MOSF was added with precursor solution of MF0.33 during MF0.33 synthesis which soaked the materials and further chemical changes occurred inside the pore. Therefore, no additional synthesis process was required for magnesium ferrite impregnated mesoporous ordered silica foam (MF0.33-MOSF) synthesis. MF0.33-MOSF showed higher morphological properties compared to other magnesium ferrite modified nanomaterials and adsorbed arsenic III [As(III)] and arsenic V [As(V)] 42.80 and 39.73 mg/g respectively. These were higher than those of other Fe-modified adsorbents at pH 7. As MOSF has no adsorption capacity, MF0.33 played key role to adsorb arsenic by MF0.33-MOSF. Data showed that MF0.33-MOSF contain about 2.5 times lower Fe and Mg than pure MF0.33 which was affected the arsenic adsorption capacity by MF0.33-MOSF. Adsorption results best fitted with Freundlich isotherm model. The possible mechanism of arsenic adsorption might be chemisorption by electrostatic attraction and inner or outer-sphere surface complex formation.Magnesium ferrite (MF Governments are investing a lot of budgets for removal of arsenic from water. Among different technologies, adsorption is an efficient technology for arsenic removal from water2. Research data proved that iron-based adsorbents performed well over other adsorbents in arsenic removal3. However, modification through impregnation, coating and functionalization of iron adsorbents with porous material improved adsorbent characteristics which enhanced As removal efficiency7.Arsenic toxicity is a well-known global problem affecting millions of people each year. Naturally arsenic comes from ground water. The use of ground water as drinking and irrigation purpose causes arsenic transfer to human health. Arsenic can cause several health dieses like keratosis, hyperkeratosis, melanosis, leucomelanosis, dorsum, and even cancer9. There were different advantages for using porous material as support or carrier material. Firstly, porous materials can reduce the aggregation of nanomaterials during adsorption process. This phenomenon decreases surface area and active sites of the adsorbent. Secondly, selectivity and catalytic activity of adsorbents for pollutant adsorption can be affected by the textural properties of the carrier material. Pollutants can form a thin layer on the pore channels or walls and be attached swiftly. Thirdly, encapsulation and impregnation of active nanomaterials into the pore and cavities of porous material increases the stability and dispersion of nanomaterials in catalysis process. Finally, for more flexibility, pore dimensions, morphology and structure of the porous materials can be designed as per requirement of the research. Activated carbon, zeolite, activated alumina can show upper three advantages but the final advantage is the specialty for mesoporous silica material which made this material more efficient over others10.Different kinds of porous materials have been used for water treatment solely or as a composite with other material so far. Sand, clay, zeolite, activated carbon and silica are typical porous materials used for arsenic removal from water through adsorption process11. Among different type of porous silica materials, macro and mesoporous silica foam (MOSF) was found most suitable for pollutant removal from water through adsorption. Yang et al.12 synthesized lanthanum coated MOSF which removed 96% phosphorus within 30 min. They further modified MOSF with γ-Fe2O3 nanoparticles and applied for As(III) and As(V) removal from water. Data revealed that maximum 34.8% γ-Fe2O3 nanoparticles dispersed on pore walls of MOSF which adsorbed 248 and 320 mg/g As(III) and As(V) [Initial conc. of As(III) and As(V) was 560 and 600 mg/l] respectively13.Porous silica material were considered as ideal material in different application because of exceptionally large surface area, thermal and chemical stability, high pore volume, low density, high selectivity and flexibility which can be modified using different organic and inorganic functional groups1. Among Fe-based adsorbents, spinel ferrites exhibited exceptional performance in pollutant removal during water treatment. However, researchers found health toxicity because of cobalt ferrites. But there were no data found on magnesium ferrite (MgFe2O4) related toxicity in literature. In addition, magnesium plays essential functional activity inside human body. Besides that, among all spinel ferrites, magnesium ferrite showed better performance in pollutant removal through adsorption because of easy availability, safe handling, large surface area, tunable porosity and economically viable properties14. However, nanomaterials have some negative properties during adsorption such as aggregation and separation. Jung et al.15 showed that MgFe2O4 /Biochar composite adsorbed higher amount of phosphate than MgFe2O4 nanoparticles. Karthikeyan et al.16 also reported that modified MgFe2O4 was performed more efficiently for toxic ions removal than single MgFe2O4 nanoparticles. Based on the literature data, it was found that the modified material showed more stable morphological properties. In addition, modification increased the adsorptive site and influenced on pHPZC that removed more toxic ions from solution. Furthermore, modification of magnesium ferrite not only increased adsorption capacity but also increased the stability and pollutant selectivity during adsorption process17. Considering the exceptional properties of MOSF in literature, present research was structured to develop a new composite material that might remove pollutants from water efficiently10.Iron was found most efficient element for arsenic removal through adsorption process2O4 modified MOSF composite material. Very few research data were found on adsorptive arsenic removal from water using magnesium ferrite nanomaterials19. In addition, MOSF might increase MgFe2O4 nanomaterials stability and adsorption capacity which was the main objective of this research13. Therefore, considering efficiency and economic value, a simple Solvothermal method was followed for magnesium ferrite impregnated MOSF synthesis in present study and applied for arsenic removal from water.Tile date, there was no literature found based on MgFe2O4 nanomaterial was synthesized by following a method with major modifications in different experimental factors20. To follow the ratio of Fe:Mg = 0.67:0.33, appropriate amount of anhydrous ferric chloride (FeCl3) and magnesium chloride (MgCl2) were dissolved in 70 ml ethanol (C2H6O). After that, 10 ml of just prepared 2.03 M sodium hydroxide-ethanol (NaOH-C2H6O) mixture added to the solution. To make a homogenized solution, the mixed solution was taken under a Sonicator for 1 h at 25 ± 5 °C temperature. Sonicator probe height, amplitude and temperature were carefully maintained for all runs. The homogenized solution mixture was taken into a Teflon-line autoclave made of stainless steel and closed it tightly. Then the autoclave was kept in a convective oven for 8 h at 200 °C temperature. The autoclave was naturally cooled to room temperature. After that, the mixture was transferred into a 100 ml beaker and washed using deionized water with the help of magnetic stirrer. The washing process was continued until the mixture was free from sodium chloride. The final precipitate was transferred to a ceramic cup and taken into the oven for 12 h at 80 °C temperature. After that time period, the ceramic cup was kept in a desiccator carefully and naturally cooled. Next, the dried materials were grounded using a ceramic mortar where a dark brown finer material produced. Finally, the material was transferred into a dark glass bottle and preserved for characterization and adsorption experiments.All the chemicals and reagents used in present study were ACS-grade and supplied by Daihan Scientific, Republic of Korea. MgFe21 with some minor changes. At first, 1 g of P123 was measured in a beaker and kept it in a water bath having 35 °C. Then, 30 ml of 0.40 M sodium sulphate (Na2SO4) solution was added and stirred continuously. Subsequently, 31 ml of 0.02 M sodium acetate-acetic acid buffer solution was added under continuous stirring. The mixture was stirred until a homogenous mixture solution formed. Next, 1.48 ml of tetramethyl orthosilicate (TMOS) solution was added to the solution mixture and stirred for 5 min further. The beaker containing solution mixture was kept in an incubator under 35 °C. After 24 h of incubation, the mixture was sealed in a teflon-lined-autoclave and leaved it in the oven for another 24 h at 100 °C. After that, the autoclave was naturally cooled and the mixture was washed with water until it became sodium free. Finally, the white precipitate was air dried and calcined at 550 ˚C temperature for 5 h in a muffle furnace. The white colored material then was cooled in a desiccator and preserved for further analysis.The method for mesoporous silica foam synthesis under present study was followed the method described by Wang et al.3 and MgCl2 were mixed with synthesized MOSF in 70 ml ethanol under sonication. After 30 min of sonication, freshly prepared NaOH solution (10 ml) was added to the mixture drop wise under continuous sonication. After 1 h of sonication, the mixture was sealed in the teflon-lined-autoclave and heated at 200 °C temperature for 8 h in the oven. Next, the naturally cooled mixture was washed with deionized water for several times until the mixture was become sodium chloride (NaCl) free with the help of 2 min continuous sonication and 10 min magnetic stirring. The drying and grinding process of the washed material was same as magnesium ferrite synthesis. The magnesium ferrite nanomaterial having molar ratio of magnesium and iron 0.33:0.67 was expressed as MF0.33 and this was incorporated into MOSF. The end material was used for characterization and adsorption process.The impregnation in this study was very simple and no further chemicals were required. Appropriate amount of anhydrous FeCl2 adsorption–desorption isotherm was recorded from micromeritics. Average pore size, shape, volume and specific surface area were obtained using Barrett-Joyner-Halenda (BJH) and Brunauer–Emmett–Teller (BET) method.Powder X-ray diffraction (XRD) pattern  of the synthesized material was determined for crystal phase identification. The average crystallographic size of synthesized material was calculated from the most intense peaks on XRD spectra using Scherrer’s equation. The elemental composition and morphology was investigated using field emission electron microscopy  functioning with an energy dispersive X-ray (EDS). Chemical composition and bonding nature were proved by Fourier transform-infrared spectroscopy . Magnetic properties of the synthesized materials were analyzed by vibrating sample magnetometer . N0.33-MOSF, nine sets  of As(III) and eight sets  of As(V) concentration were prepared from stock solution (1000 mg/l). 