxidation states. This phenomenon has been previously observed in other reports.41,42 The peaks at ∼232.6 eV and ∼235.7 eV are indexed to 3d5/2 and 3d3/2 of Mo6+, respectively.43,44 Additionally, the Ti 2p3/2 peak at ∼458.6 eV and the Ti 2p1/2 peak at ∼464.3 eV correspond to Ti4+ . Compared with the sample annealed at 450 °C, the slight shift of MoOx3− peaks for the sample annealed at 550 °C is attributed to a change of lattice constant due to the oxygen vacancies.42 Additionally, the formation of the thermodynamically stable phase of TiO2 (i.e. rutile) indicates transformation of anatase to rutile when annealed at 550 °C. Fig. S55+ and Mo4+ for sample annealed at 550 °C suggests a larger concentration of oxygen vacancies. In order to obtain a better understanding of the effect of annealing temperatures on electrical properties, Mott–Schottky plots were drawn based on capacitances derived from the imaginary part of the impedance at each potential with 1 kHz frequency. The calculated carrier densities of deposited samples annealed at 350 °C, 450 °C and 550 °C are 7.14 × 1017 cm−3, 3.12 × 1019 cm−3, 7.31 × 1019 cm−3, respectively  in the initial cycles.26 Although oxygen vacancies are capable to stabilize the crystalline structure during cycling, the suitable concentration of oxygen deficiency plays an important role in achieving good cycling performance.36,40 Hence, the sample prepared at 450 °C exhibits better cycling stability than those of others. Additionally, a fluctuation in the specific capacitance during cycling is possibly due to “activation process”52,53 and the uneven electrolyte wetting of the phases of MoO2 and MoO3.40The above-mentioned experimental results confirm the contribution of MoOs Fig. S4, we discectively . Mott–Scpability . Fig. S7mposites . The capmposites , we can 4.x3−/TiO2 nanotube composites were synthesized by galvanostatic depositions and then annealed in argon atmosphere. Electrochemical measurements confirm the contribution of MoOx3− and enhancement in capacitive performance within composite electrode materials. We also have demonstrated that the electrochemical properties of MoOx3−/TiO2 nanotube composites can be enhanced by controlling annealing temperatures. The electrode prepared at 550 °C yields highest specific capacitance of 24.74 mF cm−2 at a current density of 0.4 mA cm−2 and good capacitance retention (86.6%) even after 1000 continuous charge–discharge cycles in Na2SO4 electrolyte. The ease of synthesis and exceptional electrochemical properties suggest MoOx3−/TiO2 nanotube is a promising supercapacitor electrode material.In our study, MoOThere are no conflicts to declare.RA-008-C8RA02744G-s001


Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni  and Lai  were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1–71 amino acids) and C-terminal (72–90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues  with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.The genome of pathogenic  Leptospira is a spiral-shaped bacteria, the pathogenic species of which are known for causing leptospirosis disease in humans and a wide range of animals  harbors genetic elements of an adaptive defense system against foreign nucleic acids known as clustered regularly interspaced short palindromic repeats and their associated genes (CRISPR-Cas)  .cas genes  (cas genes encoding nucleases (L. interrogans serovars Copenhageni and Lai), there are two predetermined subtypes (I-B and I-C) of CRISPR-Cas  of subtype I-C (cas1 to cas8) of Leptospira, which lack an array component, are functionally active. Thus, in our preliminary study, out of the eight cas genes, we chose cas2 to clone, overexpress and check the nuclease activity of the purified recombinant protein.CRISPR-Cas systems involved in combatting exotic nucleic acids are cataloged into two classes, six types, and thirty-three subtypes according to the association of the signature as genes . Based oand I-U) . The CRIucleases . In the ISPR-Cas . In Lepts system . TherefoSulfolobus solfataricus), BhaCas2 , SpyCas2 (Streptococcus pyogenes), DvuCas2 (Desulfovibrio vulgaris), and TonCas2 (Thermococcus onnurineus) contains N- and C-terminal regions, with the former having a ferredoxin (βαββαβ) fold  non-catalytically into the CRISPR array  on the cut and paste mechanism  are core metallonucleases found universally in all CRISPR-bearing taxa . Althougαβ) fold . The purαβ) fold . In the αβ) fold . In E. cPR array . The actechanism . However disease . Among o and L. interrogans serovar Lai , there are two Cas2 proteins, each in the locus of CRISPR-Cas subtypes I-B and I-C, respectively. Although LinCas2B (LIC10941) and LinCas2B_Lai (LA3182) shared an identical protein sequence, the sequence similarity between LinCas2C (LIC12917) and LinCas2B (LIC10941) is 32%. Moreover, cas2c (LA0683) in Leptospira serovar Lai encodes only the first 58 amino acids (LinCas2C_Lai) because of the natural frameshift mutation  and was further compared with its homologs. This is the first report concerning the