T-cell depleted (TCD) haploidentical transplantation using CD3+/CD19+ and TCRαβ+/CD19+ depletion techniques has been increasingly used in children with hematological malignancies. We present a retrospective study aimed to compare transplant outcomes in children with leukemia receiving a TCD haploidentical transplant using either CD3+/CD19+ or TCRαβ+/CD19+ platforms.6/kg and 0.06 x106/Kg) than in the CD3+/CD19 group (24.6x106/Kg and 0.25 x106/Kg) (p=0.04 and p=0.0001), respectively. Conditioning was based on TBF. Median follow-up for survivors was 11 years  in CD3+/CD19+ group and 5 years  in the TCRαβ+/CD19+ group.A total of 159 children with leukemia (ALL=80) (AML=79) that received a TCD haploidentical transplantation using either CD3+/CD19+ (n=79) or TCRαβ+/CD19+ (n=80) platforms between 2005 and 2020 were included. Median age was 9 years in both groups. There were no differences in patient, donor, and transplant characteristics between groups except for donor KIR B genotype more frequent in the TCRαβ+/CD19+ group (91%) than in the CD3+/CD19+ group (76%) (p=0.009) and a high number of NK+ cells and lower CD19+ cells infused in the TCRαβ+/CD19+ group (35.32x10Engraftment kinetics were similar in both groups (13 days for neutrophils and 10 days for platelets). There was no difference in the incidence of acute GvHD II-IV (29 ± 5% in the CD3+/CD19+ group vs 38 ± 5% in the TCRαβ+/CD19+ group) and chronic GvHD . NRM was 23 ± 5% in the CD3+/CD19+group vs 21 ± 4% in the TCRαβ+/CD19+group. Relapse incidence was also similar, 32 ± 5% vs 34 ± 6%, respectively. DFS and OS were not different (45 ± 5% vs 45 ± 6% and 53 ± 6% vs 58 ± 6% respectively). As there were no differences on transplant outcomes between groups, we further analyzed all patients together for risk factors associated with transplant outcomes. On multivariate analysis, we identified that early disease status at transplant  (p=0.0001), presence of cGvHD  (p= 0.002), and donor KIR-B genotype  (p=0.04) were associated with better DFS.Our data suggest that there are no advantages in transplant outcomes between TCD platforms. Risk factors for survival are dependent on disease characteristic, donor KIR genotype, and chronic GvHD rather than the TCD platform used. However, such HLA identical donors are not always available for all patients, or even when having one, they do not have time to be waiting for donor availability –5. More elevance . Conside-3 and 2nd complete remission (CR) or beyond. For acute myeloid leukemia (AML) patients, transplant criteria included intermediate or poor risk characteristics at diagnosis and also 2nd CR or beyond. Details of transplantation criteria have been published elsewhere / x100), since it did not reach 0.01. At least 20 LAIP events per tube were required for the test to be considered informative.All consecutive pediatric patients who received a TCD HaploSCT with CD3+/CD19+ depletion (n=79) or TCRαβ+/CD19+ depletion (n=80) at our institution from January 2005 to January 2020 with hematological malignancies were included in this retrospective study. They were transplanted because of high-risk hematological malignancies and they lacked a suitable HLA-matched (related or unrelated) donor. Indications for allogeneic hematopoietic transplantation for children with acute lymphoblastic leukemia (ALL) included the following: poor cytogenetics, induction failure (defined as no remission at 1 month following induction treatment), persistent MRD with a cutoff point of 10lsewhere –15. MRD lsewhere  using a Patients, donors, parents, and/or their legal guardians gave written informed consent for donation and transplantation in accordance with the Helsinki Declaration. Patients, donors, and transplant characteristics are given in Donor mobilization was performed as previously described –15. Brievia a central venous catheter on day 0.T-cell depletion was performed using CliniMACS Plus device or the fully automated Prodigy device after manipulations in a laminar-flow cabinet located in a clean room certified for sterile manipulations. All immuno-magnetic procedures were performed according to the manufacturer’s standard protocol. Clinical grade reagents, disposable kits, and instrumentation were from Miltenyi Biotec . Before and after depletion procedures, the apheresis, and final product cell composition were analyzed by flow cytometry . Finally, the cell product was freshly infused www.ebi.ac.uk/ipd/kir/donor_b_content.html). Criteria for donor selection have been previously published  and the HLA-B allotypes (Bw4) were determined using high-resolution PCR-sequence-based typing. Fifteen human KIR genes and two pseudogenes were analyzed by PCR with a KIR genotyping kit . The KIR A haplotype was defined by the absence of 2DS1, 2DS2, 2DS3, and 3DS1 and the presence of 2DS4 as the only KIR-activating receptor. The KIR B haplotype was determined by the presence of any activating genes except 2DS4 . We deteublished . Donor c2/day for 5 days (day -6 to day -2), intravenous busulfan administered once daily according to patient body weight for 3 days (day -5 to day -3), intravenous thiotepa at 5 mg/kg/day for 2 days (day -3 to day -2), and intravenous methylprednisolone at 4 mg/kg/day (day -6 to day -2). Busulfan weight-based dosing was as follows: <9 kg: 1 mg/kg/dose; 9–16 kg: 1.2 mg/kg/dose; 16–23 kg:1.1 mg/kg/dose; 23–34 kg: 0.95 mg/kg/dose, and >34 kg: 0.8 mg/kg/dose , relapse incidence (RI), and non-relapse mortality (NRM). Secondary endpoints were engraftment kinetics and early and late immune reconstitution. Graft-versus-Host disease (GvHD) and infections incidence were also analyzed. GvHD was diagnosed and graded according to previously described definitions –24. Phen3/µL) and (100 x103/µL) were 10 days (range: 6-40) and 15 days  for CD3+/CD19+ group and 9 days (range: 6-27) and 13 days (range: 9-153) for the TCRαβ+/CD19+ group (p=0.07).There were no statistical differences in neutrophil and platelets engraftment between groups. The median times to neutrophil recovery were 13 days (range: 8–28) and 13 days (range: 10–26) for the CD3+/CD19+ and the TCRαβ+/CD19+ groups, respectively. Median time to platelet recovery (20 x10Primary graft failure occurred in 10 out of 79 patients (12%) in the CD3+/CD19+ group and 8 out of 80 (10%) in the TCRαβ+/CD19+ group. This difference was not statistically significant. Median age of patients with graft failure was 5 years (1-9), 14 were male. There were 7 patients (39%) diagnosed with ALL and 11 (61%) with AML. Half of them were in the early phase of the disease. Nine patients (9/18) received a second transplant and five are alive. Six patients had an autologous reconstitution and are in complete remission and three patients relapsed.The median length of hospitalization was 15 days (range: 10-127) in the CD3+/CD19+ group and 15 days (range 10–203) in the TCRαβ+/CD19+ group. Patients in the CD3+/CD19+ group received red blood transfusions for a median time of 2 days (range: 0–21) and similarly in the TCRαβ+/CD19+ group: 2 days (range: 0–24)). The platelets transfusion was also similar between groups: 3 days (range: 0–40) for the CD3+/CD19+ group and 2 days (range: 0–54)) for the TCRαβ+/CD19+ group. The median of antibiotic treatment days was 13 (range: 0–122) in the CD3+/CD19+ group and 17 days (range: 3–80) in TCRαβ+/CD19+ group (p=0.003). In the CD3+/CD19+ group, the median time needing parenteral nutrition support was 7 days  compared with 0 days (range: 0-26) in the TCRαβ+/CD19+ group (p=0.09)).Nausea and vomiting, diarrhea, and hemorrhagic cystitis were the most common toxicities in both groups. Twelve out of 80 patients developed VOD in the TCRαβ+/CD19+ group, whereas only 2 out of 79 did in a CD3+/CD19+ group (p=0.006). Data regarding toxicity profile are provided in In all, 149 infectious episodes were diagnosed using microbiological and/or clinical criteria in the CD3+/CD19+ group and 170 in the TCRαβ+/CD19+ group. All patients suffered some kind of infection during transplant admission. Taking together all infection episodes, there were no significant differences in the incidence of infection complications between groups. Viral infections were the most frequent ones in both groups (52% in the CD3+/CD19+ group vs 63% in the TCRαβ+/CD19+ group). However, there was a trend to higher incidence of viral infections in the TCRαβ+/CD19+ group with a higher incidence of HHV-6 in the TCRαβ+/CD19+ group, whereas VZV infection was more frequent in CD3+/CD19+ group.HHV-6 reactivation occurred in 23 patients and eleven developed encephalitis diagnosed by neurological symptoms, positive PCR for HHV-6 in cerebrospinal fluid, and the absence of other identified etiologies of CNS dysfunction. Main neurological symptoms were alteration of consciousness, amnesia, seizures, hypothermia, hyponatremia, and pruritus. Treatment was mainly based in foscarnet. One patient died by HHV6 disseminated disease.Clostridium species in the TCRαβ+/CD19+ group  compared with 12% in the CD3+/CD19+ group. Detailed data regarding bacterial, viral, and fungal infections are provided in Bacterial infections were the second group of infections comprising 39% of all in the CD3+/CD19+ group and 29% in the TCRαβ+/CD19+ group. However, there was a higher incidence of Immune reconstitution in the early phase after transplant was characterized in both groups by a rapid increase in NK cells on day +15  and specially on day + 30 . These differences were not statistically significant.T cells and their subsets progressively increase since day +60 in both groups become the predominant lymphocyte population on day +90 and afterward. There was a trend for a better B-cell reconstitution in the TCRαβ+/CD19+ group from day +270. Detailed data on absolute lymphocyte count (ALC), NK cells, T lymphocytes, T lymphocyte subsets, and B lymphocytes following transplant are provided in Thirty-one patients developed aGvHD in the CD3+/CD19+ group  and 40  in TCRαβ+/CD19+ group. The median time to aGvHD was 30 days  in the CD3+/CD19+ group and 29 days  in the TCRαβ+/CD19+ group. The cumulative incidence of aGvHD was 29±5% in the CD3+/CD19+ group and 38±5% in the TCRαβ+/CD19+ group. These differences were not statistically significant. Acute GvHD treatment in the first line was based on steroids and in the second line ruxolitinib and immunomodulatory therapy with photopheresis and mesenchymal cells. GvHD was the main cause of death in 7 patients.Twenty-three patients developed cGvHD  in the CD3+/CD19+ group and 17  in the TCRαβ+/CD19+ group. The median time to cGvHD was 120 days (range: 41–306 days), in the CD3+/CD19+ group and 119 days (range: 45–454 days) in the TCRαβ+/CD19+ group. The cumulative incidence of cGvHD was 32±5% in the CD3+/CD19+ group and 23±4% in TCRαβ+/CD19+ group. These differences were not statistically significant.The cumulative incidence of NRM was 23±5% in the CD3+/CD19+ group. Seventeen patients died with a median time to death of 103 days (range: 30-1391 days). In the TCRαβ+/CD19+ group, the cumulative incidence of NRM was 21±4%. Fifteen patients died with a median time to death of 86 days (range: 27-543 days). The leading cause of death was relapse of disease in both  followed by infection . These differences were not statistically significant. The primary causes of death are given in Twenty-three patients relapsed in the CD3+/CD19+ group and twenty-four in the TCRαβ+/CD19+ group. Median time to relapse was 164 days (range: 23-1286 days) in CD3+/CD19+ group and 206 days (range: 37-1876) in the TCRαβ+/CD19+ group. The cumulative incidence of relapse was 32±5% in CD3+/CD19+ group and 34±6% in the TCRαβ+/CD19+ group. This difference was not statistically significant. Nine patients underwent a second hematopoietic transplant after relapse treatment and four are alive and in complete remission.Probability of DFS was 45±5% in the CD3+/CD19+ group and 45±6% in the TCRαβ+/CD19+ group. Probability of OS was 53±6% and 58±6% for CD3+/CD19+ patients and TCRαβ+/CD19+ patients, respectively. These differences were not statistically significant.st