1.0 g/l adsorbent dose was applied. pH values were measured before and after adding adsorbent. Dilute hydrochloric acid (HCl) and NaOH were used to adjust pH 7 after adsorbents addition. The conical flasks containing arsenic solution and adsorbent were kept in a shaking incubator for 12 h. The incubator was operated at 300 ± 5 rpm and temperature was controlled at 25 °C. Next, the mixture solutions were centrifuged for 1 h at 4000 rpm to separate solids. Then, approximately 20 ml clear solution was transferred to plastic tube. To avoid further oxidation, 1 drop of concentrated HCl was added to each solution tube and stored. These solutions were used to determine equilibrium As(III) and As(V) concentration using inductively coupled plasma optical emission spectroscopy (ICP-OES). Adsorption capacity of synthesized MF0.33-MOSF nanomaterial was computed according to the equation given below22:0 (mg/l) is the initial concentration of As(III) and As(V), Ce (mg/l) is the equilibrium concentration of As(III) and As (V), V (l) for volume, m (g) for adsorbent dose and qe (mg/g) for adsorption capacity at equilibrium respectively.To observe the adsorption efficiency of MF0.33 impregnated MOSF, two adsorption isotherm models have been applied named Freundlich  adsorption isotherm model and Langmuir  adsorption isotherm model.To quantify maximum arsenic adsorption capacity and possible arsenic adsorption mechanism by synthesized MFe is the arsenic adsorption capacity by adsorbent at equilibrium (mg/g), Ce is the equilibrium arsenic concentration in water (mg/l), Kf (mg1–1/nl1/ng−1) and n is the Freundlich empirical constants which are related to the maximum arsenic adsorption and the 1/n is the heterogeneity factor of the nanoadsorbent representing the strength of adsorption. The value of n should be lie between 1 and 10 for favorable adsorption process23.The Freundlich adsorption isotherm model can be expressed as follows:L (l/mg) designated as Langmuir adsorption constant, Ce is the equilibrium arsenic concentration (mg/l), qe (mg/g) is the arsenic adsorption capacity at equilibrium and qm (mg/g) represents maximum adsorption capacity of As(III) and As(V) on MF0.33-MOSF. The affinity of arsenic toward binding site is related with KL.The Langmuir adsorption isotherm model can be expressed as follows:PZC) of MF0.33-MOSF nanomaterial was identified through pH Drift method24. In short, based on pH, 10 different sets of vial (pH 3–12) were prepared by adding 15 ml NaCl solution having 0.1 M concentration. pH 3–12 was made by using HCl or NaOH solutions . Subsequently, 0.015 g of MF0.33-MOSF nanomaterial was added to each vial. The vials were stirred 4 h using a magnetic stirrer and kept it in a temperature (25 °C) controlled incubator for 24 h. The final pH of each vials were measured and an initial pH vs pH change (ΔpH) graph was made. The line pH-ΔpH crossed the point of pH zero line is the pHPZC of the material.Point of zero charge ] cubic shape magnesium ferrite spinels which belongs to space group Fd3m . Figure 0.33-MOSF produced 6 peaks on the XRD spectra. The peak at 2θ = 22° confirmed the amorphous silica and 2θ = 31.10 (220), 35.30 (311), 43.43 (400), 56.46 (511) and 62.64 (440) were attributed to the spinel magnesium ferrite nanomaterial, which was appropriately fitted with standard XRD planes for cubic-spinel shaped MgFe2O4 nanomaterial card number ICSD-01-076-9733.The XRD spectra of synthesized MF27.Therefore, based on the comparison results, the new synthesized material contained both amorphous silica phase and magnesium ferrite spinels. These data also confirmed that magnesium ferrite crystals were well established in MOSF structure. Intense peaks of the XRD plane were used to calculate average crystallite size according to Debye–Scherrer’s formulaK (K = 0.89) is considered as Scherer’s constant, λ (Å) represents the applied wavelength of X-ray, β (radian) represents as the full width at half maximum (FWHM) of the diffraction peak and θ represents diffraction angle produced by the peak.According to the Debye–Scherrer’s formula,0.33, MOSF and MF0.33-MOSF respectively. There were no other peaks found on the XRD plane which confirmed that the material synthesized under present research is in single phase state with high purity.The calculated average crystallite size was found 5.85, 0.75 and 1.84 nm for MF0.33, MOSF and MF0.33-MOSF were measured through N2 adsorption–desorption isotherm analysis, as represented in Fig. 2 adsorption–desorption curves were matched with single mode IV which belongs to H1 hysteresis loop. Based on IUPAC classification, this type of hysteresis mode corresponding to the presence of abundant mesoporous pores28. The Barrett-Joyner-Halenda (BJH) pore size distribution pattern revealed average pore diameter of MF0.33, MOSF and MF0.33-MOSF were 4.17, 11.41 and 6.05 nm respectively  total pore volume and specific surface area of mesoporous MF0.33, MOSF and MF0.33-MOSF nanomaterials were 0.2083, 0.8012, 0.4684 cm3/g and 200.36, 412.86, 427.04 m2/g respectively. Owing to impregnation, BET surface area was increased, which indicated that the particles size of MF0.33-MOSF was decreased after impregnation. The average nanoparticle size found by BET analysis  also supported the increment of surface area. The decrease in total pore volume in impregnated material might be also resulted from the impregnated magnesium ferrite in MOSF. Therefore, the MF0.33-MOSF nanomaterial showed exceptionally large surface area, high pore volume and mesoporous pore size that would be an efficient adsorbent for pollutant removal from water.Specific surface area, average pore volume and pore size of MFly Table . MF0.33 0.33, MOSF and MF0.33-MOSF nanomaterials, FTIR analysis was performed and results showed in Fig. 0.33 nanomaterial were appeared at 3416, 1637, 1384, 1034, 590 and 433/cm region were identical to the literature data for magnesium ferrite nanomaterial29. The broader peak appeared at 3416/cm and the sharp peak turned up at 1637/cm region were associated with stretching and bending vibrations of –O–H groups and surface adsorbed water (H2O) by hydrogen bond on MF0.33 surface. The other sharp peaks produced at 1384/cm and 1034/cm region of the spectra were assigned to deformation and bending vibrations of metal hydroxide (M-OH−), which coordinated to Fe3+ or Mg2+. The intense peaks appeared at 590/cm and 433/cm were related to the innate vibrations of octahedral and tetrahedral metal oxides (M–O)29. For MOSF, the peak at 3434/cm was attributed to stretching vibration of –OH on the MOSF surface. The bending vibration of the adsorbed water on the surface absorbed the light 1629/cm. Additionally, the peak at 1091/cm and 810/cm were attributed to asymmetric and symmetric stretching vibrations of Si–O–Si bond. Furthermore, the peak produced at 465/cm was assigned to the structural SiO4 tetrahedra31. The FTIR spectra for MF0.33-MOSF nanomaterial showed almost all the chemical bonds observed in MF0.33 and MOSF. These results also confirmed the successful impregnation of MF0.33 in MOSF.To recognize the bonding types, nature and functional groups of the MF0.33, MOSF and MF0.33-MOSF nanomaterials were observed through SEM analysis  of Mg. But after impregnation of MF0.33 in MOSF by 1:1 ratio, MF0.33-MOSF contain approximately 2.37 times lower Fe and 2.67 times lower Mg than MF0.33. This lower amount of Fe and Mg in MF0.33-MOSF might affect the adsorption capacity of the impregnated material. EDS result also showed that there were no other element presents in the synthesized material, which confirmed phase purity of the nanomaterials. These results confirmed that the phase pure MF0.33 impregnated MOSF nanomaterial had been successfully synthesized in present study.For the evaluation of elemental composition in each nanomaterial, EDS analysis has been done Fig. . The EDS0.33 and MF0.33-MOSF nanomaterials were analyzed by vibrating sample magnetometer (VSM) at room temperature with an application of magnetism of − 20,000 to 20,000 Oersted (Oe)  Table . The mag0.33-MOSF can be attributed to low amount of incorporated MF0.33 in it32. The magnetic properties decreased after impregnation but magnetic properties of the MF0.33-MOSF could be tuned by controlling these two parameters.Magnetization saturation of the synthesized nanomaterials was calculated using the formula:33. Based on BET data, particles size of MF0.33 was found 29.95 nm and after impregnation, particle size was 14.05 nm for MF0.33-MOSF. Therefore, coercivity increased in MF0.33-MOSF than MF0.33. The remnant magnetization (Mr) is the magnetization left after removing external magnetic field from a material. The Mr value was found 1.30 emu/g for pure MF0.33 and 0.14 emu/g for MF0.33 impregnated MOSF. Superparamagnetic MF0.33 showed 9.29 times higher Mr than MF0.33-MOSF. The ratio of Mr and Ms is called squareness which is important properties for ferromagnetic materials. Depending on processing, Mr/Ms ratio of commercial magnets varied in the range 0.88–0.9634. The squareness ratio were found 0.08 and 0.10 for MF0.33 and MF0.33-MOSF. Because of cubic spinel shape, MF0.33 has less Mr/Ms which increased impregnated material.Coercivity depends on particle size which will increase up to certain limit with decreasing particle size35 found similar magnetization result when synthesized silver phosphate@magnesium ferrite [Ag3PO4@MgFe2O4] nanocomposites. The magnetization of pure MgFe2O4 material was found 15 emu/g which was decreased to 1.6 emu/g in Ag3PO4@MgFe2O4 (10%). They showed that non-magnetic Ag3PO4 attained good magnetic properties after composite formation with superparamagnetic MgFe2O4 that can be separated easily from water by a magnet35. Khafagy32 showed that magnetization of pure phase MgFe2O4 decreased (21.33–5.905 emu/g) when coating MgFe2O4 with polyaniline. They also found that coercivity decreased 88.66 Oe–81.60 Oe after coating because the particle size of pure MgFe2O4 (30–35 nm) increased (45 nm) after coating. Hoijang et al.17 synthesized silica-coated MgFe2O4 showing superparamagnetic properties. The magnetization of pure MgFe2O4 and silica-coated MgFe2O4 were found 37 and 27 emu/g. During synthesis, they added only 1 ml tetraethyl orthosilicate in a solution containing 200 mg of MgFe2O4. Because of higher amount of MgFe2O4, the magnetic properties was not decreased much. Therefore, magnetic properties of the nanomaterials synthesized in present study could be increased by changing MF0.33 and MOSF ratio.Zhou et al.36, synthesized silica@magnesium ferrite [SiO2@MgFe2O4] material having higher surface area and pore volume than MF0.33-MOSF synthesized in this research. But the pore size of SiO2@MgFe2O4 material approximately 1.71 times lower than MF0.33-MOSF. In addition, complex synthesis process was involved in SiO2@MgFe2O4 synthesis. At first they synthesized SiO2 and MgFe2O4 nanomaterial. After that further synthesis process was involved for SiO2@MgFe2O4 synthesis36. Whereas, the synthesis of MF0.33-MOSF material was very much simple in present study. At first MOSF synthesized and then this MOSF mixed with other precursor solutions during MF0.33 synthesis. Therefore, no further synthesis steps were involved, which could save energy and cost of the research. So, present research successfully synthesized MF0.33-MOSF nanomaterial through a simple and cost effective way having large pore size, pore volume and surface area compared to other nanomaterials found in literature  data. According to Foo and Hameed37, Langmuir adsorption isotherm is an equation based on monolayer adsorption of adsorbate onto adsorbent surface. Besides, Freundlich adsorption isotherm model is based on multilayer adsorption of adsorbate onto heterogeneous surface of the adsorbent which is not restricted to monolayer formation38. Therefore, multilayer adsorption of both arsenic species was taken place on MF0.33 and MF0.33-MOSF heterogeneous surface. The heterogeneity factor, 1/n can be found from Freundlich model. The value 0.1 ˂ 1/n ˂ 1 describes efficient adsorption of adsorbate on adsorbent surface39. In present study, the parameter 1/n calculated from Freundlich model were found 0.34, 0.32 by MF0.33 and 0.40, 0.63 by MF0.33-MOSF for As(III) and As(V) adsorption, which indicated that both species adsorbed easily on MF0.33 and MF0.33-MOSF surface40.Table del Fig. . The fit0.33 and MF0.33-MOSF nanomaterial was found in Langmuir isotherm model. Based on