YBX1) plays important roles in RNA stabilization, translation, transcriptional regulation, and mitophagy. However, its effects on porcine preimplantation embryos remain unclear. In this study, we knocked down YBX1 in the one-cell (1C) stage embryo via small interfering RNA microinjection to determine its function in porcine embryo development. The mRNA level of YBX1 was found to be highly expressed at the four-cell (4C) stage in porcine embryos compared with one-cell (1C) and two-cell (2C) stages. The number of blastocysts was reduced following YBX1 knockdown. Notably, YBX1 knockdown decreased the phosphatase and tensin homolog-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PRKN) mRNA levels. YBX1 knockdown also decreased PINK1, active mitochondria, and sirtuin 1 levels, indicating reduced mitophagy and mitochondrial biogenesis. Furthermore, YBX1 knockdown increased the levels of glucose-regulated protein 78 (GRP78) and calnexin, leading to endoplasmic reticulum (ER) stress. Additionally, YBX1 knockdown increased autophagy and apoptosis. In conclusion, knockdown of YBX1 decreases mitochondrial function, while increasing ER stress and autophagy during embryonic development.Y-box binding protein 1 ( A mitochondrion is an essential organelle that controls energy conversion and ATP production . MitochoYBX1 is necessary for cancer cell proliferation and embryonic development. In human cervical cancer HeLa cells, YBX1 affects mitochondrial oxidative phosphorylation proteins expression (PINK1) is associated with Parkinsonian disorders  to regulate mitophagy for brown adipogenesis and thermogenesis  is a cytoplasmic messenger ribonucleoprotein that can bind to RNA . It modupression . In goatpression . In mouspression . In zebrpression . Phosphaisorders . PINK1 ppathways . YBX1 knogenesis . PINK1 kpoptosis .In this study, we hypothesized that YBX1 is crucial for porcine embryonic development. To evaluate this hypothesis, we reduced the expression of YBX1 by microinjection of YBX1 siRNA to explore the role of YBX1 during the early development of porcine embryos. Additionally, we examined mitochondrial function, GRP78, calnexin, LC3, and caspase 3 following YBX1 knockdown. The results suggest that YBX1 affects embryonic development by influencing mitochondrial function, ER stress, autophagy, and apoptosis.Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich .in vitro maturation (IVM) medium  containing TCM199, 0.1 g/L sodium pyruvate, 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 10 IU/mL luteinizing hormone, and 10 IU/mL follicle-stimulating hormone. COCs were randomly divided and cultured in IVM medium (500 μL) for 44 h at 38.5°C under 5% CO2.Pre-pubertal porcine ovaries were collected from a local abattoir  and transported to the laboratory at 37°C in phosphate-buffered saline (PBS). Porcine cumulus–oocyte complexes (COCs) were aspirated from small antral follicles (diameter: 3–6 mm). Oocytes surrounded by intact cumulus layers were washed thrice with the 2, 0.05 mM MgSO4, 0.01% polyvinyl alcohol , and 0.5 mM 4-(2-hydroxyethyl) piperazine-l-ethanesulfonic acid. After that, activated oocytes were cultured with 0.4% bovine serum albumin (BSA) and 7.5 μg/mL cytochalasin B in PZM-5 for 3 h to inhibit pseudo-second polar body extrusion. Activated oocytes were washed 3 times and cultured in PZM-5 with 0.4% BSA in a 4-well plate under the same condition as IVM. Subsequently, embryos were cultured for 7 days, and then examined the blastocyst development rate (blastocyst development rate = number of blastocysts/number of embryos).Briefly, 1 mg/mL hyaluronidase was used to detach the cumulus cells. Oocyte with a polar body was selected for activation. Denuded oocytes were parthenogenetically activated using two direct-current pulses of 110 V for 60 µs in 280 mM mannitol containing 0.1 mM CaClYBX1 siRNA (50 μM) was microinjected into the cytoplasm of a parthenogenetically activated oocyte via an Eppendorf Femto-Jet  and Nikon Diaphot Eclipse TE300 inverted microscope  equipped with the Narishige MM0-202N hydraulic 3-dimensional micromanipulator . As a control, the company provided negative siRNA  was microinjected into the cytoplasm of a parthenogenetically activated oocyte under the same conditions. Embryos were cultured in PZM-5 medium for 1 or 2 or 7 d after microinjection.For the knockdown groups, the sequences of the small interfering RNAs (siRNAs) was the same with previous study . YBX1 siAccording to previous article , 4% paraFor the colocalization of mitochondria and TOM20, 4-cell stage embryos were incubated with 500 nM MitoTracker Red CMXRos  at 38.5°C for 30 min. After 3 washes with PZM-5, the staining of TOM20 was the same as in the immunofluorescence staining.60 embryos per group (control group and YBX1 knockdown group) were placed in 20 μL of ice-cold 1 × sodium dodecyl sulfate [SDS] sample buffer and incubated at 98°C for 10 min. Based on the previous article , the pro18S rRNA was used as the reference gene. All primer sequences used in this study are listed in −ΔΔCT method.RNA was extracted from a pool of 30 embryos per group (control group and YBX1 knockdown group) using the Dynabead mRNA DIRECT kit . cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Quantitative reverse transcription-PCR was performed using a fast real-time PCR system (ABI StepOnePlus). Real-time quantitative PCR (qPCR) was performed according to previous article . 18S rRNt-test was used to compare the results between groups. p < 0.05 was considered statistically significant.Each experiment was repeated at least three times and results were presented as the mean ± standard error of the mean. The GraphPad Prism 5 software  was used for statistical analysis. A YBX1 during embryonic development. We observed that the mRNA levels of YBX1 were increased from the 4C to BL stage compared with the 1C and 2C stages , four-cell , morula  and blastocyst  stage embryos. As shown in C stages , indicatYBX1 during embryonic development in pigs, we knocked down YBX1 at the 1C stage. As shown in YBX1 were decreased by approximately 62% in the YBX1 knockdown group compared to those in the control group at the 2C, 4C and blastocyst stage. Knockdown of YBX1 was verified by Western blotting  and calnexin  were all significantly increased after YBX1 knockdown. Meanwhile, protein expression levels of BiP/GRP78  and blastocyst  stage embryos of the YBX1 knockdown group compared to the control group. This result was confirmed by Western blotting . Moreover, the apoptosis related genes Caspase 3 (1 vs 1.62 ± 0.2; p < 0.05; BAX (1 vs 1.69 ± 0.24; p < 0.05; BCL2 (1 vs 1.95 ± 0.17; p < 0.05; YBX1 knockdown induces autophagy and apoptosis.Autophagy and apoptosis are important for maintaining organismal and cellular homeostasis . As mitoYBX1 decreased PINK1 and PRKN expression levels, thereby decreasing mitochondrial function and increasing ER stress autophagy as well as apoptosis during embryonic development (In this study, we found that the loss of elopment .YBX1 knockdown decreased PINK1 and PRKN mRNA levels and reduced PINK1 protein levels, indicating that YBX1 may regulate their mRNA stability and protein expression to influence mitophagy. SIRT1 is a marker of mitochondrial biogenesis (YBX1 significantly reduced SIRT1 expression and mitochondrial activity, indicating that YBX1 is important for mitochondrial biogenesis. Therefore, YBX1 is crucial for mitophagy and mitochondrial biogenesis.Mitophagy is the selective degradation of mitochondria via autophagy . It usuaogenesis . Previouogenesis . MitoTraogenesis . Additioogenesis . Here, kYBX1 knockdown increased the expression levels of calnexin and GRP78, inducing ER stress. Subsequently, increased ER stress induced UPR. Previous article indicated that YBX1 depletion induces UPR (ER serves as a crucial organelle involved in the biosynthesis of lipids, proteins, and secreted proteins as well as an important site of calcium homeostasis . ER streuces UPR , which iYBX1 knockdown increased LC3 protein level. In cancer cells and preadipocytes, YBX1 knockdown reduces LC3 protein expression (PINK1 knockdown increases autophagy and apoptosis (YBX1 knockdown increased GRP78 level and decreased PINK1 level, thereby increasing autophagy. In addition, YBX1 knockdown has been reported to upregulate the levels of apoptosis-related genes, such as FAS, tumor necrosis factor, and caspase (YBX1 expression, whereas YBX1 overexpression attenuates growth inhibition and apoptosis in PC12 cells (YBX1 knockdown induces autophagy and apoptosis.Autophagy is a crucial cellular response to stress that degrades and removes unfolded proteins and damaged organelles to protect the cells . As mitopression . In thispression , which mpression . PINK1 kpoptosis . Here, Y caspase . One stu12 cells . However12 cells . The ele12 cells . In addi12 cells . MoreoveYBX1 decreases PINK1 and PRKN expression levels and mitochondrial function and induces ER stress, thereby causing autophagy and apoptosis during embryonic development.Taken together, our results indicate that