transplant and 47 ±7% beyond (p=ns).We analyzed the patients transplanted after a previous allograft and whose OS was also similar: 60± % after the 1st CR, 28±6% in 2nd CR, and 27±6% in >2nd CR; p=0.005); amount of NK cells on day +30 , and donor kinship . However, in multivariate analysis (MVA), disease status at transplant and the amount of NK cells on day+30 were the variables associated with NRM ; MRD status at transplant : donor KIR genotype (27±4% in KIR B genotype and 52±10% in KIR A p=0.01), and cGvHD . However, relapse was associated with disease status and cGvHD in MVA  showed that NRM was associated with the following variables: disease status at transplant ; MRD status at transplant , donor KIR genotype (57±4% in KIR B genotype and 16±7% in KIR A p=0.001), and donor kinship . DFS was also influenced by the following post-transplant variables: NK cells on day+30  and chronic GvHD . In MVA, variables that impact on DFS were disease phase at time of transplantation, the presence of chronic GvHD, and donor KIR genotype  in the graft , 28–30. To our knowledge, this is the first study comparing both TCD procedures in pediatric haploidentical transplant setting. Our results clearly suggest that there are no relevant differences in terms of transplant outcomes between the two groups of patients. Patients’ characteristics are similar between groups for the most potential variables affecting transplant outcomes. It is noteworthy that in both groups there is a high proportion of patients transplanted in the advance phase of disease (more than 20%) and with MRD positive (more than 30%) at the time of transplant. Disease status at transplant is associated not only with relapse incidence but also with NRM and obviously importantly impacts on DFS. Considering that disease status and MRD status at transplant are the most important well-known variables influencing the transplant outcome, this might explain the absence of differences between groups.Engraftment kinetics are also similar in both groups, although a trend to faster platelet engraftment is observed in TCRαβ+/CD19+ likely mirroring the higher number of CD34+ cells infused in that group of patients. Graft failure incidence is also similar in both groups with a very low impact on the cause of death in both groups. Beyond HLA disparity, there are other potential risk factors for graft failure in the TCD haploidentical transplant. Recently, patient age and the pre-transplant number of CD3+CD8+ cells have been reported as risk factors in the TCD haploidentical transplant setting . The useThe toxicity profile is very similar in both owing to the fact that there were no differences in the conditioning regimen used. However, the higher incidence of SOS/VOD in TCRαβ+/CD19+ group is remarkable. There is no clear explanation for this finding as there are no differences in race/ethnicity or intensity of conditioning between groups, but it may be because the new broader diagnostic criteria of VOD in children proposed by EBMT caused a real increase in VOD incidence. It is important to the use of Busulfan TDM to decrease the incidence of VOD after transplant. Despite this, SOS/VOD has no important impact as the cause of death in both groups maybe due to defibrotide treatment .Early phase of immune reconstitution is dominated by a rapid rise of NK cell population in both groups with no differences , 15, 25.NK cells reconstitution in the early phase after transplant has been considered to have an impact on transplant outcomes , 36, 37.Relapse after transplant still represents the main cause of transplant failure. Several mechanisms of immune evasion have been described such as genomic loss of HLA, a relapse modality first described in haploidentical transplant, the transcriptional downregulation of HLA class II molecules, and the enforcement of inhibitory checkpoints between T cells and leukemia . As expeNK cells can also exert a notable GvL effect with no GvHD. However, it is not clear whether this GvL effect is mediated by NK cell alloreactivity based on donor/recipient KIR mismatching or by the presence of KIR activating receptors in donors with KIR-B genotype , 41. LikFinally, the MVA of transplant outcomes shows that patients transplanted in advanced phase of disease and those transplanted from a donor with KIR A genotype, had worse DFS due to a very high risk of relapse and mortality. Conversely, those patients that developed chronic GvHD did better in terms of DFS due to lower relapse incidence, no matter the transplant group. We acknowledge several limitations to the present study. The study limitations are mainly those derived from its retrospective nature. It covers a broad time range during which there have been several major changes in this field. In addition, being TCRαβ+/CD19+ depletion a more recent procedure, the follow-up in these patients was shorter than those transplanted with CD3+/CD19+ depletion procedure. Notwithstanding limitations inherent in all retrospective studies, we believe that the present study provides a valuable information to the hematopoietic transplant community regarding risk factors for transplant outcomes among pediatric patients receiving TCD haploidentical transplant.Maybe in the next future, combination of myeloablative conditioning with T regulatory and T conventional cells as immunotherapy might reduce relapse incidence with no increase of risk of severe GvHD , 45.In conclusion, the study suggests that there are no advantages in transplant outcomes between both TCD platforms. Risk factors for survival are clearly dependent on disease characteristic, donor KIR genotype, and chronic GvHD rather than the TCD platform used.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics committee of Hospital Niño Jesus. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.MG-V is responsible for caring patients and statistical analysis and wrote the manuscript. BM cared for the patients. IL cared for the patients and collected data. JZ, ES, JI, and JS performed the graft manipulation. JR collected the data. JV performed the HLA study. AC, LA, and MR performed the cytometry analysis. MD wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In addition, solid-state dispersion method was used for synthesizing of ZrO2/TiO2/Fe3O4 ternary nanocomposite. The ZrO2/TiO2/Fe3O4 nanocomposite was characterized by different techniques including XRD, EDX, SEM, BET, FTIR, XPS, EELS, and Photoluminescence (PL). The FTIR analysis of ZrO2/TiO2/Fe3O4 photocatalyst showed strong peaks in the range of 450 to 700 cm−1, which represent stretching vibrations of Zr–O, Ti–O, and Fe–O. The results of FTIR and XRD, XPS analyses and PL spectra confirmed that the solid-state dispersion method produced ZrO2/TiO2/Fe3O4 nanocomposites. The EELS analysis confirmed the pure samples of Fe3O4, TiO2 and ZrO2. The EDAX analysis showed that the Zr:Ti:Fe atomic ratio was 0.42:2.08:1.00. The specific surface area, pores volume and average pores size of the photocatalyst were obtained 280 m2/g, 0.92 cm3/g, and 42 nm respectively. Furthermore, the performance of ZrO2/TiO2/Fe3O4 nanocomposite was evaluated for naproxen removal using the response surface method (RSM). The four parameters such as NPX concentration, time, pH and catalyst concentration was investigated. The point of zero charge of the photocatalyst was 6. The maximum and minimum degradation of naproxen using photocatalyst were 100%  and 66.10% respectively. The stability experiment revealed that the ternary nanocatalyst demonstrates a relatively higher photocatalytic activity after 7 recycles.In this study, ZrO For the treatment of pharmaceutical wastewater and for removing pollutants before those are released into the environment, identification of the most efficient method is a challenge3. Pharmaceutical wastewater can be treated using physical4, chemical, biological5, as well as combined methods6. The Advanced oxidation processes7 including advanced oxidation based on the sulfate radicals8, ultraviolet–visible9, natural sunlight10, Fenton oxidation11, electrochemical12, nanocomposite catalysts13, and sonolysis and sono-Fenton14 have been widely used to remove pollutants from pharmaceutical wastewater with great performance. The formation of radicals during these processes leads to the oxidation of organic pollutants in aqueous solutions. In comparison to other methods, photocatalysis offers several advantages, such as high efficiency, low cost, design of suitable catalysts for specific wastewaters, and high corrosion and temperature stability17. Titanium dioxide (TiO2) has been widely used as a catalyst in the degradation of organic compounds and pharmaceutical pollutants since it is a light-sensitive semiconductor (including UV and visible light)19. The formation of valence band holes and conduction band electrons during photocatalysis produces oxidation–reduction media in wastewater. It can easily degrade organic compounds and convert them into non-toxic compounds such as CO2 and water21. Titanium dioxide is a polymorphic material with three crystalline phases: anatase, rutile, and brookite. The anatase phase is more photocatalytically active than the rutile phase23. In order to enhance the TiO2 photocatalyst activity, it is important to use smaller particles (nano size), as smaller particles have higher specific surface areas24. The removal of titanium dioxide nanoparticles after treatment reduces the benefit of this photocatalyst, and immobilization of titanium dioxide (TiO2) onto supporting materials can be performed, but immobilization reduces the specific surface area in comparison with a homogenous catalysts25. On the other hand, fast recombination of generated electron–hole pairs can decrease the activity of titanium dioxide photocatalyst26. Therefore, some other semiconductors such as ZrO2 are used to improve of the activity of TiO2. ZrO2 doping can slow down the electron–hole pair recombination, strengthen the material and increase surface area and anatase to rutile crystal phases ratio28. ZrO2-TiO2 photocatalyst has been used to degrade organic compounds, which in this compound, ZrO2 acts as support or photocatalyst in the system29.Environmental pollution by pharmaceutical compounds is considered as one of the most serious issues in recent years2 to TiO2 can increase the surface area because ZrO2 inhibits anatase to rutile phase transitions, densification, and crystallite growth by providing dissimilar boundaries30. It was observed that changes in surface chemistry, particularly acidity improve the photocatalyst activity31. In this binary catalyst, holes trap such as the hydroxyl groups, prevent electron–hole recombination and oxidation–reduction reactions, and increase quantum yield31. In addition to ZrO2 nanoparticles, it is necessary to be added another compound to these binary metal oxides to improve the surface area of the TiO2 photocatalyst as well as recovery of TiO2 from treated wastewater33. Magnetic nanoparticles, such as Fe3O4, are suitable for this purpose. A Fe3O4@TiO2 photocatalyst has been synthesized to degrade Bisphenol A under visible and long-wavelength UV light irradiation. Moderate iron loading reduced hole-pair separation effectively, and shifted the bandgap in the visible range. Further, the magnetic properties of Fe3O4 play a critical role in recovering the used catalyst from the solution, which facilitates practical applications of the Fe3O4@TiO2 photocatalyst34. Fe3O4@SiO2@g-C3N4/TiO2 nanocomposite was used to remove dye pollutants. It has been found that the 10 wt.