Herein, imidazolium-type ionic liquid- (IL-) modified rhenium bipyridine-based porous organometallic polymers (Re-POMP-IL) were designed as efficient and selective photocatalysts for visible-light CO2 photoreduction to CO based on the affinity of IL with CO2. Photoreduction studies demonstrated that CO2 photoreduction promoted by Re-POMP-IL functioning as the catalyst exhibits excellent CO selectivity up to 95.5% and generate 40.1 mmol CO/g of Re-POMP-IL1.0 Re(CO)3Cl] and 3-ethyl-1-vinyl-1H-imidazol-3-ium bromide) at 12 h, outperforming that attained with the corresponding Re-POMP analogue without IL, which highlights the crucial role of IL. Notably, CO2 adsorption, light harvesting, and transfer of photogenerated charges as key steps for CO2RR were studied by employing POMPs modified with different amounts of IL as photocatalysts, among which the CO2 affinity as an important factor for POMPs catalyzed CO2 reduction is revealed. Overall, this work provides a practical pathway to improve the CO2 photoreduction efficiency and CO selectivity by employing IL as a regulator.In the photoreduction of CO Porous organic polymers (POPs), a type of polymer porous materials with micropores and/or mesopores formed by covalent bonds of organic structural units, feature with advantages of good chemical stability and easily tailored structures  (1a) and 3-ethyl-1-vinyl-1H-imidazol-3-ium bromide (2a). For Re-POMP-IL1.0, an equivalent 1a and 2a were used. For comparison, Re-POMP without IL moiety was also synthesized by self-polymerization of 1a. The synthesized polymers, attained as yellow solids, are stable in air and insoluble in water and organic solvents . The chemical structures of Re-POMP and Re-POMP-IL1.0 were characterized by FT-IR and solid-state 13C CP/MAS NMR technique. As exhibited in −1 are ascribed to the stretching vibration of CO ligand coordinated with Re(I). Compared with Re-POMP, the occurrence of the new peak at 1157 cm−1 in the FT-IR spectrum of Re-POMP-IL1.0 arises from the C-N bond vibration of covalently linked 2a. In the solid-state 13C CP/MAS NMR spectra (2a. Similar morphologies of Re-POMP and Re-POMP-IL1.0 were observed by scanning electron microscopy (SEM)  complex. Compared with Re-POMP, both of Re 4f peaks shift to lower binding energies for Re-POMP-IL1.0, indicating the increased Re electron density after the installation of IL. The high electron density on Re atom may facilitate the interaction between Re and CO2 (vide infra). The accurate metal loading in polymers was further measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES), and the Re contents of 31.0 and 24.4 wt% in Re-POMP and Re-POMP-IL1.0 were detected, respectively, which are basically consistent with the theoretical results. It is noteworthy that this is the highest Re content detected among the reported Re-based porous materials used in CO2RR +: 209.1068; found: 209.1076.5,5'-Divinyl-2,2'-bipyridine : white s1a: yellow solid . IR : 3101, 3070, 3040, 2018, 1914, 1877, 1480, 1379, 1251, 918, and 860 cm−1. 1H NMR  δ 8.98 , 8.07 , 6.78 , 6.03 , 3.68  ppm. HRMS (ESI): m/z calcd for C17H12N2O3Re [M-Cl]+: 479.0400; found: 479.0391.H-imidazole  and bromoethane  were added. The mixture was refluxed at 70°C for 3 h and then cooled to room temperature to give the white solid. The desired 2a was obtained after drying the white solid under vacuum at 70°C for overnight.To a 50 mL Schlenk tube, 1-vinyl-12a [−1. 1H NMR  δ 9.61 , 8.23 , 7.98 , 7.32 , 5.98 , 5.41 , 4.24 , 1.45  ppm. 13C NMR  δ 134.07, 128.26, 122.55, 119.40, 109.20, 45.22, and 14.34 ppm.2a : white s1a , AIBN , and DMF (7 mL) were added to a 25 mL autoclave. Then, the autoclave was sealed and heated at 100°C for 24 h. When finished, the autoclave was cooled to room temperature and the resulting mixture was transferred to a 50 mL conical flask, followed by adding 20 mL ethanol to soak overnight. Then, the obtained mixture was filtered and washed with ethanol until the filtrate was colorless. After the filter cake was dried under vacuum at 70°C for overnight, the yellow solid (185.7 mg) was obtained and named as Re-POMP.1a , 2a , AIBN , and DMF (7 mL) were added to a 25 mL autoclave. Then, the autoclave was heated at 100°C for 24 h. When finished, the autoclave was cooled to room temperature and the resulting mixture was transferred to a 50 mL conical flask, followed by adding 20 mL ethanol to soak overnight. Then, the obtained mixture was filtered and washed with ethanol until the filtrate was colorless. After the filter cake was dried under vacuum at 70°C for overnight, the yellow solid (268.3 mg) was obtained and named as Re-POMP-IL1.0.Take the synthesis of Re-POMP-IL1.0 as an example, 2a added was adjusted to 0.16 mmol and 0.59 mmol, respectively.For the synthesis of Re-POMP-IL0.4 and Re-POMP-IL1.5, their synthetic procedures are similar to that of