model results, MF0.33 and MF0.33-MOSF adsorbed 103.94, 42.80 mg/g of As(III) and 45.52, 39.73 mg/g of As(V) at pH 7 respectively. With increasing initial arsenic concentration, adsorption capacity was also increased. At equilibrium, MF0.33 nanomaterials adsorbed approximately 2.4 times higher amount of As(III) than MF0.33-MOSF. For As(V) adsorption, MF0.33 adsorbed slightly higher amount than MF0.33-MOSF. As the MOSF showed no arsenic adsorption capacity, MF0.33 was totally responsible for arsenic adsorption. Besides that, based on EDS results, MF0.33-MOSF contained 2.37 times lower Fe and 2.67 times lower Mg than MF0.33 when MF0.33-MOSF was synthesized in 1:1 ratio of MF0.33 and MOSF. Elemental analysis data by ICP-OES also showed that MF0.33-MOSF contain 2.47 times lower Fe and 2.42 times lower Mg than pure MF0.33. Therefore, the lower arsenic adsorption capacity was due to lower Fe and Mg content in MF0.33-MOSF nanomaterial, which could be enhanced by changing the MF0.33 and MOSF ratio.The maximum arsenic adsorption capacity of MF0.33-MOSF nanomaterial can be discussed based on isotherm data and pHPZC of the material. Adsorption results were well fitted to Freundlich isotherm model indicating that arsenic adsorption occurred on heterogeneous surface of MF0.33-MOSF nanomaterial. Therefore, possible mechanism of arsenic adsorption might be through physisorption and chemisorption. In addition, the pHPZC of MF0.33-MOSF was found 9.02 which indicated that the nanomaterial surface was positively charged at pH 7 (pH ˂ pHPZC). The available forms of As(III) and As(V) under pH 7 are H3AsO3, H2AsO4- and HAsO42- which might be easily adsorbed on MF0.33-MOSF surface through electrostatic attraction, ion exchange and complex formation. There were two pHPZC (3.31 and 4.78) found for pure MOSF material  at very lower pH. In addition, after coating with magnetite the pHPZC of modified mesoporous silica changed to 8 which generated positive silica surface43.Arsenic adsorption on MF0.33 had pHPZC 10.25 which indicated that MF0.33 surface showed higher amount of positive sites at pH 7 (pH ˂ pHPZC) which favors arsenic adsorption [(103.94 and 45.52 mg/g for As(III) and As(V)]. After the impregnation of MF0.33 in MOSF, pHPZC was changed to 9.02 and adsorption capacity was 42.80 and 39.73 mg/g for As(III) and As(V) respectively. The EDS and elemental analysis data confirmed that MF0.33-MOSF contain less Fe and Mg than MF0.33, which was the main reason responsible for the lower arsenic adsorption capacity. At pH 7, Arsenous acid (H3AsO3) is the available form of As(III) and H2AsO4- and HAsO42− are two available forms of As(V) in aqueous solution. So, the MF0.33 and MF0.33-MOSF nanomaterials having positively charged surface might be easily attached the negatively charged As(V) through complex formation, ion exchange and electrostatic attraction. The possible reactions are:The adsorption experiments under present research were done at pH 7. At this pH, surface of pure MOSF can be regarded negative charged. As result, adsorption capacity of pure MOSF was very much low [(0.58 and 2.49 mg/g for As(III) and As(V) at 1–25 mg/l initial concentrations]. Conversely, pure MF3AsO3 species of As(III) has no charge which can adsorbed through complex formation. The possible adsorption reactions of As(III) are:H2AsO4- and HAsO42- adsorption through chemisorptionPossible reactions for H46.Considering above reactions, chemisorption was the dominant adsorption mechanism for As(III) and As(V) adsorption. Chemisorption process occurred through complex formation, ion exchange and electrostatic attraction. The literature data regarding arsenic oxyanions adsorption on spinal ferrites revealed that the dominant adsorption mechanism was surface complex formationFor the first time, magnesium ferrite was impregnated in MOSF which showed enhanced surface area, mesoporous pore size and high pore volume. The material was applied as adsorbent for arsenic removal at pH 7 and 25 °C. The maximum As(III) and As(V) adsorption capacity were found 42.80 and 39.73 mg/g respectively. The material adsorbed higher amount of As(III) and As(V) from water compared to other adsorbents listed in Table 47. In addition, longer equilibrium time and membrane filtration with N2 purging applied for adsorption which limited the research for further application in water treatment. Gupta and Ghosh48 synthesized Fe(III)-Ti(IV) binary oxide which removed 85 mg/g As(III) within 4.5 h at pH 7. However, the material removed very much lower amount (14 mg/g) of As(V) within 7.5 h at pH 7. In addition, they maintained pH for certain time period until equilibrium reached during adsorption. Furthermore, membrane filtration was applied for separation of adsorbent from water48. Fe3O4-MnO2 binary oxide adsorbed 32.13 mg/g As(V) at pH 549. Yu et al.6 synthesized cellulose@Fe2O3 magnetic composites and applied for arsenic removal. The nanocomposites adsorbed 23.16 mg/g As(III) at pH 7.5 and 32.11 mg/g As(V) at pH 2. 3D organized mesoporous silica coated with Fe and Al oxide was synthesized by Glocheux et al.50 which adsorbed 55 mg/g As(V) at pH 5 and 35 mg/g As(V) at pH 4 respectively. Fe2O3/SiO2 nanocomposite adsorbed only 21.50 mg/g As(III) at pH 7.5 and 14.90 mg/g As(V) at pH 4.5. In both cases, adsorption temperature was 35 °C51. Ahangari et al.52 synthesized nickel-zinc ferrite modified carbon nanotubes nanocomposite for As(V) removal from wastewater. The nickel-zinc ferrite (NZF) and carbon nanotube nickel-zinc ferrite (CNZF) nanocomposites adsorbed 56 and 66 mg/g As(V) at pH 2 and 6 g/l of adsorbent dosage52. Therefore, comparing with activated carbon, carbon nanotube, cellulose, binary oxides and other silica modified nanomaterial/nanocomposites, MF0.33-MOSF found efficient adsorbent material for As(III) and As(V) removal from water.Fe modified activated carbon material showed lower As(III) and As(V) adsorption capacity0.33 synthesis process where MOSF added with precursor materials of MF0.33 therefore, no further synthesis process had been applied. For synthesis of other modified materials  and As(V) removal from water at pH 7. MOSF having negatively charged surface area at pH 7 showed repulsion against arsenic oxyanions. However, adsorption properties were increased after impregnation with MF0.33. The magnetic MF0.33-MOSF showed better morphological properties and adsorption capacity (at pH 7) compared to other nanomaterials found in literature. Adsorption results best fitted with Freundlich isotherm and adsorption mechanism was found to chemical sorption through complex formation and electrostatic attraction on heterogeneous surface. Modification of MF0.33 with MOSF using simple, less time-consuming and cost-effective procedure was successfully confirmed to be effective. Therefore, magnetic MF0.33-MOSF composites could be competitive nanoadsorbents for arsenic removal from water.Iron based nanomaterials have drawn much attention in adsorptive removal of pollutants from water because of their availability, properties, adsorption capacity, lower cost and ecofriendly nature. Among them, magnesium ferrite nanomaterials showed greater properties and performance in water treatment process. However, being a nanomaterial, it has limitations such as stability, aggregate formation and separation. Therefore, modification of magnesium ferrite nanomaterials with well-structured materials having higher stability, surface area and easy separation from solution are very much popular in recent time. Following that, a new combination, MFSupplementary Tables.


Introduction: Osteoarthritis (OA) is a prevalent joint disorder worldwide. Sodium hyaluronate (SH) and mesenchymal stem cells (MSCs) are promising therapeutic strategies for OA. Previous studies showed they could improve knee function and clinical symptoms of OA. However, the mechanism of the therapeutic effects on the improvement of OA has not been clearly explained.Methods: In our study, we used a technique called 5-(diisopropylamino)amylamine derivatization liquid chromatography coupled with mass spectrometry to find the metabolites in OA synovial fluid under different treatments.Results and Discussion: After looking into the metabolomics, we discovered that SH and MSC treatment led to the downregulation of ω-6 polyunsaturated fatty acids (PUFAs) and the upregulation of ω-3 PUFAs. Significantly, the contents of 5(S)-HETE, PGA2, PGB2, and PGJ2 were lower in the MSC group than in the SH group after quantification using 5-(diisopropylamino)amylamine derivatization–UHPLC–QQQ-MS. This is the first report on the relationship of 11(S)-HETE, PGA2, PGB2, PGF2β, 11β-PGF2α, and DK-PGE2 with OA. Moreover, the correlation analysis of metabolites and inflammation factors showed the positive association of ω-6 PUFAs with pro-inflammation cytokines, and of ω-3 PUFAs with anti-inflammation cytokines. Our results indicated the therapeutic effect of SH and MSCs in patients with OA. In addition, this reliable metabolic approach could uncover novel biomarkers to treat OA. Osteoarthritis (OA) is one of the most prevalent joint disorders worldwide . OA patiHere, metabolomics was applied to elucidate the mechanism. Carboxylic acids were reported to be related to OA . They plScientists used different methods to find carboxylic acids in different biological samples, such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) . HoweverThus, we used this technique to profile carboxylic acids in the synovial fluid from healthy and OA guinea pigs treated with MSCs or SH, or nothing. The potential biomarkers could be determined through various statistical analyses. Then, DIAAA derivatization–UHPLC–QQQ-MS was applied to quantify these biomarkers. Moreover, the relationships of metabolites with inflammation factors were also elucidated. The results would help to explain the mechanism of the therapeutic effect of the drugs on OA and help diagnose and provide a prognosis for OA.H-1,2,3-triazolopyridinium 3-oxid hexafluorophosphate (HATU), DIAAA, 1-hydroxybenzotriazole hydrate (HOBt), and triethylamine (TEA)) were bought from Sigma-Aldrich Laboratories Inc. . Acetonitrile (HPLC grade) was obtained from Anaqua Chemicals Supply Inc., Ltd. . A Millipore water purification system  was applied to prepare deionized water. Sigma-Aldrich Laboratories Inc. provided other chemical reagents and formic acid (MS grade).We purchased all polyunsaturated fatty acid (PUFA) standards from Cayman Chemical . The chemicals (1-[bis(dimethylamino)methylene]-1The stock solutions of the PUFA mixture were obtained by dissolving the corresponding standards in 60% ACN (MS grade) at 1 μg/mL concentration. In addition, 12(S)-HETE, 5(S)-HETE, and 12(S)-HHTrE were made at different concentrations to create a wider range of linearity. Different concentrations of working solutions  were use6/T75 vials, replenishing the medium to reach the volume of 15 mL/vial. The incubation conditions of the cells were 37°C with 5% CO2. The medium was changed after 48 h of incubation and then changed every 3 days. When the cells’ confluence reached 80–90%, they were absorbed and passaged with 0.125% pancreatic enzyme. Injection therapy was performed using P5 generation cells.The adipose tissue was initially washed with 0.9% normal saline (NS). Then, the solution of type I collagenase (0.75 mg/mL) was added, and it was digested for 30 min in a thermostatic shaker at 37°C. It was isolated after centrifugation for 10 min at 900 g, and the remaining pellet, which is the stromal vascular fraction (SVF), was retrieved. The SVF was washed with 0.9% NS and mixed well. After centrifugation , the supernatant was abandoned, the residue was re-suspended in 0.9% NS, and