% g-C3N4/TiO2 composite catalyst can degrade over 91% of anionic and cationic dyes35. Therefore, it is possible to combine the three compounds to degrade toxic and harmful pollutants in wastewater. In recent years, non-steroidal anti-inflammatory drugs (NSAIDs) have been increasingly used for the treatment of COVID-1937. Therefore, it is predicted that pharmaceutical-produced wastewater containing NSAIDs such as naproxen will increase and it will be very important to develop an effective method of degradation these compounds from the wastewater. The degradation of naproxen by H2O2-modified titanate nanomaterial and Bi-modified titanate nanobulks under visible light irradiation has been successfully accomplished39. In previous works, each of these metal oxides was used separately as a catalyst to remove various contaminants43, but the interaction of the three metal oxides of Fe3O4, TiO2 and ZrO2 together and its function to remove pharmaceutical contaminants can produce a synergistic effect. The difference in physical and chemical properties of each component of these nanophotocatalyst makes it unique. A nanocomposite with magnetic, stable, and high photocatalytic properties was finally synthesized45. TiO2 has a band gap of 3.2 eV for anatase, and 3.0 eV for rutile47. The ZrO2 is used as a photocatalyst in heterogeneous reactions due to its semiconductor properties (type n). The band gap energy of ZrO2 varies from 3.25 to 5.1 eV depending on the sample preparation55. Its common band gap energy is 5 eV55. It has a wide bandgap and a highly negative flat-band potential, which can be used for hydrogen production through water decomposition56. Magnetic properties of Fe3O4 nanoparticles make recyclable nanocomposites possible. Furthermore, the excitation range of nanocomposites synthesized from Fe3O4 can be used in the visible light range due to their unique optical properties.The addition of a small amount of ZrO58. Synthesis of Fe3O4/TiO2/ZrO2 nanocomposite can improve the photocatalytic property of catalyst. The synthesis of these nanoparticles individually can have some disadvantages such as a wide energy gap, activity only in UV, lower photocatalytic activity, However, their composite synthesis will offer advantages such as higher magnetic properties, recyclable, higher stability and activity, and enhanced photocatalytic activity67. The Box–Behnken design (BBD) was used in this study due to its rotatable or nearly rotatable second-order design. The percentage of naproxen degradation was selected as the experimental design response69.The Response surface methodology (RSM) is a widely used mathematical and statistical method for modeling and analyzing a process in which a response is affected by multiple variables, and its goal is to optimize the response2/TiO2/Fe3O4 nano-photocatalyst with high photocatalytic activity and reusability for the pharmaceutical wastewater treatment. Its performance has been evaluated in the removal of naproxen from pharmaceutical wastewater under UV light. The synthesised photocatalyst has been characterized with methods such as X-ray diffraction (XRD), Energy Dispersive X-Ray (EDX), Scanning Electron Microscope (SEM), Brunauer–Emmett–Teller (BET), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS), Electron energy-loss spectroscopy (EELS), and Photoluminescence (PL). We investigated various factors including the initial concentration of naproxen, initial pH of the solution, photocatalyst dosage, and time until the optimal degradation efficiency was obtained using the RSM method.The current work is focused on the synthesis of a new ZrO2.8H2O, 98%), and Iron (II) chloride tetrahydrate (≥ 99%) and iron (III) chloride hexahydrate(≥ 99%) were used as a precursors for the synthesis of TiO2, ZrO2, and Fe3O4 respectively. Ethanol was obtained from Sigma-Aldrich and utilized without any further processing. Deionized water (DI-water) was also used in the experiment. Sulphuric acid  and sodium hydroxide  were used for the adjustment of the pH of the solution. (S)-6 methoxy α-methyl 2-naphthaleneaceticb sodium salt was purchased from Sigma Aldrich and its aqueous solution used for degradation experiments. In Table Titanium (IV) iso-propoxide 97%, zirconyl(IV) chloride octahydrate  isopropoxide was added into 60 mL ethanol. A magnetic stirrer was used to mix the solution for 30 min. 10 mL deionized water in a drop-wise fashion was added into the mixture until hydrolysis reaction occurred in the system. The obtained white gel was dried at 100 °C and later calcined at 450 °C for 2 h71.Sol–Gel method was used for the preparation of TiO3.6H2O (0.605 g) and FeCl2.4H2O (0.215 g) were dissolved in deionized water. Then, the obtained solution was placed in an ultrasonic homogenizer for 5 min. After that, 60 mL of 1 M NaOH was added dropwise into solution under ultrasonic at 70 °C. After 60 min, the brownish powder was obtained at a pH of 13.4 and it was separated from the solution using a centrifuge. Finally, it was calcined at 300 °C for 1 h72.Known amounts of FeCl2/TiO2/Fe3O4 photocatalysts were prepared using solid-state dispersion method. ZrO2, TiO2, and Fe3O4 nanoparticles were mixed at a ratio of 4:8:1 in ethanol solution. The resultant ternary oxide was stirred at a constant rate of 300 rpm for 15 min. Then, ethanol was removed from a mixture of nanocomposite by evaporation. Then, the product was dried at 110 °C and calcined at 450 °C for 6 h to obtain ZrO2/TiO2/Fe3O4 nanocomposite photocatalyst60. The mixing ratio of used components in the synthesized nanocomposite was selected based on the highest efficiency of naproxen degradation under the same operation conditions. The best ratio was achieved (4:8:1) of the ZrO2, TiO2, Fe3O4 compounds.ZrOKα radiation. SEM(AIS2100). It was used to determine the surface morphology and microstructure of ZrO2/TiO2/Fe3O4 nanocomposite photocatalyst. The Fourier-transform infrared spectroscopy  in the wavelength range from 400 to 4000 cm−1 was used to analyze the chemical bonding in a photocatalyst. Texture properties of nanoparticles was investigated using TriStar-II-Series, Micromeritics Instrument Corporation, USA. The specific surface area of photocatalysts was determined via the N2 adsorption–desorption and Brunauer–Emmett–Teller (BET) model. Barrett-Joyner–Halenda (BJH) method was used to calculate the size of pores and pore size distribution using adsorption–desorption curves. Powder samples for XPS analysis are prepared in the form of pellets in the laboratory (the powder should be large enough to cover a surface of 1.5 × 1.5 cm), then the samples should be placed in a vacuum chamber after preparation. A PHI Perkin-Elmer Model 5400 instrument was used to record XPS spectra. The EELS analysis was obtained using a sub-nanometer probe size using the GIF2000 Filter. The PL Spectroscopy was recorded by Avaspec-2048-TEC model spectrometer.The mineralogical analysis of the prepared nanoparticles was characterized by X-ray diffraction  with Cu 73. In order to determine the point of zero charge (PZC), 0.25 g of the synthesized photocatalyst was added into the solution with the same ionic strength and different pH values. The synthesized nanocatalyst was added to 50 mL of 0.1 M NaNO3 solution. The pH of solution was adjusted in the range of 3–9 using 0.1 M H2SO4 and 0.1 M NaOH. The pH of solution was measured before and after 24 h mixing and those were called pHi and pHf respectively. The plot of pH changes (ΔpH) as a function of initial pHi was used for the determination of PZC73.An amphoteric oxide method was used to determine the point of zero charge of the photocatalystIn order to optimize photocatalyst activity, the RSM has been used. Design Expert 8.0 software  was used to design experiments and analyze mathematical modeling. Each experiment was performed in duplicate. Box–Behnken design (BBD) was applied in this study because of its rotatable or nearly rotatable second-order design. The “y” was the response (the percentage of naproxen degradation). We evaluated four independent variables, including catalyst loading dosage, initial concentration of naproxen, time and pH value in order to find an optimal condition:2/TiO2/Fe3O4 Photocatalyst was used for the degradation of naproxen in a pharmaceutical synthetic solution. The experiments were conducted based on the conditions in Table 2). At the end of each experiment, centrifugation with 12,000 rpm for 20 min have been performed for each withdrawn sample followed by absorbance measurement using UV‐Vis (Shimadzu UV2401PC) at λ = 230 nm. The efficiency of designed photocatalysts for the degradation of naproxen was calculated as follows:ZrOi and Cf are the naproxen concentration before and after degradation reaction respectively.where C2/TiO2/Fe3O4 Photocatalyst. Briefly, the selected photocatalyst being used in the degradation test of naproxen was separated from solution using a 1.3 Tesla magnet. Used photocatalysts were rinsed in distilled water and irradiated under UV light for 12 h. In order to evaluate the reusability of a photocatalyst, seven successive experimental runs were conducted.Reusability tests were conducted for ZrO2, TiO2, and Fe3O4, nanoparticles, and ZrO2/TiO2/Fe3O4 nanocomposite characterized using XRD, which are depicted in Fig. 2, the primary characteristic diffraction peaks appeared at 2θ = 30°, 35°, 50.8°, 60.1°, and 63° were corresponds to crystal planes of (1 1 1), (0 0 2), (0 2 2), (3 1 1), and (2 2 2). The majority of these crystalline phases are monoclinic. The transition from tetragonal to monoclinic can occur with an increase in the calcination temperature which increases the crystallite size of the samples74. According to Fig. 2 sample, there is good agreement between the results of this study and the XRD pattern for the titanium dioxide phase reported in the literature77. The diffraction peaks at 2 thetha values of 25.32°, 37.90°, 48.09°, 54.10°, 55.15°, 62.85°, 68.99°, 70.49°, and 75.12° were belonged to crystal planes of (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 1 1), (2 0 4), (1 1 6), (2 2 0), (2 1 5)78. All the peaks observed in the diffraction pattern of TiO2 nanoparticles are in good agreement with anatase (JCPDS No. 00–001-0562). Figure 3O4. All of the diffraction peaks are related to (2 2 0), (3 1 1), (2 2 2), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) crystal planes and those were belonged to Fe3O4. The results confirmed the occurrence of inverse spinel structure in Fe3O4. The XRD peaks of impurities were not observed, which means that the synthesized Fe3O4 nanoparticles are pure79. Finally, in the XRD pattern of ZrO2/TiO2/Fe3O4 nanocomposite, the main peaks related to all three compounds were observed. The results showed that the sample has spinal reverse cube of Fe3O4 structure. The ZrO2 is amorphous phase and the phase of TiO2 is tetragonal (Anatase). The average crystallite sizes were calculated by Scherrer formula:80. The average crystallite size of TiO2, ZrO2, Fe3O4 and ZrO2/TiO2/Fe3O4 was found to be 18.75 nm, 22.29 nm, 13.27 and 33.42 nm respectively. The most important cause of difference in particle size of synthesized samples can be the difference in their synthesis method81.The constituting phase, crystalline size, and crystalline structures of the ZrOα, Kα, and Kβ of Fe and 0.52 keV is Kα of oxygen. Peaks at 2.04 (Lα) and 2.26 (Lβ) keV are for zirconium. Fe3O4 particles are larger than TiO2 particles based on the results obtained.EDAX analysis confirmed the presence of all elements, including Zr, Ti, O and Fe in the synthesized photocatalyst and there is no evidence of any other element. The inserted table in Fig. 2/TiO2/Fe3O4 nanocomposite  was calculated using the N2 isotherms. The BET-SSA, the volume of pores, the average size of pores of the photocatalyst obtained were 280 m2/g, 0.92 cm3/g, and 42 nm respectively. The sample is mesoporous as the diameter of pores is larger than 2 nm. Hysteresis loop of types IV-H1 was obtained for the sample which is related to cylindrical and spherical pores in the sample82. In terms of pore size distribution, wide pore size distribution was observed, especially, pores with size larger than 3 nm had significant volume in the sample. It was found that the pore size larger than 3 nm is appropriate for the reactants penetration into the porous media of photocatalyst84.Figure 2, TiO2, Fe3O4, and ZrO2/TiO2/Fe3O4 photocatalysts. As for ZrO2, the strong broad peak at 498–502 cm−1 region is attributed to the vibration mode of −1 are related to the Zr–O stretching vibrations in ZrO2. The IR at 1553 cm−1 attributed