crystal structure of CRISPR-Cas elements from spirochetes.L. interrogans serovars Copenhageni and Lai were retrieved from NCBI. The three-dimensional (3D) atomic coordinates of the Cas2 orthologs were downloaded from the Protein Data Bank (PDB)  (Nucleotide sequences of CRISPR-Cas I-C harbored in nk (PDB) . The gencumented  and usincumented . The phycumented . The 3D cumented , Phyre2 cumented , and thecumented . The precumented . Multiplcumented  with thecumented . Moleculcumented . The procumented  was usedcumented . LinCas2d: 5XVP) .L. interrogans serovar Copenhageni strain Fiocruz L1-130 or serovar Lai culture was maintained in Ellinghausen-McCullough-Johnson-Harris (EMJH) media at 29°C supplemented with 1×enrichment media (Difco) along with 5-fluorouracil (100 μg/ml). After 7 days of incubation, the grown culture was sub-cultured successively. Genomic DNA of L. interrogans serovars Copenhageni and Lai were isolated from a 7-day-old culture containing ∼108 cells per ml using QIAamp DNA Blood Mini Kit (Qiagen) per manufacturer protocol. E. coli strains DH5α and BL21 (DE3) were grown in Luria Bertani  broth or agar for cloning, transformation, and expression.The spirochete LIC12917  and LA0683  were amplified using the genomic DNA templates of L. interrogans serovars Copenhageni and Lai, respectively. Both full-length cas2c genes were cloned in the pCDF-1b expression vector (Novagen), and cloning was confirmed by double digestion of insert  and sequencing of plasmids.The open reading frame (ORF) of luciferase gene was synthesized using HiScribe T7 high yield RNA synthesis kit (NEB) as per the manufacturer protocol. The plasmid was isolated from a 5 ml overnight grown culture of E. coli DH5α cells using a mini-prep kit (Thermo Scientific). Single-stranded viral DNA substrate  and all enzymes used for genetic engineering were purchased commercially (NEB or Fermentas). As previously reported, short DNA oligomers of 23-mer and 50-mer were used (luciferase mRNA (0.5 µg) (2) for an hour at 37°C. DNase activity dependence for divalent metal ions (2.5 mM) was determined by substituting various divalent metal ions . All the reaction products were separated on ethidium bromide-stained 2% (w/v) agarose gel electrophoresis. The nuclease reaction containing 23- and 50-mer nucleotides were assessed on 8 M 15% urea-PAGE.Nuclease activity of rLinCas2C was investigated on various DNA and RNA substrates. RNA transcript of the ere used . The sub(0.5 µg) . The givY7A, rLinCas2CY7A+D8A, rLinCas2CR33A+F39A, and rLinCas2CY7A+D8A+R33A+F39A). In one of the mutant variants (rLinCas2CΔL2), residues involved in framing the loop L2 were deleted. All the generated constructs were outsourced for sequencing before overexpression, purification, and characterization of proteins.Using the Q5 site-directed mutagenesis kit (NEB), rLinCas2C mutant variants were generated. The mutants were generated using the template plasmid pCDF_LIC12917 and the primers used are listed in Quantitative RNase activity of rLinCas2C, rLinCas2C_Lai, and the mutant variants of rLinCas2C was done using the RNaseAlert kit . The RNaseAlert kit contains synthetic RNA oligo substrate labeled with fluorescein and a quencher at its end. When cleaved by an RNase, the substrate fluoresces green (490 nm excitation and 520 nm emission) and can be measured by a fluorometer. RNase activity was performed in black flat-bottom 96-well plates (Invitrogen) at 37°C. Fluorogenic RNA substrate (10 pmol) was incubated with rLinCas2C, its mutant variants, and LinCas2C_Lai (25 µM) in a total of 100 μl reaction buffer (25 mM Tris-Cl pH 8.0 and 100 mM KCl). Fluorescence was measured at every 5 min interval till 60 min using the Infinite M200Pro plate reader (Tecan).Streptococcus pyogenes having a sequence identity (query coverage) of 45 (98)% as a search model using the program Phaser  ion in its active site required for its activity, crystallization of the protein incubated with MgCl2 was attempted. However, a diffractive crystal could not be obtained. The details of the structure refinement and validation of the final structure models are provided in The purified protein  was screened for initial crystal hits using crystallization conditions available from Hampton Research utilizing the hanging-drop vapor-diffusion method at 4°C. Diffraction quality crystals of rLinCas2C were obtained in 0.2 M sodium citrate tribasic dihydrate pH 5.6, 5% 2-propanol, 20% polyethylene glycol (PEG) 4,000 and 0.2% low melting agarose (LMA). X-ray intensity diffraction data were collected at −173°C using the home source Rigaku MicroMax-007 HF diffractometer (operated at 40 kV and 30 mA) and R-Axis IV++ imaging-plate detector available at the central instrument facility (CIF) of the Indian Institute of Technology Guwahati, India. The crystal to detector distance was maintained at 170 mm. The diffraction data were processed and scaled using the programs iMosflm  and Aimlm Phaser . To calcdata set . The atodata set  and Refmdata set , respectdata set  and MolPdata