the suspension of the cell was filtered with a cell filter (100 μm). Then, the cell was counted and distributed evenly into two centrifuge tubes. The next step was the primary inoculation of MSCs. In brief, they were inoculated in 2 × 107, generation P5). Group C (N = 7) was injected with 100 μL SH. Then, synovial fluid samples were collected after 3 weeks. Then, the hair around the left knee joint of guinea pigs was scraped and the skin was disinfected with medical alcohol. The guinea pigs were fixed on the operating table with adhesive tape, and 100 μL of the synovial fluid was extracted from their knee by percutaneous puncture with a microsyringe (Hamilton). The samples were kept at −80°C before use.The protocol of the experiment was authorized by the Macau University of Science and Technology ethical committee. Guinea pigs  were recruited for the study. After being fed to get adapted to the environment for 1 week, these guinea pigs were randomly divided into four groups, which included the control (N = 4), model (N = 8), SH (N = 7), and MSC (N = 7) groups. For the model, SH, and MSC groups, partial meniscus resection was performed on the left knee joint. After modeling, the guinea pigs were fed for 2 weeks, and injection into the knee cavity was performed by percutaneous puncture. Group A (N = 8) was injected with normal saline (100 μL) into the joint cavity. Group B (N = 7) was injected with 100 μL adipose mesenchymal stem cell suspension , TNF-γ, interleukin 1β (IL-1β), IL-1, IL-4, matrix metalloproteinase 1 (MMP1), MMP13, ADAMTS-4, and ADAMTS-5 in the synovial fluid of guinea pigs.In all, 50 µL synovial fluid samples were extracted with 200 µL cold methanol, and then, they were centrifuged at 13,500 rpm for 5 min at 4°C. The residues were extracted twice more, and then, the supernatants were combined and dried with a nitrogen stream. The residues were kept at −20°C before DIAAA derivatization.The derivatization was accomplished according to our previously published literature . In brie−1. Mobile phase A was 0.1% formic acid-containing water, and mobile phase B was 0.1% formic acid-containing acetonitrile. The gradient was 0–0.5 min, 2%–5% B; 0.5–2.5 min, 5%–6% B; 2.5–3.5 min, 6%–7% B; 3.5–4.3 min, 7%–7.3% B; 4.3–7.3 min, 7.3%–7.8% B; 7.3–10 min, 7.8%–9% B; 10–12 min, 9%–14% B; 12–17 min, 14%–23% B; 17–18 min, 23%–26% B; 18–20 min, 26%–47% B; 20–23 min, 47%–85%; 23–24 min, 85%–94% B; 24–27.9 min, 95% B; and 28 min, 2% B.An Agilent 1,290 Infinity LC system  was employed for this study. A Waters ACQUITY UPLC HSS T3 column  was applied to separate the metabolites. The temperature of the column was kept at 40°C, and the autosampler was maintained at 4°C. The flow rate was set as 0.3 mL min−1, the dry gas temperature at 250°C, sheath gas flow at 11 L min−1, sheath gas temperature at 300°C, nebulizer pressure at 20 psig, nozzle voltage at 500 V, and capillary voltage at 5000 V. The mass spectra range was m/z 100–1,000. They used a low-frequency TOF reference mixture [containing the internal reference masses at m/z 922.0098 (C18H18F24N3O6P3)] for accurate mass measurements. Automated and targeted MS/MS were elucidated for MS/MS acquisition, and the collision cell energy (CE) was set at 30 eV.An Agilent 6550 UHD accurate-mass Q-TOF/MS system was used for mass integration. The positive (POS) ion mode was applied for analysis. These were the MS parameters used: the dry gas flow at 15 L minThe metabolites were separated by using an Agilent 1,290 Infinity LC system , which consisted of a thermostatted column compartment, an autosampler, and a binary pump. The column was Waters ACQUITY UPLC HSS T3 . The autosampler was set at 4°C. For the quantification of PUFAs, the gradient was established with the modification to shorten the analytical time .MassHunter Quantitative Analysis B.06.00  was applied to analyze the results. The MRM mode was recruited to obtain the MS/MS data. The parameters of mass were dry gas temp 250°C, dry gas flow 13 L/min; nebulizer 25 psi, sheath gas temp 275°C, sheath gas flow 11 L/min, capillary 3500 V, and nozzle voltage 500 V. The software Optimizer  was applied to optimize the specific transitions of MRM and the corresponding CE of the targeted PUFAs. The optimal parameters of MRM for each metabolite are shown in https://metlin.scripps.edu/index.php) and LIPID MAPS (http://www.lipidmaps.org/), or by analyzing the MS/MS spectra. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and biplot were recruited to evaluate the correlation of metabolites among different groups by using SIMCA-P software . The volcano plot was processed using Instant Clue (https://github.com/CJ-Chen/TBtools/releases) was used to perform the heatmap. MetaboAnalyst 5.0 was applied to construct pathway enrichment analysis. MassHunter Quantitative Analysis software was used to integrate the areas of peaks, which were measured through UHPLC/QQQ-MS. The discrimination between two different groups was calculated by using GraphPad Prism 9.0. The difference with p < 0.05 was significant . Fold change was calculated by comparison of the SH group or MSC group with the OA guinea pigs (model) group, and the healthy (control) group with the model group. Fold change with a positive value shows a relatively higher concentration in the SH group, MSC group, or control group, while a negative value indicates a relatively lower concentration compared to the control group.MassHunter software (Agilent Technologies) was used to extract and align raw data for analysis. The metabolites were confirmed by comparing their MS/MS spectra with the standards from METLIN  classification is important to assess lesion stability. Magnetic resonance imaging (MRI) is widely used to appraise OA lesions. The appearance of guinea pigs’ cartilage plateau was evaluated using ICRS and MRI . For theConsidering that the development of OA involved immune cells, which are the hot spot issue in recent years, the contents of inflammation factors, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), transforming growth factor β (TGF-β), TNF-γ, IL-1, IL-4, matrix metalloproteinase 1 (MMP1), MMP13, ADAMTS-4, and ADAMTS-5, were detected by ELISA in the synovial fluid of each group. The results showed that these inflammation factors had significant differences comparing the model group with that in the control, MSC, and SH groups . Especiap < 0.05), and the levels of 87 metabolites decreased in the SH groups, whereas others increased compared to those in the model group. Furthermore, the differential metabolites were analyzed between model and control groups, and 42 metabolites that changed in the same way in the control and SH groups were found  and explained with SIMCA-P software . We found significantly different metabolic profiles after comparing the control group levels with model and SH group levels using OPLS-DA . Furtherre found , suggestre found .To further examine the overall trends, a biplot was used to identify which metabolites contributed the most to the variance in the control, model, and SH groups. The correlation plot  manifestp < 0.05). A total of 34 metabolites  of the analytes were between 0.0007 and 0.54 ng/mL. The sensitivity was significantly increased up to 149-fold using DIAAA derivatization–UHPLC–QQQ-MS analysis compared to the literature -HETE, 12(S)-HETE, and 12(S)-HHTrE, ranged from 0.034 to 24.49 ng/mL . In addiTo further study the influence of the metabolism on the changes of inflammatory patterns in OA, the relationships of the abovementioned metabolites with inflammation factors in OA models, as well as in the control, SH, and MSC groups, were analyzed .2, PGB2, PGJ2, PGE2, and PGD2, and a negative association with ω-3 PUFAs. However, TGF-β and IL-4 were inversely associated with ω-6 PUFAs and positively related to ω-3 PUFAs, e.g., 5(S)-, 12(S)-, and 15(S)-HEPEs.A previous report showed that IL-1β and MMP13 could increase prostaglandin production in the synovium after the damage to the articular cartilage . InteresOA is a degenerative cartilage disease, and inflammation is believed to be an important factor in the development of OA . It was SH injection is a traditional treatment that can lessen pain, improve joint function, and enhance rheological properties . In addiA carboxylic acid metabolomics study was performed on the synovial fluid samples from different groups using DIAAA derivatization–UHPLC–Q-TOF/MS. Metabolomics and multivariate analyses revealed that arachidonic acid metabolism showed significant differences in the SH or MSC groups compared with that in the model group. Especially, the levels of ω-6 PUFAs, including 5(S)-, 11(S)-, 12(S)-, and 15(S)-HETEs, PGA2, PGB2, PGD2, PGE2, PGE1, DK-PGE2, PGF2α, 11β-PGF2α, and PGJ2, decreased in the synovial fluid after SH or MSC treatment. However, the trends of ω-3 PUFAs, especially 5(S)-, 12(S)-, and 15(S)-HEPEs, EPA, and DHA, increased after SH or MSC treatment in comparison with those in the model group. The levels of prostaglandins are higher in those with rheumatoid arthritis (RA) or OA . AdditioIn order to further study the therapeutic effect of SH or MSC on OA, the targeted PUFAs were quantified using the method called DIAAA derivatization–UHPLC–QQQ-MS. Finally, we found that the contents of ω-6 PUFAs, including 5(S)-, 11(S)-, 12(S)-, and 15(S)-HETEs, PGA2, PGB2, PGD2, PGE2, PGE1, DK-PGE2, PGF2α, 11β-PGF2α, and PGJ2, decreased almost 1.7–3.8-folds and they had significant differences between the model and SH groups, as well as between the model and MSC groups. In addition, the contents of 5(S)-HETE, PGA2, PGB2, and PGJ2 were lower in the MSC group compared with those in the SH group. Especially, 11(S)-HETE and 15(S)-HETE could not be detected with MSC treatment. It was well known that 15-HETE, which was metabolized from arachidonic acid through 15-LOX, showed high expression under the actions of 15-LOX in chondrocytes . 12(S)-HAs we know, OA synovial fluid contains different kinds of metabolites and inflammatory factors that affect cartilage injury and inflammation . StatistAlthough our manuscript is helpful in confirming the therapeutic effect of SH or MSC in patients with OA, there are also several limitations to our study. First, because of the lack of available clinical data, we could not further validate our results. Second, we only studied guinea pigs aged 3–4 months and created the OA injury model ourselves, without considering other factors such as natural aging. Additional studies are needed to test other conditions. In addition, a proper normalization strategy could be considered in our future work. Due to the low volume of the synovial fluid and the instability of ALOX15, COX-1, and COX-2, it is hard to detect them. We will enrich the volume and detect them immediately when we obtain the samples in future. The other potential limitation is that we investigated only male guinea pigs to reduce other influences . In future, we may study female guinea pigs as well. Finally, we should expand the sample size to further confirm our results.In our study, PUFAs had significant differences among different groups after metabolomics analysis. Especially, patients who received SH or MSC treatment showed decreased levels of pro-inflammatory ω-6 PUFAs and increased levels of anti-inflammatory ω-3 PUFAs. In addition, 11(S)-HETE, PGA2, PGB2, PGF2β, 11β-PGF2α, and DK-PGE2 were identified as OA-related for the first time. Our results showed that the mechanism of SH and MSC treatment could be elucidated through the differential changing levels of PUFAs. In other words, this reliable metabolic approach could uncover novel metabolites, which could help confirm the therapeutic effect of SH or MSC in patients with OA.


FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4β1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development.Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of  Given the large overlap in growth and guidance of the vasculature and neurons during development (We previously generated a loss-of-function mutant for the human TDP-43 gene (tardbpl) . Homozygous gene . In contous gene . The mutous gene , which pelopment , these cFN1 as a direct RNA target of TDP-43 in HUVEC by performing iCLIP experiments.We therefore aimed at identifying the molecular pathways by which TDP-43 regulates angiogenic sprouting. First, we characterized a vascular mis-patterning phenotype upon loss of TDP-43 in zebrafish and human derived endothelial cells (ECs). Second, we identified de-regulated genes mediating the vascular TDP-43 deficient phenotype by conducting next-generation RNA sequencing (NGS) in TDP-43 knockdown (KD) HUVEC. Third, we rescue the zebrafish phenotypes by reducing the levels of Vcam1, Fn, and Itgα4 in TDP-43 knockout zebrafish. Lastly, we identified Danio rerio) were kept at 28.5°C and were staged according to Zebrafish embryos  and subcloned to generate the plasmid pCS2+GW-A + sflt1-UTR for antisense probe generation.Whole mount escribed . The in hrin B2a , flt4 . Incubation with primary antibodies was conducted at 4°C ON. On the next day, samples were rinsed with PBST and washed four times for 30 min in PBST at RT. Then they were blocked two times for 30 min in NCST. Secondary antibodies were used at a dilution of 1:500 in NCST and incubated at 4°C ON. The secondary antibodies were removed and the samples rinsed once with PBST followed by at least three washing steps for 15 min in PBST. The stained embryos were kept at 4°C in PBST or PBS until imaging with the spinning disk or confocal microscopes .Embryos were fixed overnight (ON) at 4°C for 4 h at room temperature (RT). Next, they were rinsed 1x with PBS and washed three times for 5 min with PBST (PBS-Tween) at RT. For co-staining of 24 hpf Tg (fli1:EGFP)Philomath, OR, United States). Approximately 2 nL of MO were microinjected into fertilized zebrafish eggs at the 1 cell stage into the yolk.All morpholinos (MO) were purchased from Gene Tools , WB: 1:10,000AKT, 9272 , WB: 1:1,000Alexa Fluor 405 anti-mouse, A-31553 , IF 1:500Alexa Fluor 405 anti-rabbit, A-31556 , IF 1:500Alexa Fluor 488 anti-mouse, A-11029 , IF 1:500Alexa Fluor 488 anti-rabbit, A-11034 , IF 1:500Alexa Fluor 488-conjugated Isolectin-B4, I21411 , IF: 1:300Alexa Fluor 568 anti-rabbit, A-11011 , IF 1:500Alexa Fluor 622 anti-rabbit, A-21070 , IF 1:500anti-rabbit-HRP, W401B , WB 1: 10,000anti-mouse-HRP, W402B , WB 1:5,000α-dystroglycan, NCL-b-DG , IF: 1:50Calnexin, SPA-860 , WB: 1:10,000ERK1/2, 9102 , WB: 1:2000Fibronectin, HPA027066 , WB: 1:500GFP, 632375 , IF: 1:500GFP, 632592 , IF: 1:500Integrin α4, 8440 , WB: 1:1,000Integrin β1, 610467 , WB: 1:2000Tardbp, 4A12-111 , WB: 1:1 TDP-43 N-term, SAB4200006 , WB: 1:10,000–1:50,000p38, 9212 , WB: 1:1,000pAKT, 9271 , WB: 1:500pERK1/2, 9101 , WB: 1:2000, Dallas, TX, United States), WB: 1:200PLCG1, sc-426 , WB: 1:1,000pPLCG1, SAB4300082 , WB: 1:500pPI3K, 4228 , WB: 1:1,000pVEGFR2, 2478 , WB: 1:1,000VEGFR2, 9698 , WB: 1:1,000TARDBP determined by paired t-test analysis (n = 3).Zebrafish embryos were lysed in 10–15 μL Laemmli buffer per embryo, homogenized, and heated at 95°C for 5 min. Lysates were centrifuged at 13,000 rpm and supernatant was loaded on a 12% SDS-PAGE gels (7% gels for VEGFR2 and PLCG1) and transferred to a PVDF membrane  for 70 min at 400 mA. Membranes were blocked in 3% milk powder , membranes for phosphorylated epitopes were blocked in 0.2% I-Block , and membranes for FN1 were blocked in 5% BSA  for 60 min at RT. Incubation of primary antibody was performed ON at 4°C followed by 4 PBST washes and incubation in secondary antibody for 1 h at RT. After 6 washes with PBST membranes were developed with ECL Plus . The intensity of the bands was quantified using ImageJ. Each protein of interest was normalized to calnexin and significant changes between shCtr and shtardbp−/−; tardbpl−/− embryos from their wild type siblings at this stage, tardbp−/−;Tardbpl KD and their control MO injected tardbp −/− siblings were used for testing the rescue potential of the applied chemical inhibitors. The VEGFR2 inhibitor DMH4  was applied at the 16- to 17-somite stage in different concentrations: 0.1, 0.5, and 2.5 μM. 1% DMSO served as a negative control. Embryos were kept till they reached the required developmental stage that was determined by status of ISA growth according to  or TRIzol  according to the manufacturer’s protocol and reverse transcribed with random hexanucleotide primers using the M-MLV Reverse Transcription kit (Invitrogen) with RiboLock RNase inhibitor . The complementary DNA was amplified using qPCR SsoFast Evagreen Supermix  and analyzed in the C1000 Thermal Cycler (Bio-Rad). Each reaction was conducted as technical triplicates with at least 3 biological replicates. The relative expression of each gene was calculated using the ΔΔCT-method and the normalized fold expression was calculated by normalization to the reference genes The following primers were used for qPCR.2 respectively. The cells were subsequently washed twice with ice cold PBS and further processed with RIPA with 1x phosphatase and protease inhibitor for SDS-PAGE or TRIZOL for RNA analysis.HUVEC were purchased from PromoCell  and cultured in 50% Endothelial Cell Basal Medium  and 50% Medium 199  with 20% FCS. VEGF-A stimulation was performed 5 days post transduction with 25 ng/μL VEGF-A for 0, 5, 15, or 30 min at 37°C and 5% COTARDBP#1, or shTARDBP#2 on the following day with 25 μL virus mix (described below) per 10 mL culture volume in a 10 cm dish.For virus transduction, cells were grown to 80% confluence and detached with Trypsin/EDTA. They were seeded and transduced with virus containing either the sequence coding for shCtr, shin vitro Angiogenesis)”.The Tube formation assay was performed on the artificial extracellular matrix Geltrex  according to the manufacturer’s protocol “Endothelial Tube formation assay  and with the construct of interest. Before transfection, medium was replaced by 5 mL pre-warmed Optimem  with 10% FCS. Then, per three 10 cm dishes to be transfected, 108 μL Lipofectamin 2000  was mixed with 4,500 μL Optimem (L2K) and incubated for 5 min. The transfection mix contains 18.6 μg of the construct of interest, 11.0 μg pSPAX2, and 6.4 μg pVSVg with 4,500 μL Optimem. This mix was combined with the L2K and incubated for 20 min at RT. Finally, 3 mL per mix was added to one 10 cm dish. Next day, media were replaced by 10 mL high packaging media . On the second day after transfection, the supernatant of three 10 cm dishes containing virus with the same sequence of interest was pooled in 50 mL tubes and centrifuged for 5 min at 2000 rpm to remove debris. The supernatant was filtered through a 0.45 μm PES membrane filter  into 28SW ultracentrifuge tubes. The virus particles were ultracentrifuged for 2.5 h at 22,000 rpm. Supernatant was discarded and the virus particles resuspended over night at −4°C in 120 μL NB medium .rsem-prepare-reference from RSEM software. Next, rsem-calculate-expression was used to align the reads and quantify the gene abundance. Differential expression analysis was carried out using gene read counts with DESeq2 package (v1.12.4). Genes with less than 5 reads (baseMean) were filtered out. Genes with an adjusted p-value ≤0.01 and log2 fold change > |0.5| were considered to be differentially expressed.RNA-sequencing resulted in ∼25–31 million reads . Qualityp-value ≤0.01 using the Benjamini–Hochberg method for controlling the false discovery rate was set as significant for GO terms in biological processes. EnrichmentMap (v2.1.0) plugin was used in Cytoscape (v3.4.0) for enrichment visualization of top 20 terms (Gene ontology (GO) analysis was conducted using WebGestalt. An adjusted 20 terms . The ove2 in a Stratlinker 2,400 at 254 nm. TDP-43 was immunoprecipitated with protein A Dynabeads  conjugated to rabbit-anti TDP-43 . The region corresponding to 55–100 kDa complexes was excised from the membrane to isolate the RNA. High-throughput sequencing using Illumina HiSeq was done. Analysis of reproducibility of crosslink sites, identification of the significant iCLIP crosslink clusters and z-score analysis of enriched pentamers was done as described previously (http://icount.biolab.si).The iCLIP protocol was performed as described previously , with theviously  and datatardbp−/−; tardbpl−/− double homozygous loss-of-function zebrafish mutants uncovered hyperbranching and mis-patterning of EC during development  can fully phenocopy the tardbp−/−; tardbpl−/− mutant EC phenotype demonstrating the specificity and efficiency of the morpholino (MO) (We next established a TDP-43 KD model for the subsequent experiments to obtain higher numbers of phenotypic embryos compared to the 25% of double homozygous mutant embryos obtained from a ino (MO) .out of bounds (obd) to the tardbp−/−; tardbpl−/− mutant phenotype indicated a possible impairment of this signaling pathway in tardbp−/−; tardbpl−/− EC (flt1 (fms-related tyrosine kinase), is alternatively spliced into a soluble decoy receptor, sflt1, and a membrane bound version, mflt1. In obd mutants, the ratio of the soluble sflt1 and membrane bound mflt1 is shifted towards increased expression of the mflt1 splice variant, leading to an increased availability of Vegf for its pro-angiogenic receptor Kdrl . Furthe embryos , demonsttardbp−/−; tardbpl−/− mutants. If increased Kdrl signaling is indeed responsible for the sprouting phenotype, then slight reduction of Kdrl signaling with low concentrations of VEGFR2 inhibitor should be able to rescue the phenotype. To test this hypothesis, the selective VEGFR2 inhibitor DMH4 was used to slightly reduce Kdrl signaling to intermediate levels and assayed for rescue of the mutant phenotype s896 recipients expressing mCherry in the endothelium does neither alter donor derived nor recipient EC morphology, showing that the transplantation procedure per se does not affect EC morphology y1 or tardbp−/−; tardbpl−/− Tg (fli:GFP)y1 embryos are used as donors, the transplanted cells differentiating into EC within the Tg (kdrl:mCherry)s896 recipient embryos show the mutant phenotype with supernumerous sprouts at ectopic positions TARDBP targeting shRNAs, shTARDBP#1 and #2  as an unbiased approach to identify differentially expressed genes upon KD of TDP-43 in HUVECs. We identified 2,129 genes with differential expression and adjusted eads see . Pathwayeads see . Strikin.65 fold . FN1 can.65 fold , howeverll types  and therITGA4, ITGB1, FN1, and VCAM1 leads to increased protein expression. Protein expression of ITGA4 and FN1 is strongly increased upon TDP-43 KD compared to controls . We next asked if TDP-43 protein binds to FN1, ITGA4B1or VCAM1 RNA in HUVEC and thereby increases their expression upon TDP-43 KD. Consistent with previous reports we identified TDP-43 binding in coding and non-coding RNAs, introns, exons and UTRs  have been identified to be highly upregulated by RNA sequencing in HUVEC upon TDP-43 KD (Misregulation of FN1 in motor neurons from sporadic ALS patients has been previously reported and might contribute directly to toxicity by altering the ECM-interaction of motor neurons and thereby affecting their vulnerability . AlternaDP-43 KD , which mIn summary, our study identified FN1/VCAM1-ITGA4B1 as a novel, important and evolutionary conserved molecular pathway regulated by TDP-43 with a potential mechanistic link to the pathophysiology of ALS.