The as-synthesized ZrFe2O4@SiO2@Ade-Pd MNPs were methodically characterized over different physicochemical measures like VSM, EDX, Map, SEM, TEM, ICP, and FT-IR analysis. The catalytic activity of ZrFe2O4@SiO2@Ade-Pd was carefully examined for the room-temperature Carbon–Carbon coupling reaction in acetonitrile as a solvent. It is worth noting that the synthesized solid catalyst can be easily recovered with a bar magnet and reused for five cycles without decrease of catalytic activity.This work presents the simple synthesis of a green and novel Palladium based magnetic nanocatalyst with effective catalytic properties and reusability. These heterogeneous catalysts were prepared by the anchoring of Pd(0) on the surface of ZrFe Carbon–carbon and carbon–heteroatom cross-coupling reactions based on transition metal catalyst systems have revolutionized synthetic methodologies for the synthesis of simple to complex organic structures11. These types of reactions have wide applications in the synthesis of agricultural, pharmaceutical, and chemical compounds of electronic materials22.In recent years, one of the important achievements of scientists is the discovery of C–C coupling reactions in the presence of complexes containing transition metals such as copper and palladium. Among transition metal complexes, Pd is one of the best choices, due to its selectivity in coupling reactions, high activity, and simple complex preparation. The Suzuki–Miyaura cross-coupling reaction, which involves the coupling of phenylboronic acid and aryl halide, is used as an efficient and simpler method to prepare biphenyls24. Also, the development of synthetic protocols for Carbon–Nitrogen bonds have executed significant significance in each academia and industry25. The Buchwald–Hartwig reaction represents a transition metal–promoted versatile catalytic approach for the formation of Carbon–Nitrogen bonds26.Aromatic amines are ubiquitous in natural products and synthetic purposeful materials which include pharmaceuticals and drugs, agrochemicals, perfumes, and flavors31. Recently, nanostructured materials, especially magnetic nanoparticles, have been used as widely used supports in the synthesis of magnetic nanocatalysts due to their easy preparation and operation, high surface area, and easy recovery by the magnetic field, which leads to optimization of operating cost. One of the most obvious advantages of magnetic nanoparticles is their simple separation from the reaction mixture by a bar magnet. Among heterogeneous catalysts, ZrFe2O4 have been greatly favored since they are simply synthesized and surface modified using a magnet. However, various catalysts can be supported on ZrFe2O4 nanoparticles, because they can be easily separated after several consecutive uses in the reaction.Research and development of efficient and new nanomaterials as catalyst support is a big challenge in modern organic synthesisIn this research project, we report the synthesis and structural characterization of a green and novel nanocatalyst to stabilize Pd ions and to investigate their utility as a green and efficient nanocatalyst in the Buchwald–Hartwig Cross‑coupling and Suzuki–Miyaura cross-coupling reaction.2O4@SiO2@Ade-Pd nanocatalyst, first ZrFe2O4@SiO2@n-Pr-Cl were synthesized by the co-precipitation method. To synthesize the ZrFe2O4@SiO2@Ade-Pd complex, 3 g of the prepared ZrFe2O4@SiO2@n-Pr-Cl was dispersed in 50 mL toluene by sonication for 65 min. Then, 5 mmol of adenine and Et3N (10 mmol) were added into the flask and stirred at 90˚C for 60 h at 60 °C under N2 conditions. After the reaction completion, the obtained ZrFe2O4@SiO2@Ade were separated by magnetic decantation, washed with EtOAc and n-hexane, and then dried at 80 °C in an oven for 21 h. Finally, to prepare ZrFe2O4@SiO2@Ade-Pd organometallic catalytic complex, a mixture of ZrFe2O4@SiO2@Ade (2.0 g), Pd(OAc)2 (0.1 g) and 75 ml ethanol was added into the flask and, then, N2 gas was charged into the flask and, finally, it was stirred at 75 °C for 48 h under reflux conditions. Then, NaBH4 (8 mmol) was added into the flask and stirred for 8 h. Next, after completion of the reaction, the final complex ZrFe2O4@SiO2@Ade-Pd was separated and washed with H2O and ethanol and, finally, dried under vacuum at 55 °C (Scheme For the synthesis of ZrFeand Et3N 0 mmol we3N (0.5 mmol), and arylamine (0.6 mmol) were dissolved in EtOH (4 mL). The ZrFe2O4@SiO2@Ade-Pd (0.01 g) was added to the mixture, and the resultant mixture was stirred at reflux conditions. The reaction progress was monitored by TLC. Upon the reaction completion, the ZrFe2O4@SiO2@Ade-Pd was separated using a magnet, and then, EtOAc (3 × 4 mL) was added to the reaction mixture. Then the organic phase was separated and dried with the MgSO4, and then the organic solvent was removed at room temperature. The products were purified by silica gel column chromatography , Et2 (0.6 mmol), aryl halides (0.5 mmol), and K2CO3 (0.7 mmol) in the presence of ZrFe2O4@SiO2@Ade-Pd (0.01 g) was dissolved in Acetonitrile and stirred at room temperature as the mild conditions for appropriate time which is shown in Table A mixture of PhB(OH)4-Methoxy-1,1'-biphenyl:1H NMR : δH = 6.96–7.27 , 4.06  ppm.1,1'-Biphenyl]-4-amine:1H NMR : δH = 7.19–7.92 , 4.24  ppm.2-methyl-4-nitro-1,1'-biphenyl:1H NMR : δH = 7.39–7.73 ,  ppm.Diphenylamine:1H NMR : δH = 7.86–7.92 , 8.41–8.47 , 8.89  ppm.4-methoxy-N-phenylaniline:1H NMR : δH = 7.15–8.04 , 8.56 , 4.46  ppm.2O4@SiO2@Ade-Pd nanocatalyst was characterized by Map, SEM, XRD, FT-IR, EDS, ICP, and TGA techniques.The prepared ZrFe−1. The addition of each layer to the previous one leads to the observation of characteristic peaks of newly added functional groups in the spectrum which proves the formation of a new layer. The absorption bands for ZrFe2O4  below 250 °C is due to the removal of adsorbed solvents and OH groups  supported on the surface of ZrFeght loss % below 2ght loss % below 22O4@SiO2@Ade-Pd indicates the existence of Zr, O, N, Fe, Si, C, and Pd elements  with a sphere-like structure. These observations were verified with the achieved data from transmission electron microscopy images Fig. j–m.Figur2O4@SiO2@Ade-Pd were probed with N2 adsorption − desorption isotherm measurements. The obtained results show that the BET-specific surface area is 11.7 m2 g−1 for the catalyst  ZrFe2O4@SiO2@Ade-Pd was also investigated using VSM technics. As expected, the decrease in saturation magnetization from about 41 emu/g to about 29 emu/g, is related to the newly coated layer and confirmed the successful formation of the desired catalyst.As illustrated in Fig. 2O4@SiO2@Ade-Pd in the reaction between aniline with iodoarene was studied as a model reaction . Then from the intermediate reaction with an arylamine to form intermediate (II). Finally, for Buchwald − Hartwig amination reactions, the product is formed through the base-assisted elimination of X–H from intermediate (III) by latter reductive elimination and regenerates ZrFe2O4@SiO2@Ade-Pd MNPs.In the proposed mechanisms illustrated in Scheme 2O4@SiO2@Ade-Pd complex was then utilized as catalyst for the C–C cross-coupling reaction. Initial experiments with iodobenzene (1 mmol) as the substrate was conducted to optimize the reaction conditions including the amount of nanocatalysts, solvents, and base , K2CO3 (2 mmol) as the base in the Acetonitrile at 25 °C.The ZrFese Table . Also, t6H5B(OH)2 with C6H5I (Iodobenzene), the scope of the catalytic activity of ZrFe2O4@SiO2@Ade-Pd was extended to the coupling of other aryl halides with phenylboronic acid. Therefore, various aryl chlorides, aryl iodides, and aryl bromides were investigated in the cross-coupling reaction with phenylboronic acid in the presence of ZrFe2O4@SiO2@Ade-Pd MNPs  catalyst such that the Ar-X bond can be easily broken along with the formation of the Pd-Ar and Pd-X bond. Next, under the participation of the base, an organoborane compound reacts with intermediate I in transmetalation to afford intermediate II.The proposed mechanism for the Suzuki reaction in the presence of ZrFe2O4@SiO2@Ade-Pd species to give the desired product.This is followed by reductive elimination to regenerate the original ZrFe2O4@SiO2@Ade-Pd. The nanocatalyst was separated by a bar magnet from the reaction mixture and washed several times with EtOH and then with H2O several times. The nanocatalyst was used over five runs without any significant loss of activity (Fig. In another check, to investigate the recyclability of the magnetic nanocatalyst, the Iodobenzene with phenylboronic acid was examined as a model reaction in EtOH using 0.02 g of ZrFeity Fig. .Figure 92O4@SiO2@Ade-Pd we compared the results of the Suzuki reaction with the previously reported in the literature. As shown in Table 2O4@SiO2@Ade-Pd indicated significant catalytic activity in terms of the high yield and best reaction time.In the next step, to demonstrate the merit of ZrFe2O4@SiO2@Ade-Pd a green, novel, recoverable magnetic nanocatalyst. The prepared catalyst, ZrFe2O4@SiO2@Ade-Pd, was identified via EDS, SEM, VSM, TEM, XRD, TGA, and FTIR. The new nanocatalyst was used for the synthesis of the Buchwald–Hartwig Cross‑coupling and Suzuki reaction. Furthermore, this new ZrFe2O4@SiO2@Ade-Pd can be readily prepared from commercially available materials. Also, it can be mentioned good catalytic activity, easy separation, and reusability of the introduced nanocatalyst.This work reports the investigation of an efficient procedure to prepare ZrFe