to stretching of O–H groups, which indicates the adsorbed moisture85. For TiO2 nanoparticles, TiO2 network bonds and deformation vibrations of stretching mode of Ti–OH peaks were observed at 483 cm−1 and 1623 cm−1 respectively. It can be related to the absorption of water on the TiO2 surface. Asymmetrical and symmetrical stretching vibration of hydroxyl groups (–OH) was observed at 3405 cm−1. The obtained results are consistent with those reported in the literature87. According to Fig. −1. The peaks at 419 cm−1 and 589 cm−1 were attributed the stretching vibration of the Fe–O. The strong peaks in the 450–700 cm−1 range were attributed to stretching vibrations of Zr–O, Ti–O, and Fe–O in ZrO2/TiO2/Fe3O4 photocatalyst. The results confirm the presence of all three nanoparticles in the ZrO2/TiO2/Fe3O4 photocatalyst.Figure 2, TiO2 and Fe3O4 pure nanoparticles and the ZrO2/TiO2/Fe3O4 nanocomposite. The XPS spectra of pure and nanocomposite samples are shown in Fig. 3O4 nanoparticles. Three components of the Fe–O bond can be seen in the XPS spectrum. After photoexcitation, the splitting of O1s at 531.6 eV indicates the formation of a Fe–O bond. The XPS peak of C1s at 292.6 eV and O1s at 531.6 eV indicate the formation of Fe–C and Fe–O bonds following particle photoexcitation 93. In Fig. 94. In addition, two peaks at 182.52 and 184.87 eV indicate the presence of Zr-Ti chemical bonds in the composites95. In Fig. 4+ oxidation state in the nanocomposite. Meanwhile, the peak of 464.58 eV can be attributed to Zr-Ti chemical bonds96. The presence of chemical bonds Fe–O, Zr-O, Ti–O and Zr-Ti, Ti-Fe and Zr-Fe indicates a phase contact between Fe3O4, TiO2 and ZrO2. In Fig. 3O4, the major peaks at 710.96 eV and 724.59 eV are attributed to Fe3+ 2p3/2 and Fe3+ 2p1/2, respectively. The bond energy of Fe2+ 2p3/2 and Fe2+ 2p1/2 has a dual peak at 708.96 eV and 721.52 eV, respectively. Results of Fe3O4 XPS analysis are consistent with Fe2p spectrum97.For further investigation of the nature of the synthesized nanomaterial, we performed high-resolution X-ray photoelectron spectroscopy (XPS) on the synthesized ZrO3O4 and Fe2O3 spinel structures. Figure 3O4 phase, since the expected value for gamma- Fe2O3 (70.2 eV) is significantly different. As shown in Fig. 2 nanoparticles are obtained . The synthesized ZrO2 nanoparticles can be proved according to the obtained peaks from the analysis. As shown in Fig. 4+, indicates a lower oxidation state which has edges shift slightly towards the lower energy loss. Here, the blue curve is the fine structure of the Ti4+ state. EELS was obtained using a sub-nanometer probe size using the GIF2000 Filter.The electron energy-loss spectroscopy (EELS) analysis was used to identify the pure samples more accurately. The combination of EELS and statistical analysis can provide more information on the differentiation of Fea- Fe2O3 0.2 eV isa- Fe2O3 0.2 eV is2, TiO2 and Fe3O4 samples and the ZrO2/TiO2/Fe3O4 nanocomposite at room temperature in the wavelength range of 300–600 nm with an excitation wavelength of 293 nm. As shown in Fig. 2, TiO2 and Fe3O4 samples show significant PL signals at 376, 407, 478, and 480, 556, 509 and 543 nm, respectively. Among all samples, the TiO2 sample shows the highest intensity. The obtained PL spectrum for pure ZrO2 nanoparticles is almost similar to the results previously reported98. According to previous research102, the presence of these peaks in the visible range is probably due to the presence of oxygen vacancies, defects, surface states and other structural impurities. A high PL intensity of pure ZrO2 appears to indicate that the ZrO2 surface states are much lower, hence easy electron transfer can occur between VB and CB of ZrO2 even with a low-energy laser excitation source (325 nm)103. Additionally, when Fe3O4 and TiO2 are loaded with ZrO2 as ternary oxide catalysts, the PL intensity of ZrO2 decreases abruptly , (–Ti–O–Zr–) and (–Fe–O–Zr–). The peaks shown at 403, 427, and 490 nm under the excitation wavelength of 293 nm for the ZrO2/TiO2/Fe3O4 nanocomposite must be due to the interference of trap states between the gaps, such as surface defects and oxygen vacancies  was performed for PL (Photoluminescence Spectroscopy) analysis. The PL Spectroscopy was recorded by Avaspec-2048-TEC model spectrometer. The valence band (VB) and conduction band (CB) are shown in Fig. 3O4/TiO2/ZrO2 are shown in (CB) and (VB). The VB determines the energy levels of electrons in the VB of an atomic structure. The combination of three metal oxides can be improved electromagnetic separation and narrowing of the photocatalyst band gap. In Fe3O4/TiO2/ZrO2 semiconductor, electrons migrate to CB whereas; positive holes are created on VB. The OH is formed through the reaction of charge carriers with adsorbed compounds on the photocatalyst surface. In Fe3O4/TiO2/ZrO2 nanocomposite, the electron–hole separation may occur between ZrO2 and TiO2. The surface energy of Fe3O4 for VB and CB was placed in TiO2 band gap and surface energy of TiO2 was placed in ZrO2 band gap  and their intensity are closely related to its photocatalytic activity. Figure 2 values, the quadratic model was found the most accurate model for the prediction of experimental data in terms of naproxen removal. Its predicted R2 value was found to be 0.9901 which means the developed model equation is able to predict the results accurately. Therefore, the following quadratic polynomial model in terms of coded factors was proposed for investigation of naproxen degradation from pharmaceutical synthetic solution as a function of operating parameters:The experimental work was designed and the obtained actual results, as well as predicted values, were provided in Table Figure a is 4.15 for the naproxen. At pH values higher than 4.15, naproxen has a negative charge, otherwise, it has a positive charge. The neutral charge point of photocatalyst is 6. Therefore, it has a positive charge at pH below 6, whereas the surface charge of photocatalyst is negative at pH above 6. When the pH value is between 4.15 and 6, the naproxen and photocatalyst have opposite charges, so the adsorption of naproxen on the photocatalyst surface increases in this range. It is for this reason that maximum degradation occurs at pH 6 rather than pH 3 or 9. When the pH solution was 6, an increase in time from 30 to 60 min increased naproxen removal from 84.10 to 91.34%. The time and photocatalyst dosage interaction at constant initial pH and concentration of naproxen of 6 and 20 mg/L was shown in Fig. PZC, the surface charge is neutral. The surface charge can be positive or negative at pH < pHPZC and pH > pHPZC. The amount of adsorption is highly dependent on the surface charge of photocatalyst and pollutant and it can be controlled by a change in solution pH.The amount of pKp-value (< 0.001). The calculated higher F-value and lower p-value for the photocatalyst dosage (D factor) in comparison with other factors indicated that the photocatalyst dosage is the most important parameter in the system. The results in Table The analysis of variance (ANOVA) results obtained for the present model are summarized in Table An optimization was performed in order to find the optimal operating conditions for the removal of naproxen completely from pharmaceutical synthetic solutions. According to the proposed model, the following independent operating parameters are required for complete removal of naproxen (101.92%): initial naproxen concentration = 18.95 mg/L, initial pH value = 4.55, photocatalyst dosage = 0.49 g/L, time = 74.31 min. Figure The regeneration and re-use of the used or spent photocatalyst is very critical for determining the applicability of the process applied. The used photocatalyst was separated from the synthetic solution using 1.3 Tesla magnetic. Then, it was used for the treatment of pharmaceutical synthetic solution as given in Fig. 0/C) versus time (t), at different concentrations, the Langmuir–Hinshelwood (LH) kinetic model was used106:i(mg/L), the initial concentration of naproxen and C(mg/L), the concentration at time (t) after irradiation, and k is a pseudo-first-order rate constant. As shown in Fig. −1, which is about 2 times higher than the obtained reaction rate at the initial concentration of 30 mg/L naproxen. The obtained results in Fig. e (mg/g) by ZrO2/TiO2/Fe3O4 nanocomposite was calculated using the following equation , respectively. The volume of naproxen solution (ml) is V and the mass of ZrO2/TiO2/Fe3O4 nanocomposite (mg) is m. The following equation was used to calculate the percentage of naproxen adsorption (%)  Eq. :Figure 10) also gradually increased from 0.26 to 0.77 mg/L. It was shown that naproxen photocatalytic degradation occurs on ZrO2/TiO2/Fe3O4 nanocomposite surfaces and that the rate of photo degradation increases as a function of increasing of adsorption. As the initial concentration of naproxen increases, the coverage of naproxen molecules on the surface of ZrO2/TiO2/Fe3O4 nanocomposite also increases accordingly. As a result, the electron transfer efficiency of naproxen molecules to the adsorbed surface and the charge produced by the light increases, which leads to an increase in the initial optical decomposition rate (r0). As a result, the electron transfer efficiency of naproxen molecules, which leads to an increase in the initial photodegradation rate (r0), increases with increasing absorbed surface and the produced charge by light. For different initial concentrations, the kinetic constant of naproxen photo degradation gradually decreased (from 0.0514 to 0.0255 min−1), while the R2 correlation coefficient also decreased from 0.9961 to 0.9817. The relationship between the reaction rate (k) and the initial concentration of the substrate during the photocatalytic process can be generally explained by the following experimental formula (Eq. −1).As shown in Fig. Linear regression was used to analyze the relationship between the kinetic constant of NPX photo degradation and its initial concentration (5–30 mg/L). Figure 2/TiO2/Fe3O4 nanoparticles is clearly time-dependent. As shown in Fig. The results showed that the adsorption of naproxen on the surface of ZrO2/TiO2/Fe3O4 nanocomposite which is shown in Table Different kinetic models were investigated to analyze the kinetic data and determine the kinetic mechanism of naproxen adsorption on the surface of ZrO2; which was found to be 0.9202, On the other hand, the correlation coefficient value for a Langmuir–Hinshelwood (LH) kinetic model was obtained 0.9992 for NPX and greater than all other adsorption kinetic models. The calculated qe value was found to be 26.4 mg/g for NPX, which was very close to the obtained experimental values  in the degradation of naproxen at ambient temperature and pH = 3. The amount of TOC was decreased with increasing irradiation time Fig. . In thisAs shown in Table 2/TiO2/Fe3O4 nanocomposite system using a solid-state dispersion method. Furthermore, its photocatalytic activity towards naproxen degradation was investigated. The effect of operating parameters including initial naproxen concentration, initial pH, photocatalyst dosage, and time on the degradation of naproxen was investigated. Characterization results confirmed the formation of the ternary nanocomposite. The existence of cylindrical and spherical pores in the sample was proved because its N2 adsorption–desorption hysteresis followed IV-H1 type hysteresis. Based on optimization results using the RSM method, the optimal conditions for the complete removal of naproxen were determined as initial naproxen concentration of 18.95 mg/L, initial pH of 4.55, photocatalyst dosage of 0.49 g/L, and time of 74.31 min. Reusability results showed 12% reduction in naproxen degradation after 7 runs. It was found that Langmuir–Hinshelwood's (LH) kinetic model was linear. The regression coefficient (R2) was obtained 0.9992 and it was greater than all other adsorption kinetic models.In summary, we synthesized a ternary ZrO