TZQ) 1-methyl)-3-phenylquinoxalin-2(1H)-one (4) is reported. The structure has been identified with a variety of spectroscopic methods  and finally, the structure has been determined by X-ray diffraction (XRD) studies. The TZQ molecule has crystallized in the monoclinic space C2/c group with unit cell dimensions a = 41.201(2) Å, b = 10.6339(6) Å, c = 9.4997(4) Å, β = 93.904(4). The crystal structure is stabilized by intermolecular interactions (N–H ⋯ O and N–H … Cg) occurring within the molecule. The presence of these intermolecular interactions is evaluated through analysis of Hirshfeld surfaces (HS) and two-dimensional (2D) chemical fingerprints map. Additionally, energy frameworks were employed to identify the prevailing interaction energy influencing the molecular arrangement. Density Functional Theory (DFT) calculations were computed to establish concurrence between theoretical and experimental results. Furthermore, the HOMO-LUMO energy levels were determined using the B3LYP/6-31+G level of theory. Finally, molecular docking was used to predict the anti-cancer activity of the compound (4) against PFKFB3 kinase and presented noticeable hydrophilic and hydrophobic interactions at the active site region.Overall, drug design is a dynamic and evolving field, with researchers constantly working to improve their understanding of molecular interactions, develop new computational methods, and explore innovative techniques for creating effective and safe medications. The process can involve steps such as the identification of targets, the discovery of lead compounds, lead optimization, preliminary testing, human trials, regulatory approval and finally post-marketing surveillance, all aimed at bringing a new drug from concept to market. In this article, the synthesis of the novel triazolequinoxalin ( The devTriazolequinoxalin derivatives are a class of compounds that have been well studied for their various potential therapeutic applications , includiComputational chemistry has gained much attention in drug discovery due to its cost effectiveness, environment sustainablility, and zero waste of chemicals. In this study, DFT is one of the most accurate methods to determine the quantum parameters of atoms or molecules. Additionally, it has been implemented to calculate HOMO, LUMO, energy gap, hydrophilic and hydrophobic nature of triazolequinoxalin derivatives. Finally, the molecular docking study was performed to predict the anticancer potential of the titled compound by computing binding affinity and interactions with the active site residue of the target protein. Notably, further in-vitro and in-vivo experiments are crucial to improve the efficacy of the existing agents to cure cancer. As a result, effectively translating the promising relevance of such in-depth studies into cancer research has assisted in rethinking general principles regarding drug combinations and their mechanisms of action.22.14) as shown in 1H, 13C NMR, and LC-MS, alongside C, H, and N analysis. The final structure is determined using the 1H NMR spectrum of the compound (TZQ 4) as a distinctive example. This became readily apparent when the triple bond vanished in compound (1) without any difficulty. In addition to the triplet at δ 0.89 ppm which resulted from having three protons of the Methyl, then there are five methylene groups after the methyl have multiplet between δ 1.25–1.36 ppm was due to the six protons related to the three methylene groups after that quintet between δ 1.82–1.92 ppm related to the fourth methylene. Then the last methylene group in the terminal chain that connected to nitrogen of the triazole cycle exhibited triplet at δ 4.56 ppm. Furthermore, the most significant peak that differentiates the compounds (3 and 4) is the presence of a new proton of triazole ring at δ 7.64 ppm as a singlet due to its distance from the carbon chain. However, the CH group in compound (3) exhibits singlet at δ 7.95 ppm because of the near carbon chain. Finally, the multiplet between δ 7.36–8.33 ppm was attributed to the nine protons that remained in the aromatic groups. Moreover, for 13C NMR there is a notable increase in the peaks numbers related to the carbon chain that connected to the triazole cycle of the same final compound starting from 13.9 ppm for the terminal methyl group in addition of the peaks at δ 22.4, 26.2, 30.4, 31.2, 35.2 ppm for the five methylene group in the chain respectively . Ultimately, the mass spectrum becomes notable stability M+1 at m/z 388 that clearly affirmed the formation of the molecule (TZQ 4) which is the same mass as the compound (TZQ 3) .The newly synthesized compound's structure (TZQ 2.24) crystallizes within the monoclinic space group C2/c, housing a single molecule in the asymmetric unit as depicted in antiperiplanar conformations). One of the hexyl hydrogen atoms interacts with the pyrazinone ring .Compound (4) is characterized by weak C5–H5⋅⋅⋅O1i and C9–H9A⋅⋅⋅N5ii interactions  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O⋅⋅⋅π interaction is observed between atom OP1 and the phenyl part of the quinoxaline ring .The crystal packing of compound  study. The unit cell dimensions were measured as follows: a = 41.201(2) Å, b = 10.6339(6) Å, c = 9.4997(4) Å, β = 93.904(4), and Z = 8. An ORTEP diagram illustrating the title compound is provided in The monoclinic crystal system, space group 2.4dnorm descriptor. This descriptor, derived from normalized contact distances (di and de) among the closest atoms both inside and outside the surface, offers valuable insights into these interactions. Compound (4) exhibits a resulting volume of 511.86 Å³ and a surface area of 404.23 Å2. The asphericity and globularity values for its shape are 0.09 and 0.766, respectively. Collectively, dnorm surface indicate contacts shorter than the combined van der Waals radii . The solvent was evaporated under a vacuum. Following the concentration at reduced pressure, the residue was purified using column chromatography with silica gel and an eluent consisting of a 1:1:9 (v/v) mixture of ethyl acetate and hexane. The resulting solid product was then crystallized using ethanol to give both final compounds (3) as powder and (4) as off-white crystal with (30 % and 60 % yield) respectively . Anal. Calcd. for C23H25N5O(387): C, 71.29; H, 6.50; N, 18.07. Found: C, 71.22; H, 6.47; N, 18.01 %. .Eluent (hexane/ethyl acetate (90/10 %); Yield: = 60 %; F(°C) = 97–99; NMR 4.1.1.21H δ ppm: 0.87-0.91 ; 1.29–1.33 ; 1.84–1.94 ; 4.50–4.64 ; 5.63 ; 7.64 ; 7.36–8.33 ; NMR13C  δ ppm:13.9 (CH3); 22.4, 26.2, 30.4, 31.2, 35.2 (CH2); 48.7 (N–CH2); 113.3, 124.5 (C-Harom), 128.2Traizol, 129.5, 130.7, 130.7, 131.1, 131.2 (CHarom); 131.8, 133.5, 133.6, 135.5, 153.8 (Cq); 154.1 (CO); LC-MS m/z 388 [M+]. Anal. Calcd. for C23H25N5O(387): C, 71.29; H, 6.50; N, 18.07. Found: C, 71.22; H, 6.47; N, 18.01 %. .Eluent (hexane/ethyl acetate (90/10 %); Yield: = 30 %; F(°C) = 125–127; NMR4.24). Intensity data integration, correction for Lorentz and polarization effects, and empirical absorption correction were performed on the pictures using CrysAlisPRO. With the aid of Olex 2 [3), whereas non-hydrogen atoms were refined anisotropically. MoK radiation was used to capture X-ray intensity data at 293(2) K on an Agilent SuperNova diffractometer with an Eos CCD detector in order to produce a single crystal of compound  was subjected to optimization by the DFT approach with B3LYP and a 6-31+G basis set, utilizing the Gaussian 09W software package [TZQ (4) molecule was elucidated through global reactivity descriptors, MEP, and HOMO-LUMO energies.The molecular structure of  package . The ini4.44), docking studies were conducted on the target protein PFKFB3 kinase using the PDB Code: 6HVH [4) was structurally modeled in a 3D conformation using MOE's builder module and then subjected to minimization through the MMFF94x force field [In order to explore the anticancer potential of compound (de: 6HVH . Prior tde: 6HVH . The comce field . For the5.Data associated with this study has been deposited at Cambridge Crystallographic Data Centre under the accession number CCDC: 225879Nadeem Abad: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Software, Writing – review & editing. Fares Hezam Al-Ostoot: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Project administration, Writing - original draft, Writing - review & editing. Sajda Ashraf: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Writing – original draft. Karim Chkirate: Conceptualization, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft. MajedS. Aljohani: Conceptualization, Data curation, Investigation, Validation, Visualization, Writing – review & editing. HussamY. Alharbi: Conceptualization, Data curation, Methodology, Resources, Validation, Visualization, Writing – review & editing. Shafeek Buhlak: Conceptualization, Formal analysis, Resources, Software, Supervision, Validation, Writing – review & editing. Mohamed El Hafi: Formal analysis, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing. Luc Van Meervelt: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Software, Supervision, Validation, Writing – review & editing. Basheer M. Al-Maswari: Conceptualization, Formal analysis, Methodology, Supervision, Validation, Writing – review & editing. El Mokhtar Essassi: Conceptualization, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing. Youssef Ramli: Data curation, Formal analysis, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Artemisia vulgaris (AvL-EtOH) was initially analyzed by means of gas chromatography-mass spectrometry (GC–MS) to identify the important phytochemical constituents. The GC–MS analysis of AvL-EtOH exhibited 48 peaks of various secondary metabolites such as terpenoids, flavonoids, carbohydrates, coumarins, amino acids, steroids, proteins, phytosterols, and diterpenes. It was found that the treatment with increasing doses of AvL-EtOH suppressed the proliferation and migration of lung cancer cells. Furthermore, AvL-EtOH induced prominent nuclear alteration along with a reduction in mitochondrial membrane potential and increased ROS (reactive oxygen species) generation in lung cancer cells. Moreover, AvL-EtOH-treated cells exhibited increased apoptosis, demonstrated by the activation of caspase cascade. AvL-EtOH also induced downregulation of Wnt3 and β-catenin expression along with cell cycle protein cyclin D1. Thus, the results of our study elucidated the potential of bioactive components of Artemisia vulgaris in the therapeutic management of lung cancer cells.The Wnt signaling pathway is reported to be associated with lung cancer progression, metastasis and drug resistance, and thus it is an important therapeutic target for lung cancer. Plants have been shown as reservoirs of multiple potential anticancer agents. In