Specifically, H9C2 cells exposed to DOXO showed an increase in the protein expression proteins of senescence-associated genes, p21 and p16, and a decrease in the telomere binding factors TRF1 and TRF2, indicative of senescence induction. Nevertheless, A5+ pre-treatment attenuated the senescent-like cell phenotype, as evidenced by inhibition of all senescent markers and a decrease in SA-β-gal staining in DOXO-treated H9C2 cells. Importantly, A5+ restored the LC3 II/LC3 I ratio, Parkin and BNIP3 expression, therefore rescuing mitophagy, and decreased ROS production. Further, A5+ pre-treatment determined a ripolarization of the mitochondrial membrane and improved basal respiration. A5+-mediated protective effects might be related to its ability to activate mitochondrial SIRT3 in synergy with other micronutrients, but in contrast with SIRT4 activation. Accordingly, SIRT4 knockdown in H9C2 cells further increased MnSOD activity, enhanced mitophagy, and reduced ROS generation following A5+ pre-treatment and DOXO exposure compared to WT cells. Indeed, we demonstrated that A5+ protects H9C2 cells from DOXO-induced senescence, establishing a new specific role for A5+ in controlling mitochondrial quality control by restoring SIRT3 activity and mitophagy, which provided a molecular basis for the development of therapeutic strategies against cardiomyocyte senescence.Oxidative stress and impaired mitophagy are the hallmarks of cardiomyocyte senescence. Specifically, a decrease in mitophagic flux leads to the accumulation of damaged mitochondria and the development of senescence through increased ROS and other mediators. In this study, we describe the preventive role of A5 The aging process is characterized by progressive structural and functional declines of the heart, and it is by far the major risk factor for the development of cardiovascular diseases (CVD) . CellulaSeveral hypotheses on the biological basis of aging have been suggested, such as oxidative stress due to increased production of reactive oxygen species (ROS) and altered mitophagy . In partPolyphenols are naturally occurring bioactive compounds that exert important biological activities, such as antioxidant, anti-inflammatory, anti-atherogenic, and anti-aging effects, among others . Differe+ is a mixture composed primarily of Polydatin but also of Ellagic acid, Pterostilbene, Honokiol and other beneficial micronutrients. It has already been tested on different in vitro systems, showing a protective role against virus infections, neurodegeneration, and obesity mainly through anti-inflammatory effects  freshly added with protease and phosphatase inhibitors (0.1 mMol/L Dithiothreitol (DTT), 50 mM sodium fluoride (NaF), 0.1 mM sodium orthovanadate (Na3VO4), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail) . Protein concentration was determined by Bradford assay  using the 96-well plater reader GloMax®-Multi Detection System .To obtain whole cell lysates, after treatments, H9C2 were pelleted at 1500 rpm (5 min at 4 °C) and lysed with Lysis buffer [50 mM Tris-HCl pH 7.4, 5 mM ethylenediaminetetraacetic acid (EDTA), 250 mM sodium chloride (NaCl), and 0.1% Triton® TURBO; EuroClone, Milan, Italy). Densitometric analysis of the bands, relative to housekeeping proteins β-Actin or GAPDH, was determined by ImageJ Software v1.51 . Equal amounts of total cellular proteins (30 μg/lane) were boiled for 5 min at 95 °C, resolved by denaturating SDS-polyacrylamide gel electrophoresis, and transferred to a 0.45 μm nitrocellulose membrane . After blocking with 5% non-fat dry milk , membranes were incubated with the primary antibody overnight at 4 °C, followed by appropriated horseradish peroxidase-coupled secondary antibodies (Horseradish peroxidase-linked anti-mouse (NA931V) and anti-rabbit (NA934V) antibodies, GE Healthcare, Chicago, IL, USA) and developed by a chemiluminescence-based detection system ; mouse monoclonal anti-p16 ; rabbit polyclonal anti-phospho-p53 (Ser15) ; rabbit polyclonal anti-total p53 ; rabbit monoclonal anti-phospho (Ser795) Rb ; mouse monoclonal anti-Rb ; mouse monoclonal anti-TRF1 ; mouse monoclonal anti-TRF2 ; mouse monoclonal anti-cyclin D1 ; rabbit polyclonal anti-PARP1 ; rabbit monoclonal anti-SIRT1 ; rabbit monoclonal anti-SIRT2 ; rabbit monoclonal anti-SIRT3 ; rabbit polyclonal anti-SIRT4 ; mouse monoclonal anti-SIRT5 ; rabbit monoclonal anti-SIRT6 ; mouse monoclonal anti-SIRT7 ; rabbit monoclonal anti-acetyl (K122) SOD2/MnSOD ; rabbit polyclonal anti SOD2/MnSOD ; rabbit polyclonal anti LC3B ; mouse monoclonal anti-Parkin ; mouse monoclonal anti-BNIP3 ; mouse monoclonal anti β-Actin ; mouse monoclonal anti-GAPDH .+ able to promote beneficial effects without exerting toxic effects, H9C2 cells were treated with different concentrations of A5+  for 24 h. CellTiter 96® AQueous Solution Cell Proliferation Assay  was used as a metabolic assay for cell viability. Briefly, cells were seeded in 96-well plates . Following A5+ treatments, 20 µL of CellTiter 96® Aqueous Solution was added to 100 µL of culture medium. After 2 h of incubation at 37 °C, absorbance at 490 nm  was measured with the 96-well plater reader GloMax®-Multi Detection System .In order to establish the concentration of A55 H9C2 cells were seeded into 100 mm dishes and treated with increasing doses of A5+ , respectively, for 24 h, 48 h, and 72 h; in the second setting, 2 × 105 H9C2 cells were treated with 0.1 µM DOXO for 24 h or with 0.1 µM DOXO in combination with A5+ for 72 h, as described above. Subsequently, cells were harvested with trypsin-EDTA, washed twice with ice-cold PBS, centrifuged at 800× g for 5 min at 4 °C, and finally fixed with pre-cold 70% ethanol overnight at 4 °C. The following day, cells were washed with PBS and stained with 50 µg/mL Propidium Iodide  in the dark for 30 min at room temperature. Fluorescence was read by the LSRFortessa X-20 flow cytometer . The sub-G1 fraction, which represents the total amount of apoptotic cells, was determined and analyzed through BD FACSDiva software (v. 8.0.2).To evaluate cell death, the cell cycle was analyzed by flow cytometry in two different experimental sets. In the first setting, 6, 4, or 2 × 10+ for 24 h was measured using a coupled enzyme assay, following the manufacturer’s instructions . In this assay, glutamate is consumed by GDH, generating NADH as a reaction product, which, in turn, reacts with a probe to generate a colorimetric (450 nm) product proportional to the GDH activity present in the sample. One unit of GDH is the amount of enzyme that will generate 1.0 µmol of NADH per minute at pH 7.6 at 37 °C. Briefly, a total of 1 × 106 cells were lysed in 40 μL of GDH assay buffer and kept for 10 min on ice. Afterward, lysates were centrifuged, and 10 μL of the supernatant was added to 40 μL of GDH buffer and 100 μL of a mix containing GDH assay buffer, developer, and glutamate. The whole mix was transferred into a 96-well plate and incubated at 37 °C for 3 min. Finally, absorbance was read at 450 nm using a Glomax multidetection system . The absorbance of the assay buffer was subtracted from each experimental sample.Glutamate dehydrogenase (GDH) activity in control cells or in H9C2 cells treated with 10 µM, 25 µM, and 50 µM A5Here, 5,5,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) dye was used for monitoring the mitochondrial membrane potential (Δψm) as an indicator of mitochondrial health . In mitochondria, this lipophilic cationic probe can enter and accumulate in a monomeric or aggregated form, depending on mitochondrial membrane potential (∆Ψm). In healthy cells, where ∆Ψm is high and mitochondria are energized and negatively charged, JC-1 spontaneously polymerizes to form red fluorescent J-aggregates. On the contrary, in unhealthy cells, where mitochondrial integrity is compromised and ∆Ψm assumes a lower value, JC-1 remains in a monomeric form (J-monomers), showing a green fluorescence emission. The fluorescence shift from red to green is an indicator of mitochondrial depolarization. 4 cells were grown on glass coverslips and treated with 25 µM A5+ or 50 µM A5+ for 72 h, with 0.1 µM DOXO for 24 h, and with 0.1 µM DOXO for 24 h in combination with A5+ for 72 h, as described above. Here, 72 h cultured, untreated cells were used as controls. After treatments, cell culture medium was removed and then replaced with warm medium (37 °C) containing 2 μM  of JC-1 dye. After a 20 min incubation at 37 °C with 5% CO2, cells were washed in warm PBS (37 °C), and fluorescence was quickly observed by LSM 510 confocal microscopy .For confocal microscopy monitoring of mitochondrial membrane potential, 5 × 10+ for 48 h, culture medium was replaced with unbuffered DMEM medium, supplemented with 2 mM L-glutamine, 11 mM glucose, and 1.2 mM pyruvate, adjusted to pH 7.35 and then incubated for 30 min at 37 °C in a CO2-free incubator. Oxygen Consumption Rates (OCR), proportional to mitochondrial respiration, were measured for the basal state and following the sequential injection of oligomycin (1 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (3 μM), and a mix of antimycin A (2 μM) and rotenone   in each well, according to the Seahorse Mito Stress Test protocol [Mitochondrial respiration was measured by plate respirometry using a Seahorse XFp Extracellular Flux Analyzer , as previously published . Brieflyprotocol . Data we5 H9C2 wt, shControl, and shSIRT4 cells were seeded in 100 mm diameter dishes and treated with 0.1 µM DOXO for 24 h or with 0.1 µM DOXO in combination with A5+ for 72 h, as described above. After treatments, the medium was removed and the H9C2 were fixed for 15 min with β-galactosidase fixative  at room temperature and then washed three times in PBS at pH 6 at room temperature. Subsequently, the cells were incubated with freshly prepared pH 6 SA-β-gal staining solution  per ml of dimethylformamide/40 mM citric acid/sodium phosphate, 5 mM potassium ferrocyanide/5 mM potassium ferricyanide/150 mM NaCl/2 mM MgCl2) and incubated in a dry incubator at 37 °C without CO2 overnight. After staining, cells were washed twice with pH 6 PBS. Senescence was quantitated by visual inspection of blue/green stained cells using an inverted Zeiss IM35  with a 100× magnification and a digital camera . The percentage of positive cells was calculated by counting the blue-stained cells out of the total cells in 20 randomized low-power fields for each sample.To detect senescent cells, senescence-associated β-galactosidase activity  associated with increased lysosomal content was investigated. Staining for SA-β-gal was performed with a Senescence Cell Histochemical Staining kit , according to the manufacturer’s protocols. Briefly, 2 × 106 H9C2 cells were seeded for 24 h before treatment with doxorubicin, A5+, or doxorubicin+A5+. After treatments, the medium was removed and the cells were fixed with 2.5% glutaraldehyde in PBS for 48 h, as previously described [For ultrastructural analysis, 1 × 10escribed .5 H9C2 wt, shControl, and shSIRT4 cells were seeded in 100 mm diameter dishes and treated with 0.1 µM DOXO for 24 h or with 0.1 µM DOXO in combination with A5+ for 72 h, as described above. After treatments, culture medium was discarded and replaced with fresh medium containing a final concentration of 25 µM H2DCF-DA dissolved in DMSO. After a 30 min incubation at 37 °C and 5% CO2, cells were collected and washed twice in HBSS/Ca/Mg . Subsequently, fluorescence was read by a CytoFlex flow cytometer , and median fluorescence intensity (MFI) has been considered for graphic analysis.ROS intracellular level was estimated using the cell-permanent 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) fluorescent dye , according to the manufacturer’s instructions. H2DCF-DA is a non-fluorescent chemically reduced form of fluorescein, but in the presence of intracellular ROS, it is oxidized and converted, upon cleavage, to highly fluorescent dichlorofluorescein (DCF). Briefly, 2 × 107 cells for each treatment were harvested and pelleted at 850× g for 5 min. Then, Mitochondria Isolation Reagent A, supplemented with protease inhibitors, was added to the cells, followed, after a few minutes of incubation on ice, by the addition of 10 µL of Mitochondria Isolation Reagent B. Cells were then incubated on ice for 5 min, vortexing at maximum speed every minute, before adding Mitochondria Isolation Reagent C. Cells were centrifuged at 700× g for 10 min at 4 °C, and the supernatant was transferred to a new tube for an additional centrifugation step at 3000× g for 15 min at 4 °C to separate the cytosol fraction (supernatant) and the isolated mitochondria (pellet). In a final step, isolated mitochondria were washed with Mitochondria Isolation Reagent C and centrifuged at 12,000× g for 5 min to obtain a more purified fraction of mitochondria. Mitochondria were then lysated with RIPA buffer  Triton® X-100, 0.5% (v/v) Sodium Deoxycholate, 0.1% (v/v) SDS, pH 7.4) freshly added with protease and phosphatase inhibitors, and protein content was determined by a Bradford assay , as described previously. Mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells , following the manufacturer’s instructions. Briefly, ~1 × 10Freshly lysated mitochondria were immediately prepared for measuring the MnSOD activity using the Superoxide Dismutase (SOD) Colorimetric Activity Kit , based on the manufacturer’s recommendation. Briefly, 10 μL of mitochondrial lysate or standards  and 50 μL of substrate were added into a 96-well plate, and the absorption was recorded at 450 nm (background absorbance) using a Glomax multidetection system . Then, 25 µL of Xanthine Oxidase (a chromogenic detection reagent) were added to each well, and the plate was incubated at room temperature for 20 min before being read again at 450 nm. The total MnSOD activity (mU/mL) was calculated in relation to a SOD standard curve and normalized by the protein abundance in each sample.t-test for two groups; two-way analysis of variance (ANOVA) was applied for multiple comparisons with Bonferroni post hoc analysis. Differences between groups were considered statistically significant at values of p < 0.05. Results are expressed as mean ± S.E.M. Data were analyzed on GraphPad version 9 for Windows .Parametric statistical analysis was performed using the Student’s In this study, we used an established cell model of doxorubicin-induced cell senescence . SpecifiTo verify the induction of senescence, the protein expression of senescence-associated proteins p21 and p16 was determined by WB. Both markers were markedly increased at 24 h in the DOXO treatment group compared to the control a,b. In aSince sirtuins are essential factors that delay cellular senescence , we deteBased on the data obtained for senescence induction following DOXO treatment, 0.1 μM of DOXO for 24 h exposure was optimized for subsequent experiments.+ intervention was estimated using Cell Titer and flow cytometry assays in vitro.Firstly, viability after A5+ for 24 h had almost no cytotoxicity to H9C2 cells. A significant decrease in cell viability was detected only at 200 and 500 µM compared to control cells  of A5ol cells a.+ concentrations . The results of the sub-G1 assay by flow cytometry demonstrated that A5+ treatment did not cause a sub-G1 peak reflective of DNA fragmentation except at the latest time point, i.e., 72 h, at the highest concentration, i.e., 100 µM.  using different A5 100 µM. b,c.+ for all subsequent experiments.According to these results, we decided to use 50 µM A5+ to modulate sirtuins, we analyzed the protein expression by WB analysis of all sirtuins following incubation of H9C2 cells with A5+ at 50 µM for 24, 48, and 72 h. A5+ treatment resulted in a significant down-modulation of SIRT1 and SIRT5 and up-regulation of mitochondrial SIRT3 and SIRT4 after 72 h of treatment  enzymes, limiting the metabolism of glutamate and glutamine and, therefore, reducing ATP production . We measreatment b.+ on the cytosolic autophagosome marker LC3 B, the mitophagy kinase Parkin, and the mitophagy receptor BCL2/adenovirusE1B 19 kDa interacting protein 3 (BNIP3) in H9C2 cells compared to controls at all time points. As showed by WB analysis, stimulation with A5+ remarkably increased the ratio of LC3 II/LC3 I  seemed to decrease, whereas the mass of the mitochondria with a high membrane potential (red fluorescence) seemed to increase in treated cells compared to the control.We next studied the changes in the mitochondria in terms of their inner membrane potential using the mitochondria-specific fluorescent probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). JC-1 is a dichromatic dye that exhibits potential-dependent accumulation in mitochondria. It exists as either a green-fluorescent monomer at depolarizing mitochondria or a red-fluorescent aggregate at polarizing mitochondria. The ratio of green to red fluorescence allows for the assessment of mitochondrial polarization states . Represe+ might induce attenuation in cardiac senescence, maintaining integral mitochondrial function through mitophagy enhancement.These results suggest that A5+ mitigates DOXO-induced cell senescence, H9C2 cells were exposed to DOXO (0.1 μM) for 24 h in the absence or presence of 48 h pre-treatment with A5+ (50 μM).To investigate whether A5+ pre-treatment   and maximal respiration  when compared to vehicle (CTRL). The addition of A5+ improved basal respiration  but not maximal respiration (p = n.s.). A5+ alone caused a decrease of 37.08 ± 10.31% of maximal respiration when compared to vehicle (p = 0.04), with no effect on basal respiration (p = n.s.).Importantly, mitochondrial respiration was affected by DOXO h. Twenty+-induced attenuation in cardiac senescence may be related to its protective effects on mitochondria and that these effects might be mediated by mitochondrial sirtuin activation.These results suggest that A5+ pre-treatment attenuated this increase    markedlyshSIRT4) a, while shSIRT4) . Then, w+ following DOXO exposure was more significant in shSIRT4 H9C2 cells compared to shControl cells -induced cardiac hypertrophy in mice, it was demonstrated that SIRT4 inhibited the binding of MnSOD to SIRT3, resulting in increased MnSOD acetylation and elevated ROS accumulation upon Ang II stimulation [+ pre-treatment followed by DOXO exposure, a significant decrease in MnSOD acetylation compared to shControl cells, confirming the inhibitory effect of SIRT4 on SIRT3-mediated deacetylation of MnSOD. Importantly, in shSIRT4 cells, MnSOD activity was already higher in basal conditions compared to shControl cells, increased following A5+ treatment, and remained high after A5+ pre-treatment and DOXO exposure. Accordingly, the inhibition of ROS production was more significant in SIRT4-knockdown H9C2 cells compared to shControl cells.The activation of mitophagy was paralleled by an enhancement in the expression of the mitochondrial sirtuins SIRT3 and SIRT4 and of their targets. Interestingly, it has been widely demonstrated that polyphenols like resveratrol, Pterostilbene, Polydatin and Honokiol can activate anti-inflammatory and antioxidant sirtuin pathways through the activation of autophagy and mitophagy ,46. In tmulation . Taking + and DOXO showed a more significant decrease in the amount of cellular ROS and no significant increase in autophagy compared to shControl cells. Together, these results suggested that SIRT4 might have an important role in managing the players involved in the antioxidant response in senescent H9C2 cells, in particular by increasing ROS production through inhibition of SIRT3-mediated MnSOD deacetylation and through a decrease in mitophagy. Noteworthy, SIRT4 silencing also determined further activation of mitophagy, as evidenced by enhanced expression of Parkin and BNIP3. SIRT4 has already been reported to negatively regulate Parkin-mediated mitophagy. Specifically, moderate overexpression of SIRT4 in fibroblast models of senescence increased the levels of the inner-membrane-bound long form of the GTPase OPA1 that promoted mitochondrial dysfunction and decreased Parkin-associated mitophagy . FurtherOne of the limitations of the present work is that the H9C2 cell line is phenotypically distinct from cardiac myocytes, even though they share several properties ,56. This+ has shown the benefit of ROS scavenging and mitophagy stimulation in preventing H9C2 cell senescence but has also evidenced that simultaneous activation of mitochondrial SIRT3 and SIRT4 might not always be synergistic, as evidenced in our in vitro model (+ and DOXO stimulation in shControl cells, even though to a lesser extent, and these effects, most likely, might be due to the potential synergistic effects of SIRT3 with other micronutrients present in A5+. For instance, zinc has already been demonstrated to induce mitophagy, leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation in cardiac cells [In conclusion, A5ro model . Neverthac cells . Seleniuac cells . Neverthac cells ,63.