Powder X-ray diffraction data show that all samples are phase pure, with no detectable traces of impurity. The diffraction peaks shift to lower angle values with increase in doping (x), which is consistent with larger ionic radii of Na+ and W6+ ions. X-ray photoelectron spectroscopy data reveal that a mixture of Co2+, Co3+ and Co4+ valence states are present in all samples. It has been observed that electrical resistivity (ρ), Seebeck coefficient (S) and thermal conductivity (κ) are all improved with dual doping of Na and W in Ca3Co4O9 system. A maximum power factor (PF) of 2.71 × 10−4 W m−1 K−2 has been obtained for x = 0.025 sample at 1000 K. The corresponding thermoelectric figure of merit (zT) for x = 0.025 sample is calculated to be 0.21 at 1000 K, which is ∼2.3 times higher than zT value of the undoped sample. These results suggest that Na and W dual doping is a promising approach for improving thermoelectric properties of Ca3Co4O9 system.The detailed crystal structures and high temperature thermoelectric properties of polycrystalline Ca x3−2Nax2Cox4−WxO9 (0 ≤ x ≤ 0.075) samples have been investigated.The detailed crystal structures and high temperature thermoelectric properties of polycrystalline Ca Therefore robust TE materials with large thermoelectric power factor: PF = S2/ρ and smaller thermal conductivity are required. In addition, TE materials must be stable in air at high operating temperatures over a long period of time and should be made of earth-abundant low cost elements. Conventional thermoelectric materials such as Bi2Te3 (Tmax = 550 K), SiGe (Tmax > 1300 K expensive and oxidation sensitive) and half-Heusler compounds (Tmax = 850 K) do not meet all requirements for high temperature thermoelectric applications.5–7 Transition metal oxides are promising candidates and have been explored for their potential applications in high temperature thermoelectric devices. A number of transition metal oxides such as CaMnO3,2 Al-doped ZnO8 and Ta-doped SrTiO3  for automotive applications.12 Among the transition metal oxides, misfit-layered cobaltites such as NaxCoO2,13 Ca3Co4O9,14 CuAlO2 (2Sr2Co2Ox (3Co4O9 (abbreviated as C-349 in the following text) cobaltite is especially an interesting candidate material due to its good thermoelectric performance , and its high thermal and chemical stabilities in air.17,18For practical high waste heat to electricity conversion efficiency devices, d SrTiO3  show goo4 CuAlO2  and Bi2SSr2Co2Ox  are cons3Co4O9 cobaltite has a monoclinic misfit structure with superspace group (X2/m(0b0)s0) crystal symmetry. C-349 compound is generally described as [Ca2CoO3][CoO2]1.61 and its high performance is linked with its unique layered crystal structure.14 It consists of two subsystems: a NaCl-type rocksalt (RS) Ca2CoO3 layer [subsystem 1] sandwiched between two CdI2-type (H) CoO2 hexagonal layers [subsystem 2].19 These two subsystems share the same a, c and β lattice parameters, and stack alternatively along the c axis. The mismatch of two unit cells results in dissimilar lattice parameters along the b axis i.e., b1 [subsystem 1] and b2 [subsystem 2] with a ratio b1(RS)/b2(H) ∼ 1.61. The Ca2CoO3 (RS)-type block is an insulating layer whereas CoO2 (H) sheet is conductive.20Ca21 hot pressing,22 auto-combustion synthesis and sol–gel based electrospinning followed by SPS23,24etc. Chemical substitution of alternate metal cations at both Ca- and Co-sites of Ca3Co4O9 is another approach that has been used to fine tune the electrical and thermal transport properties of TE oxides. These studies include partial substitution of Na, Bi, Y, Ag, Nd, Sr and Pb ions25–32 at Ca-sites, which adjusts the carrier concentration without changing much the band structure of materials, and substitution of Fe, Mn, Cu, Ti, Ga, Mo, W and In ions33–38 at Co-sites with significant changes in the band structure and transport mechanism. In another research work, it was reported that doping of Na ions at Ca-sites resulted in decrease of electrical resistivity and as a consequence increase of thermoelectric power factor to ∼5.5 × 10−4 W m−1 K−2 at 1000 K, though thermal conductivity (κ) of these samples was still too high (4.0 W m−1 K−1), impeded the further improvement of zT values.25 On the other hand, high valence 4d and 5d transition metal-doped C-349 samples exhibited much smaller thermal conductivity with reasonably good zT values.39 There are some research studies on simultaneous substitution of two different metal cations at Ca- and Co-sites in C-349 system with significant improvement in TE properties with zT values of ∼0.20–0.25.40–42 However, there are no reports published on dual doping of Na and W metals in Ca3Co4O9 cobaltite as yet.Recently, a number of research studies have focused on improving the TE performance of C-349 polycrystalline materials by using innovative synthesis methods such as spark plasma sintering (SPS),x3−2Nax2Cox4−WxO9 (0 ≤ x ≤ 0.075) oxides by the conventional solid-state reaction method, and investigate their structural and high-temperature thermoelectric properties. We anticipated that Na and W dual doping in C-349 system would increase the Seebeck coefficient and electrical conductivity while thermal conductivity would decrease due to the W substitution. In this way, we expected to achieve much better zT values for these materials.This prompted us to prepare a series of Ca2.x3−2Nax2Cox4−WxO9 (0 ≤ x ≤ 0.075) series were prepared by the conventional solid-state reaction method. Stoichiometric quantities of CaCO3 , Co3O4  and Na2WO4·2H2O  were ground, thoroughly mixed and pressed into pellets and initially sintered at 700 °C for 8 h. Sintered pellets were reground, pressed into pellets again and sintered twice at 900 °C for 8 h, with intermediate grinding and pelletizing, at a heating rate of 10 °C min−1 in air and slowly cooled down to room temperature.Polycrystalline samples of Caθ ≤ 60° with a step size of 0.02° using a Bruker D8 Advanced diffractometer at room temperature with Cu Kα (λ = 1.5406 Å) radiation. Rietveld refinements of XRD data were performed using a computer program JANA2006.43 Surface morphology of samples was studied using a FEI Nova NanoSEM 450 scanning electron microscope (SEM). X-ray photoelectron spectroscopy  was used to examine the oxidation states of Co and W ions in C-349 based materials. Analyses were performed using a monochromatic (Al-Kα) X-ray source at room temperature with a take-off angle of 90° from the surface plane. High-resolution Co 2p and W 4f XPS spectra were recorded using 50 eV detector pass energy and 10 scans. The binding energies were assessed by referencing to the Au 4f peak at 84.0 eV. Hall measurements were carried out at room temperature by using van der Pauw method with a (5.08 T) superconducting magnet.Powder X-ray diffraction (XRD) data were collected in 2 theta range 5° ≤ 2S) and electrical resistivity (ρ) were simultaneously measured from room temperature to 1000 K with an ULVAC-RIKO ZEM3 under a low pressure helium atmosphere. The thermal diffusivity (α) was measured with (NETZSCH LFA-457) laser flash system under vacuum. The heat capacity (Cp) was estimated using temperature independent Dulong–Petit law. The thermal conductivity (κ) was calculated using equation (κ = α·ρ·Cp), where Cp, ρ and α are the specific heat capacity, mass density and thermal diffusivity, respectively. Mass density of the samples was measured by Archimedes method using water with a few drops of surfactant.The Seebeck coefficient (3.3.1.x3−2Nax2Cox4−WxO9 (0 ≤ x ≤ 0.075) samples were analyzed by collecting the powder X-ray diffraction data at room temperature. The diffraction peaks in XRD patterns of all samples (44 indicating the formation of phase pure compounds. The enlarged portions of (0020) diffraction peaks are presented in inset of θ values with increase in doping content (x). The XRD data was Rietveld refined using a computer program JANA 2006 (X2/m(0b0)s0 and the resulting structural parameters are listed in x = 0.05 sample is shown in a, b1, c and unit cell volumes (V1 and V2) all slightly increase with increase in doping content (x), which is consistent with the observed shifting of diffraction peaks to lower 2θ values. However, the values of b2 decrease with increase in doping suggesting that there is an increase of crystallographic distortion. This trend in the lattice parameters with doping is in agreement with the observation that ionic radii of Na+ (1.02 Å) and W6+ (0.6 Å) ions are larger than the corresponding Ca2+ (1.0 Å) and Co3+ (0.545 Å)/Co4+ (0.53 Å) ions in the six coordination number,45 respectively. The quality factors like GOF, Rwp and Rp  and more compact than crystal grains of other samples (0.83–1.45 μm). The measured densities for all samples are in the range ∼86–94% of the theoretical density  and perpendicular (⊥p), to the pellets pressure axis  were stu density .3/2 and 2p1/2 with an intensity ratio of approximately (2 : 1) due to the spin–orbit coupling.47 The line shapes of both Co 2p3/2 and 2p1/2 spectra are similar to the reported results in literature.48 The main peaks corresponding to the Co 2p3/2 energy are located at 778.8 eV, 779.69 eV and 781.0 eV corresponding to x = 0.0, 0.025 and 0.075 samples, respectively. Shake-up satellite peaks due to the metal-to-ligand charge transfer processes at higher binding energies than the 2p3/2 and 2p1/2 main peaks are also detected. The observed variations in Co 2p3/2 binding energies can be explained by the larger electronegativity of tungsten  than cobalt .49 With careful analysis of XPS data, we can predict that Co ions have three types of valence states: Co2+, Co3+ and Co4+ in all samples. However, the average valence state of Co is most likely between 3+ and 4+ as reported elsewhere.50,51 The observed increase in binding energies of Co 2p3/2 peaks suggests that the relative population of Co3+ ions is decreasing with increase in doping. The 4f5 and 4f7 peaks for W ions closely resemble with the reference peaks of WO3, indicating that they are present in W6+ valence states in all samples.The binding energies of Co 2p and W 4f sub-shells of selected samples were estimated from the high resolution XPS measurements as shown in 3.2.ρ(T), as a function of Na and W co-doping (x) are shown in ρ(T) curve for x = 0.0 sample exhibits a semiconducting like behavior (dρ/dT < 0) from room temperature to around 500 K, and then shows a transition to metallic like behavior (dρ/dT > 0) from 600 K onwards. This kind of behavior in resistivity of C-349 system has been previously attributed to the spin-state transition,52 removal of oxygen atoms from porous layered cobaltites38 and structural distortion in Ca2CoO3 sheets.53 On the other hand, all doped samples show metallic behavior at low temperatures before showing a transition to semiconducting behavior above 400 K. The absolute values of resistivity at 1000 K for x = 0.025 and 0.05 samples are smaller than that of undoped sample, but higher for x = 0.075. This shows that dual doping of small amounts of Na and W has beneficial effect on resistivity of our samples.The temperature dependent electrical resistivities, 54 which is given by the following relation:ρ0 is a constant factor called the residual resistivity, kB is the Boltzmann constant and Ea is the activation energy of electrical conductivity for polaron hopping.We can describe the high temperature electrical resistivity of cobaltites using the small polaron hopping model,ρ/T) versus 1/T above 600 K, as shown in Ea/kB) were used to estimate the activation energies for all samples as listed in Ea values for doped samples are relatively higher than the pristine C-349 system. This suggests that energy demand for carriers to jump from the top of valence band to the bottom of conduction band, in general, increases with doping in our samples. However, this variation could also be due to the creation of some in-gap states which would change with doping. As discussed elsewhere, hoping of carriers occurs between Co3+ and Co4+ in CoO2 layer and as a consequent the ratio of Co3+ and Co4+ ions directly affects the hopping distance in these materials.55 We anticipate that concentration