the present investigation, the ethanolic leaf extract of  Lung cancer is the most common carcinoma and the main cause of cancer-associated morbidity globally. Lung cancer contributes 11.4% to the global cancer burden, with an estimated 2,206,771 new cases in 2020. Furthermore, the recent report of Global Cancer Observatory also stated that lung cancer resulted in the death of 1,796,144 individuals, which constituted 18% of the 9,958,133 deaths reported from different types of cancer  ELISA kit . The absorbance of each treatment group was read at 450 nm on a microplate reader and compared with that of the untreated A549 cells.The levels of cleaved PARP in the A549 cells exposed to varying 7 A549 cells seeded in a 6-well plate were subjected to AvL-EtOH  treatment for 24 h, and then, the total RNA was extracted using a HiPurATM Total RNA Miniprep Purification Kit, Himedia, India  following the manufacturer’s protocol. Furthermore, as per the manufacturer’s instructions, the Verso cDNA synthesis kit, Thermo Scientific, USA  was used to prepare cDNA synthesized from 2 μg of isolated RNA. The primers used in this study are listed in In total, 1 × 10p-value of <0.05 was considered statistically significant. The levels of significance were * p < 0.05, ** p < 0.01, and *** p < 0.001. Data are presented as the mean ± standard error of the values obtained in three discrete experiments performed in triplicate. The statistical significance among different treatment groups as compared with the untreated control was determined through one-way analysis of variance followed by the Dunnett post hoc test. A A. vulgaris L. showed the presence of several bioactive compounds  on the basis of these above-mentioned concentrations. The findings of the MTT assay demonstrated that AvL-EtOH  substantially inhibited the viability of the A549 cells, which were found to be 84.42 ± 2.71%, 63.04 ± 4.33%, and 34.52 ± 2.66%, respectively. Thus, AvL-EtOH exhibited an inhibitory effect by suppressing the growth of these cells , as evaluated by the MTT assay   .AvL-EtOH in A549 cells. It was noted that AvL-EtOH exhibited a dose-dependent increase in the number of apoptotic cells by 27.91 ± 3.31% (at 100 µg/mL), 62.61 ± 4.39% (at 200 µg/mL) and 87.15 ± 5.33% (at 400 µg/mL) as compared to the untreated control (8.13 ± 2.02%)  A,B.AvL-EtOH-induced apoptosis, the activity of different caspases was determined  increased the activities of caspase-8, -9, and -3 by 23.54 ± 2.81%, 47.39 ± 3.83%, and 77.02 ± 2.94%; 53.98 ± 3.36%, 74.85 ± 2.11%, and 96.53 ± 2.37%; and 70.03 ± 3.38%, 96.92 ± 2.98%, and 124.38 ± 3.73%, respectively. Moreover, AvL-EtOH dose-dependently induced PARP cleavage  markedly increased the expression of proteins involved in apoptosis onset (Bax and Bad) by 1.47 ± 0.15-, 2.24 ± 0.05-, and 2.53 ± 0.05-fold and 1.34 ± 0.04-, 1.54 ± 0.03-, and 1.86 ± 0.05-fold, and decreased the expression of anti-apoptotic genes (Bcl-XL and Bcl-2) by 0.82 ± 0.02-, 0.73 ± 0.07-, and 0.52 ± 0.03-fold and 0.82 ± 0.06-, 0.68 ± 0.15-, and 0.47 ± 0.14-fold, respectively, in A549 cells  in escalating intracellular ROS by 26.68 ± 2.94%, 52.85 ± 3.43%, and 68.39 ± 3.40%, respectively, as compared with the control (17.06 ± 1.21%) (The mean fluorescence intensity of DCF-DA estimated through flow cytometry presented in ± 1.21%) A,B.8-OHdG is a ubiquitous marker of oxidative stress and contributes significantly to carcinogenesis. As observed in AvL-EtOH on the mRNA expression of the key components of this pathway. Wnt3 expression was significantly downregulated after treatment with AvL-EtOH. The Wnt3 expression levels were found to be 0.85 ± 0.05-, 0.61 ± 0.04-, and 0.43 ± 0.04-fold after treatment with 100, 200, and 400 μg/mL of AvL-EtOH, respectively, compared with those in the control. Similarly, AvL-EtOH inhibited the expression of β-catenin mRNA. The fold change in the expression of β-catenin mRNA was significantly reduced to 0.91 ± 0.04-, 0.74 ± 0.03-, and 0.53 ± 0.04-fold after exposure to 100, 200, and 400 μg/ mL AvL-EtOH, respectively, compared with the control. Furthermore, we evaluated the effect of AvL-EtOH on the expression level of β-catenin target protein mRNAs, such as cyclin D1 and c-myc, in the NSCLC cells. The mRNA expression of c-myc and cyclin D1 was substantially downregulated by 0.88 ± 0.06-, 0.72 ± 0.03-, 0.48 ± 0.03-, 0.90 ± 0.02-, 0.72 ± 0.04-, and 0.42 ± 0.05-fold with 100, 200, and 400 μg/mL AvL-EtOH treatment, respectively, in A549 NSCLC cells  and dead cells. These results are supported by the fluorescent micrographs of Hoechst/PI staining and an increased amount of cleaved PARP in the AvL-EtOH-treated A549 NSCLC cells.ROS generation leads to intensive DNA damage and induces DNA damage-dependent pathways. DNA damage beyond repair triggers cell death. A study reported that apoptosis is triggered by various DNA lesions ,43,44. AAvL-EtOH in the induction of ROS production, which is associated with numerous cellular events, resulting in apoptosis. Mitochondria are the main site for intracellular ROS generation [AvL-EtOH treatment, which finally triggered the caspase cascade through caspase-3 activation, which is a key regulator of apoptosis progression, as it subsequently functionally activates crucial cellular proteins such as endonucleases [AvL-EtOH substantially promotes ROS generation in A549 cells. ROS overproduction can induce oxidative stress, leading to apoptosis [AvL-EtOH might lead to apoptosis, thus confirming the anticancer properties of AvL-EtOH.Studies have suggested that ROS directly affect DNA damage, leading to apoptosis ,46,47. Fneration , and theneration . We founucleases . Thus, Apoptosis . Thus, wA. vulgaris exerted a cytotoxic effect on HepG-2 with an IC50 value of 41.0 ± 1.0 μg/mL [A. vulgaris exhibited anticancer efficacy against HL-60 leukemic cell line by inducing 94.8% apoptosis at 2.0 μg/mL via the release of cytochrome c and the activation of caspases-3, -9, and -8 [A. vulgaris showed substantial cytotoxicity against HeLa and MCF-7 cell lines via reducing their viability by 20% and 18%, respectively, at 140 μg/mL [Our above results are in accordance with a previous observation where the ethanolic extract of .0 μg/mL . Further, and -8 . In addi40 μg/mL .AvL-EtOH treatment inhibits the mRNA expression of Wnt3 in lung cancer cells. The binding of Wnt3/3a to specific receptors was found to inhibit glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation of β-catenin through the removal of GSK-3β from the β-catenin degradation complex. The accumulation of β-catenin molecules in the cytoplasm triggers their translocation in the nucleus and culminates in the activation of genes related to cellular proliferation, stem cell characteristics , metastasis , and angiogenesis  [AvL-EtOH-mediated inhibition of the β-catenin signaling pathway further decreases the mRNA expression of cyclin D1 and c-myc, thereby obstructing the growth and proliferation of human NSCLC cells. Thus, our study demonstrated that apoptosis in lung cancer cells treated with AvL-EtOH was mediated by inhibiting the Wnt/β-catenin signaling cascade. The proposed molecular mechanism of AvL-EtOH-mediated inhibition of the β-catenin signaling pathway in NSCLC is depicted in The canonical Wnt signaling cascade involves β-catenin-mediated activation of downstream pathways through different Wnt ligands. Intriguingly, Wnt3 and Wnt3a are crucial ligands upregulated in NSCLC ,53. Thes factor) . We obseAvL-EtOH against NSCLC A549 cells. AvL-EtOH induced caspase-mediated apoptosis, as evidenced by nuclear fragmentation and condensation, the loss of ΔΨm, cytochrome-c release, and PARP cleavage. These potential anticancer effects of AvL-EtOH are plausibly mediated through the downregulation of the canonical Wnt/β-catenin signaling cascade. Our findings provide a basis for studying the anticancer effects of various bioactive compounds AvL-EtOH against NSCLC cells. AvL-EtOH may serve as a novel adjuvant modality to the conventional chemotherapeutic regimen, with a significant therapeutic index and reduced side effects. However, in-depth mechanistic studies with in vivo models are warranted to confirm the preventive effect of AvL-EtOH against NSCLC.This study demonstrated the antiproliferative and pro-apoptotic activities of


Bacillus amyloliquefaciens by observation of phenotypic characteristics, physiological and biochemical tests, 16S rDNA and gyrB gene sequencing. In vitro experiments showed that the strain possessed biocontrol activity against a variety of pathogens including Sclerotium rolfsii. The strain has the ability to produce hydrolytic enzymes, as well as volatile organic compounds with antagonistic and probiotic effects such as ethyleneglycol and 2,3-butanediol. In addition, HSE-12 showed potassium solubilizing (10.54 ± 0.19 mg/L), phosphorus solubilization (168.34 ± 8.06 mg/L) and nitrogen fixation (17.35 ± 2.34 mg/g) abilities, and was able to secrete siderophores [(Ar-A)/Ar × 100%: 56%] which promoted plant growth. After inoculating peanut with HSE-12, the available phosphorus content in rhizosphere soil increased by 27%, urease activity increased by 43%, catalase activity increased by 70% and sucrase activity increased by 50% (p < 0.05). The dry weight, fresh weight and the height of the first pair of lateral branches of peanuts increased by 24.7, 41.9, and 36.4%, respectively, compared with uninoculated peanuts. In addition, compared with the blank control, it increased the diversity and richness of peanut rhizosphere bacteria and changed the community structure of bacteria and fungi. The relative abundance of beneficial microorganisms such as Sphingomonas, Arthrobacter, RB41, and Micromonospora in rhizosphere soil was increased, while the relative abundance of pathogenic microorganisms such as Aspergillus, Neocosmospora, and Rhizoctonia was decreased.The HSE-12 strain isolated from peanut rhizosphere soil was identified as  On the  enzymes . Bacillu control . Zhang eut roots .Bacillus amyloliquefaciens HSE-12 was isolated from peanut rhizosphere, which can promote plant growth by producing siderophore, fixing nitrogen, dissolving phosphorus and potassium, and can also produce protease, cellulase and chitinase to inhibit fungal growth, and has the potential of biological control. The effects of HSE-12 on peanut growth, soil chemical properties, soil enzymes, rhizosphere microbial diversity and community structure were studied. It is expected to provide theoretical basis and strain support for the development of peanut special microbial fertilizer.In this study, 2.2.1.Plant growth promoting rhizobacteria were isolated from peanut rhizosphere soil in Yishui County, Linyi City, Shandong Province, China . The soil suspension was prepared by adding 1 g of soil to 99 mL of sterile water and