Iron sulfide-based heterostructure in situ hybridized with nitrogen-doped carbon nanotubes was prepared through a successive pyrolysis and sulfidation approach.7S8/FeS2/NCNT electrode exhibits superior sodium storage performance in both ester and ether-based electrolytes.The as-prepared FeThe structure advantages of the electrode contribute to high electrochemical performance in the ester-based electrolyte, while fast ionic diffusion and favorable capacitive behavior result in the robust sodium storage performance in the ether-based electrolyte.The online version contains supplementary material available at 10.1007/s40820-023-01082-w. 7S8/FeS2/NCNT) have been prepared through a successive pyrolysis and sulfidation approach. The Fe7S8/FeS2/NCNT heterostructure delivered a high reversible capacity of 403.2 mAh g−1 up to 100 cycles at 1.0 A g−1 and superior rate capability (273.4 mAh g−1 at 20.0 A g−1) in ester-based electrolyte. Meanwhile, the electrodes also demonstrated long-term cycling stability  and outstanding rate capability (536.5 mAh g−1 at 20.0 A g−1) in ether-based electrolyte. This outstanding performance could be mainly attributed to the fast sodium-ion diffusion kinetics, high capacitive contribution, and convenient interfacial dynamics in ether-based electrolyte. Heterostructure engineering combined with carbonaceous materials shows great promise toward promoting sluggish kinetics, improving electronic conductivity, and mitigating the huge expansion of transition metal sulfide electrodes for high-performance sodium storage. Herein, the iron sulfide-based heterostructures in situ hybridized with nitrogen-doped carbon nanotubes (FeThe online version contains supplementary material available at 10.1007/s40820-023-01082-w. Sodium-ion batteries (SIBs) with a similar operating mechanism to lithium-ion batteries and abundant sodium resources are considered as one of the most promising candidates for low-cost renewable energy storage –5. Howev2 with a high theoretical capacity (894 mAh g−1), low cost, and environmental friendliness, is a promising and effective sodium storage electrode material [+ insertion/extraction, large electrochemical polarization, electrode degradation, and hence poor rate capability and limited cycling life [Transition metal sulfides (TMSs) are considered as promising electrode candidates due to their high theoretical capacities –15. The material –19. Howeing life –23.+ diffusion kinetics, improve the capacitive contribution of charge storage, and form robust solid electrolyte interface- (SEI) layer. Liu et al. [2 nanocrystals wrapped in a turbostratic carbon framework (FeS2–C) via a mechanical alloying process. The FeS2–C electrode achieved rapid electrons/ions transport and robust structural stability, thus enabling improved rate capability and long cycling performance for SIBs. Chen et al. [1−xS/MoS2 heterostructure with abundant active sites, and the corresponding electrode exhibited good rate capability and stable cycling performance. In addition to improving the sodium storage performance from the perspective of material synthesis, it was also found that different electrolytes can greatly affect the structural transformation and electrochemical performance of FeS2-based anodes during cycling. Zhou et al. [2@C electrode coupled with ether-based electrolyte could realize high ionic diffusion and electrochemical reversibility. These individual modification strategies can indeed modify the performance of iron chalcogenide-based electrodes. Therefore, the combination of the above three strategies will simultaneously endow the electrodes with high conductivity, rapid charge transfer, and favorable interfacial behaviors.To overcome the above mentioned challenges, many efforts have been made, mainly focusing on several aspects –27. Thesu et al.  designedn et al.  designedu et al.  demonstr7S8/FeS2/NCNT) via an in situ pyrolysis and sulfidation strategy. The as-prepared Fe7S8/FeS2/NCNT heterostructures inherit substantial lattice defects and sufficient buffer space offered by the NCNT, which endow the Fe7S8/FeS2/NCNT electrode with high sodium storage activity, rapid ionic diffusion kinetics, and excellent electrochemical reversibility. Interestingly, the electrochemical performance of Fe7S8/FeS2/NCNT electrode can be further improved in ether-based electrolyte. We also systematically investigated the ion diffusion kinetics, charge storage mechanism, and SEI composition of the Fe7S8/FeS2/NCNT electrodes in different electrolytes.Herein, we rationally designed and prepared iron sulfide-based heterostructures encapsulated in N-doped carbon nanotubes (Fe3) was added into the above aqueous solution and stirred for 1 h at room temperature. The mixture was then dried in 80 °C water bath to obtain light red powder (DICY-Fe). After that, the product was ground and calcined at 800 °C for 2 h under argon to receive the desired Fe/NCNT.A facile pyrolysis method was applied to prepare the Fe/NCNT precursor. Typically, 10 mL of deionized water and 250 mg dicyandiamide (DICY) were mixed together under stirring, and then 50 mg ferric chloride (FeCl−1) for 2 h under argon atmosphere to get the Fe7S8/NCNT. Then the Fe7S8/NCNT and sulfur powder with a mass ratio of 1:1 were calcinated at 400 °C (heating rate of 2 °C min−1) for 1 h under argon to obtain the Fe7S8/FeS2/NCNT. For comparison, the Fe7S8/NCNT and sulfur powder with a mass ratio of 1:3 were calcinated at 400 °C for 4 h under argon to obtain the FeS2/NCNT.The obtained Fe/NCNT and sulfur powder with a mass ratio of 1:3 were placed in an alumina boat, then annealed at 600 °C , where the lattice fringes with d-spacings of 0.261 and 0.268 nm correspond to (2 2 1) plane of Fe7S8 and (2 0 0) planes of FeS2, respectively, can be detected  manifests that the clear diffraction rings can be well indexed to the (4 1 1), and (2 2 0) planes of cubic FeS2 and (− 2 2 1) and (− 6 2 3) planes of monoclinic Fe7S8 [7S8/FeS2/NCNT are displayed in Fig. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to investigate the morphology features of the as-prepared samples. The SEM image of Fe/NCNT Fig. S1a) reveals regular nanotube morphology with diameters of about tens of nanometers, indicating the conversion from the DICY-Fe complexes to Fe species confined into the carbon nanotubes after carbonization. The SEM images of Fea revealslectrode . More sttransfer , 38. Besic Fe7S8 , 37. The7S8/FeS2/NCNT manifests the characteristic peaks of monoclinic Fe7S8 (JCPDS No. 52–1516) and cubic FeS2 (JCPDS No. 42–1340) [7S8, while the diffraction peaks centered at around 33.1°, 37.1°, 40.7°, 47.4° and 56.3° can be ascribed to the (2 0 0), (2 1 0), (2 1 1), (2 2 0) and (3 1 1) planes of cubic FeS2, respectively, thereby indicating the successful fabrication of the target heterostructure [7S8/NCNT and FeS2/NCNT can be identified with monoclinic Fe7S8 and cubic FeS2, respectively. While no diffraction peak related to the NCNT can be observed, mainly due to their amorphous state and low content. To confirm the existence of NCNT, Raman spectra are performed  and disordered domain (D bands) in the carbonaceous skeleton, respectively [ ID/IG value of Fe7S8/FeS2/NCNT (0.86) than those of Fe7S8/NCNT (0.81) and FeS2/NCNT (0.80) means that the more defects generated in the heterostructure, offering extra active sites and contributing to improving the sodium storage performance [7S8/FeS2/NCNT is characterized by thermogravimetric (TG) analysis. As shown in Fig. S5, the weight loss is attributed to the oxidation of Fe7S8/FeS2 and N-doped carbon in air. Based on the fact that the sulfidation process will not change the mass ratio of Fe element to NCNT from Fe/NCNT to Fe7S8/FeS2/NCNT, thus the content of NCNT in Fe7S8/FeS2/NCNT is calculated to be 30.4% [To preliminarily determine the phase structures of as-prepared samples, X-ray powder diffraction (XRD) measurements were performed. Particularly, the diffraction peaks of the as-synthesized Fe/NCNT  can be well indexed to the cubic Fe (JCPDS No. 03–1050), indicating the successful conversion from the DICY-Fe to Fe/NCNT . In Fig.42–1340) . Specifitructure . For comectively . Particuformance . Meanwhi7S8/NCNT, Fe7S8/FeS2/NCNT, and FeS2/NCNT samples were characterized through X-ray photoelectron spectroscopy (XPS). The XPS survey spectra of all samples shown in Fig. S6 verify the co-existence of elements Fe, S, C, and N in the target samples. Note that the appearance of O 1s peak should be ascribed to the