of Co4+ ions would decrease with increase in doping of W6+ ions resulting in increase of hopping distance and therefore increase in activation energies with doping. Similar results have been reported for the activation energies of Fe, Ag, Gd and Y doped misfit layered cobaltites.41,55,56The linear fits of ln(n300 K) initially increases from 5.12 × 1019 cm−3 (x = 0.0) to 6.59 × 1019 cm−3 (x = 0.025) with doping and then decreases again to 4.48 × 1019 cm−3 (x = 0.075) with further increase in doping content as shown in + for Ca2+ ions in C-349 system which results in increase of the number of hole carriers. With further increase in doping content (x), structural distortions and electron-doping like behavior of W6+ ions, due to the higher valence state of W6+ than Co3+ and Co4+ ions, start to dominate and result in decrease of carrier concentration. The carrier mobilities (μ300 K) also follow the same trend and decrease to a value of 0.65 cm2 V−1 s−1 with increase in doping after showing the maximum value for x = 0.025 sample  of the Cax3−2Nax2Cox4−WxO9 samples. The sign of S is positive for all samples suggesting that holes are the majority charge carriers. The values of S increase with increase in temperature for all samples. It is also evident from x). The maximum value of 216 μV K−1 at 1000 K for x = 0.075 sample is higher than previously reported S values for Na doped C-349 system (187 μV K−1) at this temperature.25,40 As discussed above, x = 0.025 sample shows the largest values of n300 K and μ300 K, and then values of these quantities decrease with further increase in doping. This is consistent with the observed behavior of S with doping in our samples.26,28 The contribution of carrier concentration and carrier mobility, μ(ε) in describing S is given by the Mott's formula (originated from the Sommerfeld expansion).57σ = enμ(ε)] in Ce = (π2kB2T/3e)Ψ(ε) and n, μ(ε) Ce and Ψ(ε) are the carrier concentration, energy correlated carrier mobility, electronic specific heat and density of state (DOS), respectively. There are three possible reasons associated with this increase in S values: (1) the Drude model predicts that first term  in above equation is dominant.58 In Na and W dual doped samples, carrier concentration (n) decreases with increase in doping (x) and therefore thermopower (S) increases. However, x = 0.0 sample does not follow this trend suggesting that the electronic specific heat (Ce) and second term in above equation are the dominated factors here. Wang et al., reported that Fe doping in C-349 system increases the carrier concentration (n) and the electronic specific heat (Ce), but the effect of Ce dominates over n which results in larger S values for doped samples.59 (2) We could assume that slope of the density of states at Fermi level is the main contribution to the second part of the above equation for undoped sample. (3) Partial substitution of W6+ for Co3+/Co4+ ions decreases hole carriers and thus results in an increase of thermopower.By using [kB, h, q and m* are the Boltzmann constant, Plank's constant, unit charge of electron and effective mass of carriers respectively. We have calculated a value of m*/me ∼ 0.9 for all samples by plotting room temperature S values vs. n−2/3 as shown in inset of n and estimated m* values as described by the following equations:60F1/2(ξ) is the Fermi integral, ξ is the reduced electrochemical potential, λ is a scattering parameter and its value is taken 0 for acoustic phonon scattering, 1 for optical phonons scattering, and 2 for ionized impurity scattering.60 The calculated S values at room temperature as a function of carrier concentration (n) are shown in S match very well when we take λ = 0, which suggests that the acoustic phonon scattering is the dominant scattering mechanism for all samples.According to the Pisarenko relation for degenerated semiconductors:S2/ρ for all samples as shown in x = 0.025 sample exhibits the highest PF of 2.71 × 10−4 W m−1 K−2 at 1000 K which is about 2.3 times more than PF, 1.27 × 10−4 W m−1 K−2, of undoped sample. The PF obtained in this work is higher than the previously reported value of ∼2.1 × 10−4 W m−1 K−2 at 1073 K for Ca3Co3.97Cu0.03O9 sample prepared by the conventional solid state reaction method.61We have used the electrical resistivity and thermopower values to calculate the thermoelectric power factor PF = 3.2.1.κTotal) for Cax3−2Nax2Cox4−WxO9 samples is shown in κ(T) decreases with increase in temperature for all samples in the measured temperature range. For x = 0.0 sample, measured value of κ at 1000 K is 1.36 W m−1 K−1 and it decreases to 1.26 W m−1 K−1 for x = 0.025 sample. On further increase in doping, κ1000 K slightly increases again but its value remains lower than the pristine C-349 system. In order to understand the observed changes, we have investigated the contribution of electronic (κel) and lattice (κLattice) parts of the thermal conductivity, separately. κel as determined from the experimentally measured ρ values by using the Wiedemann–Franz law (κel = LT/ρ), where L is the Lorentz number and its value is 2.44 × 10−8 W Ω K−2 for free electrons.3 The values of κLattice were calculated using the relation κLattice = κTotal − κel and are shown in κLattice, and not κel, is the major contributing factor to total thermal conductivity of our samples. Hence, changes in κTotal with increase in doping content (x) are mainly originated from the changes in κLattice.33 We can attribute these changes in κLattice to the larger ionic radius of W6+ than Co3+/Co4+ ions resulting in structural distortions, and therefore increase in phonon scattering. However, we cannot rule out some other unexplained microstructural aspects of these materials that can also be responsible for irregular behavior in thermal conductivity of doped samples.The temperature dependent total thermal conductivity (3.2.2.zT = S2/κρ) for Cax3−2Nax2Cox4−WxO9 samples as a function of temperature and doping content (x) are shown in zT values of doped samples are significantly higher than the pristine C-349 system. The x = 0.025 has the highest zT value of 0.21 at 1000 K among all samples, which is about 2.3 times higher than zT value of the undoped sample. This increase in zT value is due to increase in Seebeck coefficient, and decrease in electrical resistivity and thermal conductivity of this sample. zT values of other doped samples are also reasonably good as listed in zT value of our x = 0.025 sample is comparable or slightly better than previously reported results of Na doped Ca2.5Na0.5Co4O9 (zT1000 K = 0.18),25 Bi and Na dual doped C-349 system (zT1073 K = 0.18),62 Ca2.8La0.2Co3.8Cu0.2O9 (zT773 K = 0.203),40 Bi and Na substituted Ca3Co4O9 system (zT1000 K = 0.32),26 Gd and Y dual doped Ca2.7Gd0.15Y0.15Co4Oδ9+ (zT973 K = 0.26),63 NaF doped Cax3−NaxCo4Ox9−Fx (zT873 K = 0.13)64 and Ca2.9Y0.1Co3.97Fe0.03O9 (zT1000 K = 0.22) compound.42 It has been observed that electrical resistivity of our samples is still higher than most of the previously reported results, and therefore zT values of these materials are moderate. We believe that zT values of these samples can be further improved by preparing more compact materials under optimized synthesis conditions.The thermoelectric figure of merit (4.x3−2Nax2Cox4−WxO9 (0 ≤ x ≤ 0.075) have been synthesized by the conventional solid state reaction method. Powder X-ray diffraction data revealed that Na+ and W6+ ions enter into the Ca2CoO3 and CoO2 layers of Ca3Co4O9 system respectively. High resolution XPS data showed that the average valence state of Co in our samples is between 3+ and 4+. Significant improvements in ρ, S and κ values of Na and W dual doped samples have been observed. These results are also supported by the carrier concentrations (n) and carrier mobility (μ) as confirmed by the Hall effect measurements. The observed power factor (PF) and thermoelectric figure of merit (zT) of 2.71 × 10−4 W m−1 K−2 and 0.21 respectively at 1000 K for x = 0.025 sample are comparable or slightly higher than most of the reported results of C-349 based materials, prepared by the conventional solid state reaction method. These results show that Na and W dual doping is an effective route for improving thermoelectric properties of Ca3Co4O9 system.Polycrystalline samples of CaThere are no conflicts to declare.


Corallococcus silvisoli c25j21 GDMCC 1.1387, which is predatory to plant pathogenic bacteria. In this study, we identified an endolysin-like GH19 glycoside hydrolase, C25GH19B, from the genome of c25j21. After its heterologous expression and purification from E. coli, the enzymatic properties of C25GH19B were characterized. C25GH19B showed lysozyme activity with the optimal reaction conditions at 40 °C and pH 4.5–5.0. Moreover, C25GH19B showed bacteriolytic activity against both Gram-positive and Gram-negative plant pathogenic bacteria. Our research provides not only a candidate enzyme for the development of novel biocontrol agents but also an experimental basis for further study on the function and mechanisms of extracellular enzymes in myxobacterial predation.Myxobacteria, as predatory bacteria, have good application potential in the biocontrol of pathogenic microorganisms. Extracellular enzymes are thought to play an important role in their predation and also provide resources for discovering new antibacterial molecules. We previously isolated a myxobacterium, The online version contains supplementary material available at 10.1186/s13568-022-01393-y. Fusarium roseum and Spodoptera litura  includes chitinases from plants or bacteria, and lysozymes or endolysins from phages. Several bacteriophage-derived endolysins have been biochemically characterized and reported to have antimicrobial activity against Gram-positive or Gram-negative bacteria, and are considered to be ‘enzybiotics’ with good application potential  in c25j21 genome was identified and expressed in E. coli. Furthermore, the enzyme activity, biochemical characteristics, and bacteriolytic activity against Gram-positive and negative phytopathogens were evaluated.We previously isolated a myxobacterium strain, C. silvisoli c25j21 was inoculated on VY/2 agar plates  incubated at 30 °C for 5 days. Strain Curtobacterium flaccumfaciens GDMCC 1.343 and Pseudomonas syringae GDMCC 1.330 were inoculated in NB liquid medium and shaken at 160 rpm for 3 days at 30 °C. The freshly cultured pathogen was collected by centrifugation, washed, and resuspended in TPM buffer  to adjust the optical density to OD600 = 5. Then a spot of 150 µL pathogen bacterial suspension was inoculated on the center of TPM agar . A 5 mm diameter plug strain of c25j21 was inoculated on the center of the pathogen lawn and the plates were incubated at 30 °C. The zones of predation were observed with a stereomicroscope .NcoI and XhoI restriction sites by GENEWIZ . The construct was transformed into E. coli BL21 (DE3) to express the protein. The resultant E. coli BL21 (DE3) cells were cultured at 37 °C and 200 rpm in Luria–Bertani (LB) medium containing kanamycin (50 μg/mL) until the OD600 was approximately 0.6. 