serially diluting 10 times. The isolated bacteria were inoculated with 200 μL of the dilution in Luria Bertani medium (LB)  and then incubated in the plates at 28°C for 24 h. After purification the different colonies were selected by repeated purification in LB at least three times, depending on the shape, color, size, regular or irregular, convex or equal morphological characteristics of the colonies. The purified strains were stored in a 30% glycerol solution at −80°C.2.2.Sclerotium rolfsii, Fusarium oxysporum, Rhizoctonia solani, Fusarium graminearum, Fusarium pseudograminearum, Alternaria alternata, Botryosphaeria dothidea, Bipolaris sorokiniana, and Cryphonectria parasitica. After 4 days, the diameters of the zone of fungal growth inhibition around the bacteria were measured and recorded.The antagonistic abilities of the isolates were determined in dual-plate confrontation assays against the plant pathogens 2.3.The isolates were characterized by observing the phenotypic characteristics such as colony morphology, color, size, edge and Gram staining. In addition, the experiments of glucose oxidative fermentation, contact enzyme, nitrate reduction, starch hydrolysis, methyl red, V-P, gelatin liquefaction, citrate utilization, hydrogen sulfide, oxidase, and indole were carried out. All tests were made in triplicate.2O for 50 μL. Reaction conditions: 94°C for 1 min, 33 cycles of 94°C for 20 s, 54°C for 20 s and 72°C for 2 min, followed closely by a final extension at 72°C for 10 min.16S rDNA and gyrB sequence analysis was used to identify the strain. The strain was inoculated in LB liquid medium and shaken at 37°C (200 rpm) for 24 h. Bacterial genomic DNA was extracted from each strain using the bacterial genomic DNA isolation kit DP302 . Genomic DNA was extracted from each strain using 16S rDNA gene primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5’-TACGACTTAACCCCAATCGC-3′) for amplification. The reaction was performed with 25 μL of 2 × taq PCR mix, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of template DNA and 22 μL of ddHThe gyrB sequence of bacteria was amplified by using primers G3F (5’-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′) and G3R (5’-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′). Reaction conditions: 94°C for 1 min, 35 cycles of 94°C for 20 s, 58°C for 20 s and 72°C for 2 min, followed closely by a final extension at 72°C for 10 min. Reaction products were qualified by 0.8% agarose gel electrophoresis and then sent for sequencing to Sangon Biotech (Shanghai) Co. The NCBI Blast server2.4.Sclerotium rolfsii was tested using a double plate antagonism assay  and antagonism to the plant pathogen sm assay . The typ2.5.2.5.1.2PO4 0.2 g, MgSO4 0.2 g, NaCl 0.2 g, K2SO4 0.3 g, CaCO3 5 g, agar 15 g, distilled water 1,000 mL), and incubated at 30°C for 3 d to observe the growth of colonies and the formation of hyaline circles. The determination of nitrogen fixation efficiency was referred to the method of Fresh cultures of the strains were spot inoculated on ashby medium 2SO4 1 g, Na2HPO4 2 g, MgSO4∙7H2O 0.5 g, CaCO3 1 g, potassium feldspar powder 1 g, agar 15 g, distilled water 1,000 mL), and incubated at 30°C for 3 d to observe the growth of colonies and the formation of hyaline circles.Potassium-solubilization efficiency was estimated by flame photometry 2SO4 0.5 g, Ca3(PO4)2 5 g, agar 15 g, distilled water 1,000 mL), and incubated at 30°C for 3 d to observe the growth of colonies and the formation of hyaline circles. The solubility of the strains for inorganic phosphorus was determined using the molybdenum blue colorimetric method , and incubated at 30°C for 3 d to observe the growth of colonies and the formation of hyaline circles. Siderophores were quantified using spectrophotometry , the height of the first pair of lateral branches (LBH), the fresh weight (FW), dry weight (DW) of the above-ground parts and the number of pods (NP) were measured for 18 seedlings from each treatment.2.7.4.The activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) activities, according to the method of 2.7.5.Available nitrogen (AN) determination using diffusion method, available phosphorus (AP) using sodium bicarbonate extraction method, available potassium (AK) using flame spectrophotometry determination, the determination of organic matter (OM) using potassium dichromate oxidation outside heating method . The act2.8.To study the effect of HSE-12 strain on soil microbial community structure, soil genomic DNA was extracted from control and HSE-12-treated soil using a soil DNA kit . The V3-V4 region of the bacterial 16S rRNA gene was amplified with bacterial universal primers 338F and 806R, and the fungal-specific primers ITS1F and ITS2R were used to amplify the ITS1 region of the fungus. High-throughput sequencing was performed by Majorbio Bio-Pharm Technology Co., Ltd.  on the Illumina Miseq PE300 platform .2.9.After removing the adapters and primer sequences, the original sequences were assembled for each sample based on unique barcodes using QIIME . Split sequences from each sample were combined using Flash v.1.2.11. The valid labels of all samples were clustered using the Uparse software. Valuable sequences were classified into operational taxonomic units (OTUs) using USEARCH software, with 97% identification threshold as the cut-off point. For species annotation, representative bacterial sequences were matched against the SILVA database (version 138) and representative fungal sequences were classified using the UNITE database (version 8.0). Alpha diversity analysis was performed using Mothur software. Principal coordinate analysis (PCoA) and unweighted pairwise grouping algorithm clustering were performed using the R language. Tax4Fun was used to analyze the function of bacterial community. Fungal ecosystem analyses were performed using the FUNGuild database. Raw illumina sequencing data from the current study were submitted to the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA8889472.10.p < 0.05.All experimental data were analyzed using Microsoft Excel 2019 and SAS 8.0. Statistical significance was set at 3.3.1.In vitro antagonism tests were carried out on bacteria isolated from peanut rhizosphere soil. Strain HSE-12 was able to inhibit the growth of Sclerotium rolfsii, Fusarium oxysporum, Rhizoctonia solani, Fusarium graminearum, Fusarium pseudograminearum, Alternaria alternata, Botryosphaeria dothidea, Bipolaris sorokiniana and Cryphonectria parasitica, as shown in Bacillus amyloliquefaciens (MH 447524.1) was 99%, and the homology of gyrB sequence with Bacillus amyloliquefaciens (CP 053376.1) was 99%. HSE-12 strain was identified as Bacillus amyloliquefaciens.3.2.3.2.1.The activity of antagonist-related enzymes was determined for the production of strain HSE-12. After fermentation, chitinase, protease, cellulase and xylanase levels reached 17.64 ± 0.79 U/mL, 15.88 ± 0.27 U/mL, 35.95 ± 0.27 U/mL, and 44.69 ± 1.68 U/mL, respectively.3.2.2.Staphylococcus aureus and Streptococcus pyogenes , phosphorus solubilizing (168.34 ± 8.06 mg/L) and nitrogen fixation (17.35 ± 2.34 mg/g) ability, and was able to secrete siderophore [(Ar-A)/Ar × 100%: 56%] which might induce plant growth.3.4.5 after adding bacterial liquid for 10 days, and it tended to be 104 after 20 days, HSE-12 could colonize peanut rhizosphere.The colonization of HSE-12 in peanut rhizosphere was determined by double antibiotic labeling method and plate counting method. As shown in 3.5.Compare to CK, the dry weight, fresh weight and the length of the first lateral branch of peanut seedlings were significantly increased after treatment with HSE-12, which increased by 24.7, 41.9, and 36.4%, respectively, . The act3.6.HSE-12 significantly increased the contents of alkali-hydrolyzable nitrogen, available phosphorus and the activities of sucrase, urease and catalase in peanut rhizosphere soil by 27.2, 36.5, 43.3, 50, and 69.8%, respectively .3.7.The diversity of the peanut rhizosphere soil bacterial and fungi community was shown in For bacteria, at the phylum level, the dominant bacterial composition of the CK and HSE-12 groups was essentially the similar, being Actinobacteriota , Proteobacteria , Acidobacteriota , and Chloroflexi , with the relative abundance of Actinobacteriota being the greatest in each treatment group .norrank_o_gaiellales (8.3%), Bacillus (3.7%), and Burkholderia-Caballeria-Paraburkholderia (3.4%). The top three genera in HSE-12 group were norrank_o_gaiellales (5.5%), Sphingomonas (3.5%), and Bacillus (3.5%)  was greater than 1%. The top three genera of CK were s (3.5%) .Talaromyces (36.8%), Aspergillus (8.8%), and Penicillium (4.3%). The top three genera in HSE-12 group were Talaromyces (27.7%), Alternaria (7.5%), and Mortierella (5.8%) , Basidiomycota , Mortierellomycota , and Chytridiomycota  . At the a (5.8%) . HSE-12 3.8.Principal coordinate analysis (PCoA) was used to analyse the differences in microbial diversity between treatments . The res3.9.The function of bacterial community was predicted by FAPROTAX software . The resFunction of fungal community was annotated by FUNGuild . In CK t4.Azospirillum, Azotobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus, Enterobacter, Klebsiella, Pseudomonas, and Serratia have a plant growth promoting effect , thereby promoting plant growth and facilitating biological control , strain BsA3MX reduced damage caused by M. phaseolina and had a significant pro-growth effect  production and cleavage through extracellular enzymes. Ghadamgahi et al. found that  control . Rangel-h effect . In thisThe main function of soil enzymes is to decompose and convert nutrient forms that cannot be directly utilized by plants into forms that are easily absorbed and utilized by plants. They are also involved in many biochemical processes, including the synthesis and decomposition of humus, the hydrolysis of animal and plant residues, and various redox reactions of organic and inorganic compounds in soil . Here, wSphingomonas, Arthrobacter, RB41, and Micromonospora. Sphingomonas is a genus of bacteria that is widely distributed in nature, commonly found in soil and aquatic environments, and has been shown to inhibit infection with Pseudomonas syringae, Fusarium oxysporum and Fusarium insipidum and to promote plant growth  can be found at: HL: Data curation, Writing – original draft, Writing – review & editing, Project administration. CL: Data curation, Project administration, Writing – original draft, Writing – review & editing. XS: Data curation, Writing – review & editing. JL: Writing – review & editing. PZ: Writing – review & editing. FS: Writing – review & editing. ZG: Writing – review & editing. XL: Conceptualization, Funding acquisition, Resources, Writing – original draft, Writing – review & editing.