exposure to air [p spectrum of Fe7S8/FeS2/NCNT exhibits two peaks at 706.2 and 718.6 eV, respectively, which confirms the existence of Fe2+, and the characteristic signals for Fe3+ can be observed at around 710.6 and 724.6 eV [p spectrum shown in Fig. p3/2 species can be detected at around 161.6 and 162.7 eV. In addition, the presence of S 2p1/2 can be confirmed by the peak detected at 163.8 eV, and two peaks at around 167.5 and 168.6 eV correspond to oxidized sulfur, which may be attributed to the oxidization of metal sulfides in air [s spectrum .The sodium storage performances of as-prepared samples were investigated in ester-based electrolyte. The comparison of initial cyclic voltammetry (CV) curves of Feectively . Subsequ+ to Fe0 . As for  NaxFeS2 , 41. To n of SEI . Afterwatability . In cont7S8/FeS2/NCNT as anode material for SIBs, the comparison of cycling stability of the as-fabricated electrodes was conducted , 448.5 (1.0 A g−1), 406.6 (2.0 A g−1), 359.7 (5.0 A g−1) and 316.7 (10.0 A g−1) mAh g−1, respectively . For the Fe7S8/NCNT and FeS2/NCNT electrodes, the gradually degraded plateaus and decreased capacities suggest their poor structure stability  [To further highlight the structural advantage of the Feted Fig. b. The FeS10b, c) .7S8/FeS2/NCNT electrode, CV curves collected from 0.2 to 0.8 mV s−1 scan rates have been performed , the CV shape of Fe7S8/FeS2/NCNT is well-maintained with increasing scan rate, indicating its weak electrochemical polarization and excellent electrochemical reversibility. It is well known that both diffusion and capacitive behaviors contribute to the charge stored in electrode, and the response current and scan rate obey the following equation [a is a variable parameter, and the value of b is a typical index to judge the reaction kinetics, and it can be received from the slope after plotting log (i) versus log (v). Generally, the value of b close to 1.0 corresponds to a pseudocapacitive process, while the value approaching to 0.5 means that the electrochemical process is dominated by diffusion [b values after fitting for the oxidation peaks are 0.68 (peak 1), 0.84 (peak 2), and 0.85 (peak 3), and the corresponding values of the reduction peaks are 0.87 (peak 4), 1.00 (peak 5) and 0.79 (peak 6), indicating that the charge stored in the Fe7S8/FeS2/NCNT electrode is dominated by both diffusion and capacitive behaviors [−1 manifest that the Fe7S8/FeS2/NCNT electrode displays a lower charge transfer resistance (93.5 Ω) compared with those of Fe7S8/NCNT (130.1 Ω) and FeS2/NCNT (134.4 Ω), indicating better electrochemical kinetics of the special designed Fe7S8/FeS2/NCNT heterostructure , and the corresponding contour plot in the range of 30° to 36° can observe the phase change more clearly , suggesting that Fe7S8/FeS2/NCNT experiences the Na+ insertion and phase transition upon discharge [2S and Fe, gradually weaken, combined with the appearance of NaxFeS2, suggesting the deintercalation of Na+. Finally, the formation of FeS2 and Fe7S8 suggests the reverse phase transition, highlighting its superior electrochemical reversibility [7S8/FeS2/NCNT electrode during sodiation/desodiation process. It undergoes a two-step reaction mechanism upon sodium storage: the Na+ intercalation and phase transformation during the discharge process; the reverse conversion reaction and Na+ extraction during the charging process [Furthermore, in order to reveal the working mechanism of the Fethe FeS2 , 22. Aftischarge . During sibility . Figure  process .7S8/FeS2/NCNT electrodes in ester/ether-based electrolytes. Figure 7S8/FeS2/NCNT electrode in those two electrolytes. Particularly, an impressive capacity of 466.7 mAh g−1 is maintained after 1,000 cycles at 5.0 A g−1 in the ether-based electrolytes, demonstrating its robust cycle life. For comparison, negligible capacity can be detected of Fe7S8/FeS2/NCNT electrode after 500 cycles in the ester-based electrolyte. In addition, the initial Coulombic efficiency (ICE) of 73.4% can be obtained in the ether-based electrolyte, which is much higher than that in the ester-based electrolyte (60.5%). This could be attributed to the high electrochemical reversibility of electrode in ether-based electrolyte. Besides, a superior CE can be detected in ether-based electrolyte (~ 100%) than that in ester-based electrolyte . In general, the excellent CE in the ether-based electrolyte could be attributed to the uniform and stable SEI generated on the electrode surfaces, which can efficiently prevent further consumption of electrolyte and thus improve electrochemical reversibility [In addition to improving electrochemical performance from material synthesis perspective, it has been found that electrolytes can greatly influence the structural evolution and electrochemical performance of iron sulfide-based electrodes in SIBs. Figure sibility , 50.Fig.7S8/FeS2/NCNT electrodes in those two electrolytes. Even at 20 A g−1, a high capacity of 536.5 mAh g−1 can be maintained in the ether-based electrolyte, and the capacity retention rate is about 81.7% for the reversible capacity at 0.5 A g−1. However, only 52.4% of the capacity can be retained in the ester-based electrolyte. Figure S14 shows discharge–charge profiles of the Fe7S8/FeS2/NCNT electrode at different current densities in the ether-based electrolyte. The charge/discharge curves at different current densities are almost consistent and show longer charge potential plateaus, contributing to the outstanding rate capability and high specific capacity [7S8/FeS2/NCNT can be also confirmed through the comparison with other related studies (Table S1), from where the target Fe7S8/FeS2/NCNT shows excellent sodium storage, mainly resulting from the heterostructure design and combination with NCNT [Figure capacity . Besidesith NCNT .7S8/FeS2/NCNT electrode shows the typical tubular structure, which indicates that the electrode fabrication process plays little effect on its morphology  have been investigated through the CV measurements  [7S8/FeS2/NCNT in ether-based electrolyte are higher than those in ester-based electrolyte, which indicate that the electrode coupled with ether-based electrolyte features rapid interfacial kinetics and larger capacitive contribution for charge storage , and galvanostatic intermittent titration (GITT) were conducted to evaluate Nants Fig. a, which rization . Meanwhiig. S15) , 55. Furage Fig. b [30, 32iffusion , 57. As 7S8/FeS2/NCNT electrode at 1st, 10th, 20th, and 50th cycles at 0.1 A g−1 in ether/ester-based electrolytes have been illustrated in Fig. Rct) and Warburg factor, respectively [Rct values in ether-based electrolyte indicate rapid charge transfer and stable interface [Rct value increases significantly from 34.6 (1st cycle) to 93.5 Ω (50th cycle), which can be attributed to the formation of a thick and unstable SEI layer caused by the ester-based electrolyte and then slow down the interfacial kinetics [The EIS comparison of the Feectively . As shownterface . For thekinetics .+ diffusion coefficient in different electrolytes. In general, the diffusion coefficient of sodium ion can be evaluated based on the following equation [Mb, mb, Vm, S and τ represent the molecular weight, the mass loading of active material, the molar volume of the electrode material, the surface area of the electrode, and the applied current, respectively, while ΔEs and ΔEτ correspond to the steady-state voltage change after the discharge pulse and the voltage variation during the discharge pulse [7S8/FeS2/NCNT electrode in ether-based and ester-based electrolytes (mainly ranging from 10−11 to 10−7 cm2 s−1). Specifically, the average diffusion coefficients of Na+ upon the first discharge and charge in the ether-based electrolyte are 4.71 × 10−9 and 2.66 × 10−8 cm2 s−1, respectively. As a comparison, the ester-based electrolyte exhibits relatively low diffusion coefficients, obtaining 1.24 × 10−9 and 1.62 × 10−8 cm2 s−1 during the first discharge and charge, respectively. Similarly, the variation trend of Na+ diffusion coefficients in the second cycle of the two electrolyte is similar to that in the first cycle , suggesting good electrochemical reversibility and stable ion transport channels [Besides, GITT investigation was carried out to compare the Naequation , 60:3\doge pulse , 60. Figchannels , 60.7S8/FeS2/NCNT heterostructures have been successfully fabricated via an in situ pyrolysis and sulfidation strategy. The as-prepared Fe7S8/FeS2/NCNT features Fe7S8/FeS2 heterostructure embedded in the hollow carbon nanotubes. The unique nano-architecture provides abundant lattice defects, superior electronic conductivity, and sufficient buffer space to maintain the integrity of the electrode during cycling. Consequently, the Fe7S8/FeS2/NCNT electrode delivered high sodium storage capacity, rapid ionic diffusion kinetics, and remarkable electrochemical reversibility. In particular, the Fe7S8/FeS2/NCNT electrode exhibits even more stable long-term cycling performance  and rate capability (536.5 mAh g−1 at 20 A g−1) in ether-based electrolyte, which endows the battery with rapid ion diffusion kinetics, low charge-transfer resistance, high capacitive contribution to charge storage and robust SEI layer. The advanced Fe7S8/FeS2/NCNT heterostructure design and in-depth investigation on the origin of the superior electrochemical performances in ether-based electrolyte have paved the way for the development of TMS-based electrodes in sodium storage system.In summary, the rationally designed FeSupplementary file1 (PDF 2050 kb)Below is the link to the electronic supplementary material.