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at 22 °C with 200 rpm shaking overnight to induce protein expression. The cells were harvested by centrifugation at 4 °C, and then suspended in lysis buffer . After sonication, the cell suspension was centrifuged (4 °C) at 12,000 rpm for 20 min, and the supernatant was subjected to nickel-chelating chromatography. Fractions containing C25GH19B were pooled, concentrated, and dialyzed against 50 mM acetate buffer (pH 5.0) for further analysis.The sequence of C25GH19B (WP_161664743.1) was analyzed by the tools SignalP  were calculated from the plots of ln  versus time  of the enzymes was calculated by the equation: t1/2 = ln2/ kinact.The purified C25GH19B (1 mg/ml) were incubated at 40 °C and sampled at different intervals to measure the residual activities at 30 °C. The average thermal inactivation rate constants  in the concentration range of 0.1–10 mM were added to the reaction mixture, and the relative activity of C25GH19B (0.2 mg/mL) was determined.As the above-mentioned enzyme activity assays, divalent metal ions (FeCurtobacterium flaccumfaciens GDMCC 1.343 and Pseudomonas syringae GDMCC 1.330 as substrates.The bacteriolytic activity of C25GH19B on the pathogen was determined by the turbidimetric method as described above using the lyophilized C. flaccumfaciens GDMCC 1.343 and P. syringae GDMCC 1.330 at the mid-logarithmic phase were washed and resuspended in 50 mM acetate buffer (pH 6.0) containing C25GH19B (0.5 mg/mL). Controls were incubated in the absence of C25GH19B. The mixture was incubated at 30 °C for 2 h, serially diluted, and the dilutions were spread on nutrient agar plates. The colony-forming units (CFU) were obtained after incubating the agar plates at 30 °C for 72 h. All assays were performed in triplicate and the results are the means of three independent experiments.Viable plate counts were also used to test the bacteriolytic activity of C25GH19B on the pathogen. C. flaccumfaciens GDMCC 1.343 and P. syringae GDMCC 1.330 at the mid-logarithmic phase were washed and resuspended in 50 mM acetate buffer (pH 6.0) containing the C25GH19B (0.5 mg/mL). Controls were incubated in the absence of protein. The mixture was incubated at 30 °C for 2 h or 6 h, then the cells were observed using an H7650 TEM microscope .C. flaccumfaciens GDMCC 1.343 and P. syringae GDMCC 1.330, respectively, according to the previously described method    (Sievers and Higgins https://itol.embl.de/) (Letunic and Bork The protein sequences of the characterized GH19 enzymes were obtained from the CAZy database (https://swissmodel.expasy.org/)  to express the enzyme protein. The purified C25GH19B was about 20 kDa (Fig. M. luteus, indicating that it is a lysozyme (Fig. The homology of C25GH19B with bacteriophage-derived lysozyme and endolysin encouraged us to explore its bacteriolytic activity. The gene of C25GH19B without the signal peptide was cloned into the pET-28a vector and transformed into li BL21DE to expreli BL21DE to expreT5015 value, the temperature when half of the enzyme activity is retained after 15 min incubation, was calculated to be 48.7 °C. The 40 °C-thermal inactivation profile showed that more than 50% of the enzyme activity was maintained after 130 h of incubation (Fig. To investigate the optimal catalytic reaction conditions of C25GH19B, we first assayed the effect of pH on its activity. As shown in Fig. We further investigated the effect of divalent metal ions on the enzyme activity of C25GH19B Fig. E. The teM. luteus, we would like to investigate whether it can lyse plant pathogenic bacteria. C. flaccumfaciens and P. syringae were used as representatives of Gram-positive and Gram-negative bacteria, respectively, to test the bacteriolytic activity of C25GH19B. As shown in Fig. C. flaccumfaciens, which is consistent with its lytic activity against M. luteus, indicating that the enzyme has a bacteriolytic activity on Gram-positive bacteria. In addition, although slightly less active, C25GH19B showed lytic activity against P. syringae (Fig. Since C25GH19B has lysozyme activity against C. flaccumfaciens and P. syringae cells were killed by 88% and 95%, respectively. Transmission electron microscopy (Fig. C. flaccumfaciens treated by C25GH19B. While P. syringae treated with C25GH19B showed morphological changes and loss of cytoplasmic contents. Moreover, the longer the treatment time, the more obvious the bacteriolytic effect. Altogether, these results suggest bacteriolytic activity of C25GH19B against both Gram-positive and negative phytopathogens.The antimicrobial activity of C25GH19B was further tested using plate count assays (Fig. opy Fig.  showed tM. luteus and phytopathogenic bacteria. We speculated that it might act on peptidoglycan of the bacterial cell wall. To test this speculation, we studied its binding ability to the cell wall peptidoglycan of C. flaccumfaciens and P. syringae. As shown in Fig. C25GH19B showed bacteriolytic activity against C. silvisoli c25j21 is a soil indigenous myxobacterium strain that preys on a variety of plant pathogens and could be a potential biocontrol agent. A GH19 family enzyme derived from c25j21, C25GH19B, was characterized in this study. Sequence analysis revealed that C25GH19B is homologous to bacteriophage-derived endolysin and lysozyme, but not bacterial chitinases. Biochemical characterization showed that C25GH19B had peptidoglycan-hydrolysis but not chitin-hydrolysis activity, which further confirmed that it was a myxobacterial lysozyme. C25GH19B prefers a weakly acidic environment with an optimal pH of 4.5–5.0, which is different from the previously reported endolysins, such as LYS177 and LYS188, whose optimal pH is 6–7 (Orlando et al. M. xanthus, which only lyses Gram-positive bacteria (Arend et al. C. flaccumfaciens and P. syringae. Due to the outer membrane barrier of Gram-negative bacteria, the enzyme does not easily access its peptidoglycan, which is reflected in the lower activity of C25GH19B against P. syringae when measured by turbidimetry. However, the partial destruction of the cell wall can cause leakage of the cell contents and kill the bacteria, so we could see that C25GH19B also has good bacteriolytic activity against Gram-negative bacteria P. syringae in the plate counting experiment and scanning electron microscope observation. The results of the substrate binding experiments indicated that C25GH19B acts on the cell wall peptidoglycan of phytopathogenic bacteria. These results suggested that C25GH19B has the potential to be developed as a component of novel biocontrol agents.Unlike LlpM from M. xanthus (Thiery and Kaimer Corallococcus sp., Vitiosangium sp., and Pyxidicoccus sp., etc., are of great significance for the development of novel biocontrol strategies.Myxobacteria have a broad predatory spectrum, but most of them have not been well studied except for the model organism C. silvisoli c25j21. C25GH19B is an endolysin-like enzyme with bacteriolytic activity against both Gram-positive and Gram-negative phytopathogens. Our results provide an experimental basis for the development of myxobacterial extracellular enzymes as novel biocontrol agents.In summary, we characterized the GH19 enzyme, C25GH19B, from the myxobacterium strain Additional file 1: Figure S1. Phylogeny of C25GH19B and the GH19 proteins in the GH19ED database. The members of the chitinases and endolysins sub-families in GH19ED database are shown in blue and red, respectively. Figure S2. Ramachandran plot of the structure model of C25GH19B. Figure S3. Verify_3D evaluation of the structure model of C25GH19B.


Zn-based alloys are considered as new kind of potential biodegradable implanted biomaterials recently. The difficulty of metal implanted biomaterials and bone tissue integration seriously affects the applications of metal implanted scaffolds in bone tissue-related fields. Herein, we self-designed Zn0.8Mn and Zn0.8Mn0.1Li alloys and CaP coated Zn0.8Mn and Zn0.8Mn0.1Li alloys, then evaluated the degradation property and cytocompatibility. The results demonstrated that the Zn0.8Mn0.1Li alloys had profoundly modified the degradation property and cytocompatibility, but Zn0.8Mn0.1Li alloys had particularly adverse effects on the surface morphology of osteoblasts. The results furtherly showed that the CaP-coated Zn0.8Mn and Zn0.8Mn0.1Li alloys scaffold had better biocompatibility, which would further guarantee the biosafety of this new kind of biodegradable Zn-based alloys implants for future clinical applications. In recent years, biodegradable ferroalloy and magnesium alloy as the main representatives of medical metal materials with biodegradable properties have attracted extensive attention . The uniin vitro degradation, and cytocompatibility evaluations of ZnLi alloys, which had well optimal scaffold performance 2. Then, placed the reaction beaker in an ultrasonic cleaner for 60 min. In the last, the samples were cleaned with deionized water and dried at room temperature.The pure Zn, Zn0.8Mn alloy, and Zn0.8Mn0.1Li alloy were cut into a disk with a diameter of 10 mm and a thickness of 1 mm, then grounded with 240–5,000 silicon carbide sandpaper, and polished to obtain a smooth surface. Ultrasonic cleaning was then performed in acetone, alcohol, and deionized water. As The phase composition of the samples was measured by XRD . The surface morphology of all samples was watched by FE-SEM , and the microstructure was observed by FE-SEM after etching with 4% nitric acid solution. Under the condition of 300 g load and 10 s loading time, the microhardness of the materials was measured by microhardness tester. For the accuracy of the results, we have tested at least five parallel samples. The wetting angle was measured with a contact angle meter to evaluate the surface wettability of the sample .XRD was employed to detect the crystal structure of the coatings. The surface morphology of the CaP coatings was manifested using FE-SEM. The surface chemical constituent was determined using the FE-SEM equipped with EDS. The surface wettability was evaluated by DSA100S, and the surface roughness was analyzed by atomic force microscope .2. Before measurement, the samples were soaked in SBF solution for 30 min to obtain a stable surface state. The open circuit potential was set to 1,800 s to achieve stability. The potential dynamic polarization (PDP) test was performed in the range of ±0.3 V. Constant scanning rate was 1 mVs−1. Electrochemical impedance spectroscopy (EIS) was performed in the frequency range of 105 Hz to 10–2 Hz. ZSimpWin software was used to analyze the impedance data and fit it with the equivalent curve.The electrochemical properties of the materials were tested by an electrochemical analyzer . Working electrode, placed on a specific mold, with an exposed study surface area of 0.237 cmEarly corrosion behaviors of Zn, Zn0.8Mn, and Zn0.8Mn0.1Li were researched in SBF at 37 ± 0.5°C for 14 and 28 days. Microscopical images and chemical compositions were obtained using FE-SEM equipped with EDS. The corrosion products were analyzed by XRD with radiation and 4°/min scanning speed.Before the tests, the samples were polished and soaked in 75% ethanol solution for 20 min, followed by UV irradiation for 20 min.2 atmosphere.The osteoblast precursor cell line (MC3T3-E1) was selected for cell tests. The culture medium was α-DMEM  supplemented with 10% FBS , 1% L-glutamine, and 10% penicillin-streptomycin at 37°C in 5% CO2 at 37°C for 3 h. The cellular activity was assessed on a microtiter plate reader .After the cells were cultured on the scaffold for 1, 3, and 7 days, 10% CCK-8 reagent was added and placed in an incubator with 5% CO5 MC3T3-E1 cells were inoculated for 3 days and fixed in 4% paraformaldehyde (Sangon Biotech) for 30 min. Washed three times and then dehydrated with 30%, 50%, 75%, 90%, 95%, and 100% gradient ethanol (Biogenic organism) solutions for 5 min each time. Finally, it dried slowly at room temperature. The sample morphology was observed by FE-SEM. And cell adhesion numbers were performed with DAPI (Sigma) after cultured for 3 days.The 2 × 105 MC3T3-E1 cells were inoculated for 1, 3, and 7 days. Added 2 µl of Calcein-AM Solution (2 mM) and 6 µl of Propidium iodide  to 1×buffer and mixed well. Then added 200 µl mixture to the surface of the Zn-based alloys and stained it for 20 min without light. Finally viewed under a fluorescence microscope .The 2 × 105 MC3T3-E1 cells were inoculated for 1, 3, and 7 days. Then, the cells were fixed with 4% paraformaldehyde for 15 min and infiltrated with 0.1% Triton for 20 min. Finally, 200 μl podophyllotoxin dye and 100 μl 1% bovine serum albumin were dyed at room temperature for 30 min.The 2 × 10Osteogenic differentiation was induced by adding osteogenic induction solution  into the complete medium. After 7 days of culture, alkaline phosphatase (ALP) activity was detected by BCIP/NBT ALP Color Development Kit (Beyotime), and after 14 days of culture, extracellular matrix mineralization was detected by alizarin red S(Sigma) staining.To further investigate the effects of different scaffold materials on the osteogenic expression of MC3T3E1, the expression of RUNX2 and COL-1 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) after the cells were cultured on the scaffold for 7 days. Total RNA was extracted by TRIzol reagent (Life Technologies). Complementary DNA was synthesized from 1,000 ng of total RNA using the SensiFAST™ cDNA Synthesis Kit  according to the manufacturer’s protocol. The gene expression was then quantified using an SYBR Green quantitative polymerase chain reaction kit (Invitrogen) in a 7500 RT-PCR system  with a procedure of 95°C for 3 min and 95°C for 3 s, followed by 40 cycles at 60°C for 30 s. Gene expression was normalized to GAPDH using the ΔΔCt method. The primer sequences were shown in p < 0.05.Using SPSS 22.0 statistical software, The data were demonstrated as means ± standard deviation (SD). Using one-way ANOVA to analyze the statistical difference. The distinction was significant at in vitro. 