set . As the cas2c (ORF id: LIC12917) gene of size 273 bp in L. interrogans serovar Copenhageni is illustrated  gene in the CRISPR-Cas I-C locus (nucleotides coordinate 686432–693873), appeared to be absent (Leptospira predicts the cas2c (LA0683) gene to be of 272 bp size with a natural deletion of one nucleotide (adenine108) that may result in partial ORF translation. Thus, Xiao and co-workers  might encode for truncated (58 amino acids and frameshift after 35th residue) and inactive LinCas2C_Lai  in the CRISPR I-C locus of L. interrogans serovar Lai  and cas2c_Lai  were cloned, and the recombinant proteins (rLinCas2C and rLinCas2C_Lai) were purified using Ni-NTA affinity chromatography to investigate the nuclease activity  was used to investigate the nuclease activity on various DNA substrates. We excluded LinCas2B_Lai in our analysis as it had 100% sequence similarity to the well-studied LinCas2B. In a nuclease assay, increasing concentrations (5–25 µM) of each rLinCas2C and rLinCas2C_Lai were taken to optimize the cleavage of the dsDNA . Around 25 µM of each rLinCas2C and rLinCas2C_Lai could completely cleave the DNA (0.5 µg) in an hour at 37°C . The DNA2+ ion was substituted with other divalent metal ions  to explore their (rLinCas2C and rLinCas2C_Lai) preference for DNase activity. Both the nucleases (rLinCas2C and rLinCas2C_Lai) displayed higher affinity towards Mg2+, followed by Mn2+ and Fe2+ as a cofactor for its DNase activity. LinCas2B and BhaCas2 also preferred Mg2+ over other metal ions  . In agretal ions .2 and −73 kcal/mol, respectively. There are two loops named loop L1 and L2 connecting β1-α1 and α2-β4, respectively, found in all Cas2 orthologs. Loops L1 and L2 are speculated to recognize DNA and RNA substrates, respectively . LinCas2C crystal is composed of a total of three α-helices (α1–α3) and five anti-parallel β-strands (β1–β5) , as descectively . Structuectively . The modectively . Structuectively . Intact Structural homology search based on scores of selected parameters [Z-score and root mean square deviation (rmsd)] of the web server DALI  revealedIn addition, the crystal structure of rLinCas2C was superimposed with the structures of Cas2 orthologs  and the putative DNA (L1) and RNA (L2) binding loops were compared . SpyCas2A multiple sequence alignment of LinCas2C with its orthologs also displays the variation in the residues responsible for constituting the loop L1 and L2 . Notably2, forming 29–32 hydrogen bonds between the two protomers , BhaCas2 (10.6 Å), DvuCas2 (15.4 Å), and HpyVapD (12.6 Å) measured uneven distance between the conserved equivalent aspartate residue , BhaCas2 (39 and 100%), DvuCas2 (37 and 100%), and SsoCas2 (30 and 68%). Several conserved residues  and motifs (RVQ and SVF) in LinCas2C were identified , XabCas2 of entified . We haveentified . Thus, ientified . A modelentified . Two othentified ], and weentified .Y7A) and double (rLinCas2CY7A+D8A) mutants demonstrated reduced DNase activity, where a change in conformation of plasmid was evident due to a partial nick in DNA (R33A+F39A and rLinCas2CY7A+D8A+R33A+F39A) exhibited complete abolition in DNase activity (ΔL2) was generated to analyze the DNase and RNase activity. To our surprise, rLinCas2CΔL2 displayed a complete loss of DNase activity  reduced moderately  in a divalent metal- and pH-dependent manner. However, these nucleases were inert toward small DNA oligos (23-50-mer). In a recent study, LinCas2C_Linhai, a Cas2C ortholog, prefers Mgactivity . In cont of Mn2+ . The DNa of Mn2+ , SpyCas2 of Mn2+ , XorCas2s oryzae , and Lins oryzae . Consists oryzae . The recactivity . DetectiIn a recent study, the virulence-associated protein D (VapD) of toxin-antitoxin systems (TA) was shown to possess a ribonuclease fold similar to Cas2 proteins . The Vap2+ ion. For these aspartates to bind a bridging metal, the rLinCas2C would need to undergo either a major conformational change of the β1 or ferredoxin fold region or altogether the dimer orientation. Similarly, the uneven distance between the conserved equivalent aspartate residue was observed for the protomers of SpyCas2, BhaCas2, DvuCas2, and HpyVapD. ; however, dissimilar to that of SsoCas2. In SsoCas2, the coordination between Mg2+ and two Asp10 residues from two dimer subunits is mandated for initiating the phosphodiester cleavage. These two Asp10 residues from SsoCas2 dimer molecules can create coordination with an Mg2+ ion, as the distance between the side chains from the two residues is only 6.5 Å. However, in the case of HpyVapD or rLinCas2C, the distance between the side chains of two instances of Asp7 or Asp8 are greater than 10 Å indicating its inability to coordinate the metal ion. HpyVapD showed ribonuclease activity without metal ions (Metal-independent RNase activity of rLinCas2C functionally corroborates with that of HpyVapD of tal ions . At thistal ions . This metal ions .The closest homolog SpyCas2 is a metal- and pH-dependent dsDNase and shares standard functional features with the BhaCas2 . Mutagen