Desulfovibrio vulgaris has been a primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe0 have been proposed. We investigated Fe0 oxidation with a mutant of D. vulgaris in which hydrogenase genes were deleted. The hydrogenase mutant grew as well as the parental strain with lactate as the electron donor, but unlike the parental strain, it was not able to grow on H2. The parental strain reduced sulfate with Fe0 as the sole electron donor, but the hydrogenase mutant did not. H2 accumulated over time in Fe0 cultures of the hydrogenase mutant and sterile controls but not in parental strain cultures. Sulfide stimulated H2 production in uninoculated controls apparently by both reacting with Fe0 to generate H2 and facilitating electron transfer from Fe0 to H+. Parental strain supernatants did not accelerate H2 production from Fe0, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe0 and either riboflavin or anthraquinone-2,6-disulfonate, and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe0 via H2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H2-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. Microbial corrosion of iron-containing metals is a substantial economic problem, but the mechanisms are poorly understood –3. Funda–This reaction must be coupled with an electron accepting reaction to proceed. Thus, microorganisms can stimulate iron corrosion by either accelerating reaction 1 or by contributing to one or more reactions that consume the electrons from reaction 1.Desulfovibrio spp. have been the most studied pure culture isolates for investigating iron corrosion under sulfate-reducing conditions, dating back to the some of the earliest studies on microbial corrosion Fe0 action 2 .Desulfovibrio species to participate in iron corrosion is direct microbial electron uptake from Fe0 (11–D. ferrophilus (previously known as strain IS5) can grow with H2 as the sole electron donor, but it was inferred to directly consume electrons from Fe0 based on the observation that it reduced sulfate faster than several other H2-oxidizing sulfate reducers  eliminating the possibility that H2 was serving as an electron shuttle between Fe0 and cells and (ii) demonstrating with gene deletions that outer-surface c-type cytochromes were required for electron uptake from Fe0. In contrast, no studies have previously been reported on D. vulgaris corrosion with strains that were unable to use H2   , an abil Fe(III) .0 reduces:D. vulgaris oxidizing the reduced riboflavin with the reduction of sulfate:Higher rates of corrosion following the addition of riboflavin , 28, 29 0 could donate electrons to riboflavin or whether reduced riboflavin can serve as an electron donor for sulfate reduction. The alternative possibility that riboflavin might stimulate other aspects of microbial metabolism was also not evaluated. Furthermore, electron transfer via H2 was still possible in those studies.However, those studies did not determine whether Fe2 serving as an intermediary electron carrier is to determine whether strains unable to use H2 as an electron donor can respire with Fe0 as the sole electron donor . In contrast, the parental strain maintained low H2 concentrations , which would require 13.6 mM H2 . Only about half that much H2 accumulated in the hydrogenase mutant cultures  was more thermodynamically favorable, possibly accelerating H2 generation over that in the hydrogenase mutant cultures in which H2 accumulated. We could not devise an experimental approach to abiotically mimic the expected rapid removal of H2 at the Fe0 surface. However, the alternative possibility that sulfide produced during the growth of the parental strain on Fe0 accelerated H2 production could be evaluated.However, the quantity of Hcultures . One postrations , H2 prod0 as the electron donor is also likely to have promoted H2 production  in which there is a 1:1 stoichiometry for sulfide reacted and H2 produced. However, within 300 h the addition of 1.25 mM sulfide produced 2.7 mmol/L H2 (0 to H+ (reaction 1), leading to additional H2 formation. The addition of 10-fold more sulfide only increased H2 an additional ~2-fold   , 35. Ribon donor . The midendments , 28, 29 D. vulgaris growing on Fe0 was very low compared to the turbidity in H2-grown cultures when a comparable amount of sulfate had been reduced , delay attachment to glass surfaces  and resupplying a reactant (H+) near the Fe0 surface. Sulfide produced at the Fe0 surface can further accelerate H2 production.The turbidity of  reduced . A portisurfaces , but confaces  in a modification of the previously described NBAF medium 2 · 12H2O, 0.005 g of H3BO3, 0.09 g of Na2MoO4, 0.11 g of NiSO4 · 6H2O, and 0.02 g of Na2WO4 · 2H2O. The medium pH was 6.7. Cells were routinely grown with sodium l-lactate as the electron donor (20 mM) and sodium sulfate (20 mM) as the electron acceptor. Growth was monitored by inserting culture tubes directly into a spectrophotometer and determining the A600 value. Growth with H2 as the sole electron donor was evaluated with 5 mM sodium acetate as a carbon source and H2 (140 kPa) as the sole electron donor. Cultures were incubated horizontally with shaking at 25 rpm and were routinely repressurized with H2 to compensate for any H2 consumption.Cultures were routinely grown anaerobically at 37°C in 10 mL of medium in 28-mL anaerobic pressure tubes  under NF medium , designasolution , and 10.0 as the potential electron donor, cells were grown in NB medium with Fe0 granules  as the sole electron donor, 5 mM sulfate as the electron acceptor, and 5 mM sodium acetate as a carbon source. A 10% inoculum of a mid-log-phase culture of lactate-grown cells served as the inoculum. When specified, 50 μM riboflavin or 50 μM AQDS was added from concentrated anaerobic stock solutions. For studies with 316L stainless steel as the potential electron donor for sulfate reduction, five stainless steel cubes (5 mm × 3 mm × 3 mm) replaced the pure Fe0. The stainless-steel cubes were polished with sand paper, and the pure Fe0 and stainless steel were presterilized with ethanol as previously described  through a 0.2 μM PES filter in a Coy anaerobic glove bag  into pressure tubes with 2 g of Fe0. Tubes were resealed and flushed with N2/CO2 (80:20) for 5 min. Controls were sterile NB medium.A final concentration of either 1.25 or 12.5 mM sodium sulfide was added to sterile Fe2 concentrations in the headspace were monitored on an Agilent 6890 gas chromatograph fitted with a thermal conductivity detector. The column was a Supelco Carboxen 1010 plot capillary column (30 m × 0.53 mm) with N2 carrier gas and 0.5-mL injections. The oven temperature was 40°C, the inlet was splitless at 5.5 lb/in2 and 225°C, and the detector had a makeup flow of 7 mL/min and a temperature of 225°C.For sulfate determinations, culture aliquots (0.1 mL) were anaerobically withdrawn with a syringe and needle, filtered , and analyzed with a Dionex ICS-1000 with an AS22 column and AG22 guard with an eluent of 4.1 mM sodium carbonate and 1 mM sodium bicarbonate at 1.2 mL/min. H0 was gently removed from the pressure tube, soaked in isotonic wash buffer for 10 min, drained, stained for 10 min ; 1 mL staining with 3 μL of each stain per mL), and destained for 10 min in isotonic wash buffer. Fe0 pieces were then mounted on petri plates with an antifade/glycerol mixture. Cells were visualized with a 100× objective on a Nikon A1R-SIMe confocal microscope with NIS-Elements software.For confocal microscopy, Fe