13 phase was detected in Zn0.8Mn, and MnZn13 and LiZn4 phases were detected in Zn0.8Mn0.1Li alloys. 13 and LiZn4 were detected in the Zn0.8Mn0.1Li alloy. LiZn4 particles attached to MnZn13 particles and formed MnZn13/LiZn4 compound structure. By comparing the two figures, the addition of Li refined the microstructure significantly. Systematically, the microhardness of the alloy was significantly increased from 20.94 HV of pure Zn to 61.2 HV of Zn0.8Mn0.1Li alloy 2, CaZn2(PO3)2(H2O), H(Zn4(PO4)3)H2O, and Zn2(PO4)(OH). The CaP Zn0.8Mn0.1Li consisted a small amount of LiZn(PO4)2 phase. The diffraction peak intensity of CaP Zn was obviously stronger than CaP Zn0.8Mn and CaP Zn0.8Mn0.1Li. The surface morphologies of CaP Zn, CaP Zn0.8Mn and CaP Zn0.8Mn0.1Li were shown in As can be seen from AFM test showed that the roughness of the samples surface increased significantly with the surface morphology . WettabiGood proliferation and adhesion of cells on scaffolds were two important factors in the construction of bone tissue engineering scaffolds. For the sake of estimating the proliferation ability of MC3T3-E1 cells cultured on scaffolds, CCK-8 was measured on days 1, 3, and 7 after cells inoculation. Results were shown in For the sake of the early stage osteogenic activity of scaffolds, we observed the ALP staining of MC3T3-E1 cells cultured on different scaffolds for 7 days. As shown in 13 particles stimulated the recrystallization of Zn grains at room temperature and released the internal stress during the tensile test 2, CaCO3, Zn3(PO4)2, and ZnCO3, which were specific corrosion products of Zn and its alloys in physiological environment  can respectively attract with OH−, HCO3−, HPO42-, and Cl− in the SBF as follows:The ions released from the alloy were primarily Zn2 in the water environment, forming Li2O in the outer corrosive layer  is low in the alloy, it is preferentially released during degradation due to its high chemical reactivity. This can effectively regulate the release of Zn2+ and avoid the adverse effect of excessive local zinc ion concentration on the osteogenic effect.It was considered that the coexistence of MnE1 cells . The ost2+ moderately. Therefore, Zn0.8Mn0.1Li alloy had good comprehensive properties and was expected to become a candidate material for the development of biodegradable alloys.In the future design of Zn-based alloys for orthopedic, we should select elements with osteogenic effects and low electrode potential to balance their synergistic effects and reduce the release rate of ZnThe physicochemical properties of the material surface were the key factors to regulate the bone-implant bonding. The adhesion, proliferation, and differentiation of cells were intensely dependent on the surface of materials, including surface composition, charge, and hydrophilicity. Such as the effect of roughness on protein adsorption will directly affect cell adhesion and diffusion . It has All in all, to satisfy the clinical demands of osteogenic ability, biocompatibility, and biodegradability of medical degradable metal materials, a high-strength Zn0.8Mn0.1Li alloy was designed. To further improve its cytocompatibility, we modified its surface and prepared calcium-phosphorus coatings. It decreased the ion release and subsequently also decreased toxicity and enhanced MC3T3-E1 cells proliferation and adhesion. Therefore, this study will propose an innovative strategy for the surface design of biomedical implants.


Conopeptides are peptides in the venom of marine cone snails that are used for capturing prey or as a defense against predators. A new cysteine-poor conopeptide, Czon1107, has exhibited non-competitive inhibition with an undefined allosteric mechanism in the human (h) α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, the binding mode of Czon1107 to hα3β4 nAChR was investigated using molecular dynamics simulations coupled with mutagenesis studies of the peptide and electrophysiology studies on heterologous hα3β4 nAChRs. Overall, this study clarifies the structure–activity relationship of Czon1107 and hα3β4 nAChR and provides an important experimental and theoretical basis for the development of new peptide drugs. Acetylcholine acts on nicotinic acetylcholine receptors (nAChRs) , which aNeuronal nAChRs are associated with cognitive activity, pain perception, learning and memory formation, and neuronal development in humans, thus therapeutics that act on nicotinic synaptic transmission may be useful for the treatment of pain, memory disorders, Parkinson’s disease, schizophrenia, and nicotine addiction ,10.The α3β4 nAChR  is the pConus snails  could be explained by the formation of hydrogen bonds with the carbonyl and hydroxyl groups of T262 -scan mutagenesis on Czon1107. The replacement of residues at positions 1 and 7 with the positively charged Arg slightly enhanced the potency of Czon1107 in inhibiting the hα3β4 nAChRs . Both th of T262 D. Furthe of T262 F with thp < 0.05, n = 7–9). The hydrophobic interactions formed between F2 and P264 and P271 were removed by the introduction of arginine at this position, resulting in a significant decrease in activity for Czon1107[F2R]. Similarly, the F9R mutation resulted in a decrease in inhibitory activity due to the removal of the hydrophobic interactions and the VDW between F9 and L468. In addition, as the S4 residue is embedded in the binding pocket, S4R mutation could introduce steric hindrance, which could be responsible for the significant decrease in activity for Czon1107[S4R] in comparison to the wild-type peptide. No significant change in activity at hα3β4 nAChRs was observed for Czon1107[P5R] and Czon1107[P8R].In contrast, Arg substitution of residues F2, S4, and F9 reduced the inhibitory activity of the peptide at hα3β4 nAChRs. In comparison to Czon1107, all three analogues inhibited the ACh-evoked current amplitude by 5–20% (50 of 77.2 µM) [In addition to the hα3β4 nAChRs, Czon1107 inhibited nicotine-evoked currents at hα7 nAChRs (IC77.2 µM) . A compa1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethylu-ronium hexafluoro-phosphate  and N, N-diisopropylethylamine  were added in DMF for 1 h at room temperature and washed three times with DCM and DMF. After the completion of the chemical reaction, 20% piperidine solution (30 min) was used to remove the last Fmoc protective group and washed three times with DCM and DMF. Addition of 10 mL trifluoroacetic acid (TFA): triisopropylsilane: water (9:0.5:0.5) were reacted at a constant temperature on a shaker for 3 h to remove the peptide from the resin. After washing the resin with DCM three times, the filtrate was combined. After removal of excess TFA, the peptide was then precipitated in cold diethyl ether, washed three times with diethyl ether, and dissolved in an H2O/acetonitrile (1:1) mixture at a peptide concentration of 0.1 mg/L. Then, the reaction was purified by reversed-phased high-performance liquid chromatography (RP-HPLC) , using a gradient of buffer A  and B  of 100% to 40% for 1 h with a flow rate of 6 mL/ min. Subsequently, the peptide was oxidized by adding 5 mg/mL iodoacetonitrile under a closed environment at 28 °C for about 2 h. After the reaction was finished, an ascorbic acid buffer was added to quench the reaction. The oxidized product was purified by RP-HPLC, and the separation conditions were the same as above. This was followed by concentration under vacuum and lyophilization of the peptide as a white powdery solid; the product was stored at −20 °C.Peptides were synthesized using the standard solid-phase peptide synthesis (SPPS) as reported previously . After sX. laevis were sourced from Nasco  and a maximum of four frogs were kept in a 15 L aquarium at 20–26°C with a 12 h light/dark cycle. Oocytes  were removed from X. laevis by surgical laparotomy and defolliculated with 1.5 mg/mL collagenase Type II . Oocytes were obtained from three frogs. Defolliculation was done at room temperature (21–24 °C) for 1–2 h in OR-2 solution containing (in mmol/L) 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4. Plasmid pT7TS constructs of hα3 and hβ4 were linearized with XbaI  for in vitro T7 mMessage mMachine®-cRNA transcription . Oocytes were injected with 5 ng of hα3β4 cRNAs at α3 to β4 ratio of 1:1  using glass pipettes as described previously [2, 1 MgCl2, and 5 HEPES at pH 7.4, supplemented with 5% fetal bovine serum (FBS), 50 mg/L gentamicin  and 10,000 U/mL penicillin-streptomycin (GIBCO).All procedures were approved by the University of Wollongong Animal Ethics Committees (project number AE2003). Female eviously . OocytesX. laevis oocytes expressing hα3β4 nAChRs. Voltage clamp recordings were performed using a GeneClamp 500B amplifier and pClamp9 software interface  at a holding potential of −80 mV at room temperature. Oocytes were impaled with two microelectrodes filled with 3 mol/L KCl. Voltage recording and current injection electrodes were both made of GC150T-7.5 borosilicate cate glass  and had a resistance of 0.3–1 MΩ when filled with 3 mol/L KCl. Oocytes were continuously perfused with ND96 solution (2 mL/min). Three ACh applications were then performed with 300 µM ACh solution  [50 of wild-type Czon1107 at hα3β4 nAChRs [ACh+peptide /IACh ) were applied to assess peptide activity at hα3β4 nAChRs. The activity of the peptides was compared using an unpaired Student’s t-test , and it was deemed statistically significant with values of p < 0.05.Two-electrode voltage clamp recordings were carried out on 4 nAChR) . After p4 nAChRs . The pephttp://codockpp.schanglab.org.cn/, accessed on 17 November 2021). The produced top 10 docked conformations of the Czon1107-WT were selected for further analysis and those with the optimum binding modes were used for subsequent MD simulations to further optimize the structure.The crystal structure of the protein was retrieved from the Protein Data Bank (PDB). Czon1107-WT was docked to the hα3β4 nAChR in the CoDockPP web server  [http://www.ks.uiuc.edu/, accessed on 26 January 2022) was used to analyze the motion trajectory after MD simulations and calculate the RMSD value of the resulting conformation.MD simulations were carried out in AMBER 20 software using ff19SB force field . The hα3ry 2022)  and enerry 2022)  (http://Conopeptides such as AuIB , RegIIA This study gives insight into the structure–activity relationship of Czon1107 and hα3β4 nAChR. Through molecular docking and MD simulations, the favorable binding site for Czon1107 is putatively located at the α3 subunit. The MD simulations of the interactions also suggest that Czon1107 residue F2 forms hydrophobic interactions with residues P264 and P271 of the α3 subunit. Furthermore, Czon1107 residue R3 interacts with residues S225 and T280 of the receptor via hydrogen bonds. These findings provide an experimental and theoretical basis for the structure optimization and mechanisms of action of Czon1107 and its analogues. Based on the current experimental results, multi-site mutation of Czon1107 and α3 subunit is a promising research direction for elucidating a potent conopeptide at the α3β4 nAChR.