The physical properties of the prepared products were examined using SEM, XRD, and UV–visible. The XRD data indicated the formation of pure CuO, Fe2O3 and MgO NPs, as well as nanocomposite formation with Fe2O3 (cubic), MgO (cubic), and CuO (monoclinic). The crystallite size of all the prepared samples was calculated via Scherrer's formula. The energy bandgap of CuO, Fe2O3 and MgO and Fe2O3–MgO–CuO NCs were computed from UV–visible spectroscopy as following 2.13, 2.29, 5.43 and 2.96 eV, respectively. The results showed that Fe2O3–MgO–CuO NCs is an alternative material for a wide range of applications as optoelectronics devices due to their outstanding properties.In this paper, tri-phase Fe The NCs are composed of two or more nano-oxides and possessing properties which depend on the concentration of each constituent oxide in the mixture8. They are useful in a variety of applications, including solar cells, photovoltaic instruments, battery materials, gas sensors, and fuel cells15. Copper oxide (CuO) is a p-type semiconductor with a narrow bandgap of 1.2 eV8. It has unique optical and structural properties with low-cost preparation. It has attracted considerable attention due to its potential applications in superconductivity, gas sensing, solar cell and supercapacitor17. Furthermore, it is a non-toxic and readily available semiconductor19. Magnesium oxide (MgO), with a direct bandgap of 5.2–7 eV, is an n-type semiconductor that displays noticeable structural, catalytic, optical, and chemical properties22. Iron(III) oxide (Fe2O3) is a narrow bandgap of nearly 2 eV. It is associated with certain features, like the low toxicity, low cost, magnetic behavior and high solubility24. Hence, it is engaged in various applications involving biomedicine, cosmetics, diagnostics, sensors, radiology, and vaccines26.Due to their unique optical, electrical, thermal, photocatalytic, mechanical, adsorbent and structural properties, metal oxide (MO) nanocomposites (NCs) have attracted much attention in recent years27. Mixed metal oxide NCs can be fabricated via different approaches such as the co-precipitation28, sonochemical7, solution combustion29, microwave technique10, ultrasonic-assisted30 and green methods11.By combining the different metal oxides (MOs) to form new NCs, various properties of individual oxide could significantly enhanced and, consequently, open up a new avenue of research for optoelectronics, electrical, thermal, photo-catalysis, and biological applications2O3–MgO–CuO NCs and pure CuO, Fe2O3 and MgO NPs were prepared using sol–gel method. It has the advantages of being environmentally friendly, simple, cheap and fast to perform without any special equipment. Herein, the novelty lies in the designed combination of the three metal oxides in one NC, which supposedly could lead to enhanced properties and potential applications. The obtained oxides were characterized for their structural and optical properties using XRD, UV–visible, and SEM.In this work, tri-phase Fe3)2·6H2O; 97%), Iron nitrate nonahydrate (Fe(NO3)3·9H2O; 97%), copper nitrate trihydrate (Cu(NO3)2·3 and absolute ethanol were purchased from BDH and used as received without additional treatment.Magnesium nitrate hexahydrate 2·3(H2O) (3.382 g in 20 mL ethanol), Fe(NO3)3·9(H2O) (5.65 g in 20 mL ethanol) and Mg(NO3)2·6(H2O) (3.589 g in 20 mL ethanol) with constant molar ratio (1:1:1) were synthesized as three separate solutions. Each solution was stirred for 10 min at 23 ± 2 °C to obtain a homogeneous solution. The solutions were mixed under constant stirring for 70 min at 80 °C until gel was obtained. After that, the gel burns to create xerogel, which grinded to fine powder and annealed at 800 °C for 90 min. The individual pure oxides  were separately prepared following similar steps as composite, using the corresponding salt.The sol–gel methodThe optical properties of the synthesized materials were investigated using UV–Vis spectrophotometer (Hitachi U3900 with a software of Varian Cary 50). The structural properties were investigated by X-ray diffraction (XRD) using a Shimadzu EDX-720 (China) with CuKα radiation (λ = 0.154 nm). Morphological properties were assessed using SEM machine from JEOL .32. However, such high temperature could stimulate production of pure substances with better performance.The structural integrity of the synthesized metal oxides is confirmed via powder X-ray crystallography. The targeted substances were obtained via sol–gel route followed by calcination at 800 °C. The annealing temperature of 800 °C suggests high crystalline products as reported elsewhere2O3–MgO–CuO NCs. The observed diffraction peaks of pure oxides are close to the diffraction patterns reported in the X-ray database of JCPDS CuO (45-0937), Fe2O3 (33-0664) and MgO (45-0946). Similarly, in Fe2O3–MgO–CuO NCs, the diffraction patterns of CuO (48-1548), Fe2O3 (39-1346), and MgO (45-0946) match well with their respective standard reference cards. The diffracted peaks in composite were assigned for MgO (cubic), Fe2O3 (cubic) and CuO (monoclinic) phases. The characteristic diffraction peaks of CuO, Fe2O3 and MgO are well specified with no peaks relating to secondary or impurity segments or hydroxide in the sample, confirming the successful growth of Fe2O3–MgO–CuO NCs. The crystalline nature of the sample is assessed based on the sharp and strong diffraction peaks in Fig. 2O3 hexagonal phase and Fe2O3–MgO–CuO nanocomposite were calculated36 and listed in Table The crystalline arrangements and phase of the prepared nanopowder are estimated by XRD. Figure f CuO 48-548, Fe2Of CuO 48-548, Fe2O37 was utilized to compute the crystallite size (D) of CuO, Fe2O3, MgO and Fe2O3–MgO–CuO NCs. Then, their dislocation density was also calculated39, Table 2O3. The Fe2O3 particles act as nucleation sites for the CuO and MgO particles, resulting in aggregation into larger clusters. This phenomenon is known as the Ostwald ripening effect, where smaller particles dissolve and re-deposit on larger particles, resulting in an increase in their size. Hence, the presence of Fe2O3 in the NCs leads to an increase in the particle size of CuO and MgO.The Scherrer equation2O3, MgO and Fe2O3–MgO–CuO NCs. It is seen that the formed nanostructures have spherical shapes with hardly distinct morphology. Furthermore, due to the low resolution of the presented SEM images, the non-size and thus, particle sizes and distribution are difficult to be counted. Nevertheless, the XRD data supported the claimed nanostructures. To improve the seen, and thus the suggested nanostructures, a higher magnification of the SEM image was presented as an insert within the corresponding image. The resulting magnified view is simply support that the particles are in nanometer range. In addition, some nanoparticles are well separated and thus could be counted. For example, by counting of the obviously countable particles of the composite image  value can be computed via the following equation 34.The transmission spectra of all the synthesized materials showed almost an opposite behavior to that seen in Fig. 2O3, MgO and Fe2O3–MgO–CuO NCs is presented in Fig. 43.The change in α (λ) for CuO, Fe2O3, MgO and Fe2O3–MgO–CuO NCs is presented in Fig. The change in 2O3, MgO and Fe2O3–MgO–CuO NCs for the direct electronic transition between the valence band (VB) and conduction band (CB) can be computed via Tauc's relation45 as shown in Fig. 2O3, MgO NPs were calculated to about 2.13, 2.29, and 5.43 eV, respectively. However, Fe2O3–MgO–CuO NCs displayed the 2O3, MgO NPs, Fe2O3–MgO–CuO displayed significant increased absorbance in the visible region due to incorporation of three metal oxide. The reason for the change in the bandgap energy of the NCs compared to the individual metal oxides is likely due to the formation of new energy states at the interfaces between the different metal oxides. This can result in a shift in the electronic structure and a change in the bandgap energy. Additionally, the presence of multiple metal oxides in the NCs can also lead to increased electron–hole separation and improved charge transport properties, which can further affect the bandgap energy. This result was in good agreement with the literature46, which showed the possibility of using the prepared materials in some optical application.The energy bandgap (Eg) values of CuO, Fe2O3–MgO–CuO NCs and pure CuO, Fe2O3 and MgO NPs were successfully fabricated using a sol–gel approach. The XRD emphasized the formation of pure CuO, Fe2O3 and MgO NPs and CuO–Fe2O3–MgO NCs. The variation in the average crystallite size (D) and lattice constant were observed due to the interaction of the corresponding metal oxides. The optical bandgap was reached 2.13, 5.43, 2.29 and 2.96 eV for CuO, MgO, Fe2O3 and Fe2O3–MgO–CuO NCs, respectively.In Summary, tri-phase Fe