Re-POMP-IL1.0, except that the amount of 2a , AIBN , and ethanol (5 mL) were added to a 50 mL Schlenk flask under Ar atmosphere. Then, the reaction was conducted at 80°C for 16 h. When finished, the resultant mixture was cooled to room temperature, followed by adding 20 mL ethanol to soak overnight. Then, the obtained mixture was filtered and washed with ethanol (5 × 20 mL). After the filter cake was dried under vacuum at 70°C for overnight, PIL (420.2 mg) was obtained as white solid.2 through the freeze-pump-thaw method. Then, the reaction tube was sealed and placed under a 500 W long-arc Xenon lamp (λ ≥ 400 nm) for 12 h at room temperature. The light source was cooled by flowing cooling water before irradiating the reaction tube and the number of moles of photons absorbed by the photocatalyst was measured as 1.4 × 10−8 einstein/s . After the reaction, partial gaseous products (1 mL) were taken from the tube using a syringe and then analyzed by gas chromatography with a TCD detector.To a 25 mL Schlenk tube, photocatalyst (1 mg), TEOA  and MeCN (3 mL) were successively added. Then, the reaction mixture was sonicated to allow the polymer to disperse evenly in the whole solution. Air in the Schlenk tube was replaced by CO


The iron-loading value in the labeled granulocytes, as obtained by MRI, was 6.40 ± 0.18 pg/cell. Similar values were found with the ICP-MS and NIRF imaging techniques. Therefore, our study shows that it is possible to isolate granulocytes with high purity and yield and labeling with M-SPIONs provides a high internalized iron load and low toxicity to cells. Therefore, these M-SPION-labeled granulocytes could be a promising candidate for future use in inflammation/infection detection by optical and MRI techniques.This in vitro study aimed to find the best method of granulocyte isolation for subsequent labeling with multimodal nanoparticles (magnetic and fluorescent properties) to enable detection by optical and magnetic resonance imaging (MRI) techniques. The granulocytes were obtained from venous blood samples from 12 healthy volunteers. To achieve high purity and yield, four different methods of granulocyte isolation were evaluated. The isolated granulocytes were labeled with multimodal superparamagnetic iron oxide nanoparticles (M-SPIONs) coated with dextran, and the iron load was evaluated qualitatively and quantitatively by MRI, near-infrared fluorescence (NIRF) and inductively coupled plasma mass spectrometry (ICP-MS). The best method of granulocyte isolation was Percoll with Ficoll, which showed 95.92% purity and 94% viability. After labeling with M-SPIONs, the granulocytes showed 98.0% purity with a yield of 3.5 × 10 Inflammation is the immune system′s response to pathological agents (infection), damaged cells and toxins, and it aims to maintain integrity, defense against microorganisms and cellular and tissue repair ,2,3. AcuHard-to-reach or occult inflammation and infection processes are clinically challenging. Their clinical diagnoses involve biochemical and radiological examinations, but these methods can produce false negatives ,13,14,15Proinflammatory effects help leukocytes migrate from the blood vessel to the site of injury, due to increased vascular permeability through stimulation by cytokines and interleukins. There is an initial accumulation of granulocytes, mainly neutrophils, in the inflamed area and later of lymphocytes and macrophages ,23,24. GThe noninvasive evaluation of acute inflammatory processes makes it possible to analyze molecular and cellular processes in vivo, usually based on targeting specialized cells such as macrophages, lymphocytes or granulocytes to the desired target . NoninvaOne of the possibilities for contrast agents is nanoparticles, which have the potential to be used by various molecular imaging techniques due to their range of magnetic, fluorescent and radiative properties, among others . The mosSuperparamagnetic iron oxide nanoparticles (SPIONs) are outstanding MRI contrast agents due to their excellent magnetic properties and low toxicity, which allow the use of special surface coatings with organic or inorganic molecules, including surfactants, drugs, proteins, starches, enzymes, antibodies, nucleotides, nonionic detergents and polyelectrolytes , in addiMost studies employing exogenously labeled granulocytes to analyze infection or inflammation processes have used SPIONs with different strategies ,60,61,62Thus, this in vitro study aimed to find the best (highest purity and yield) granulocyte isolation technique, with the aim of labeling the granulocytes with multimodal nanoparticles detectable by MRI-NIRF imaging, for qualitative and quantitative evaluations of their internalized M-SPION loads, a promising candidate for future research use in detecting inflammation/infection processes by optical and MRI techniques.p < 0.001). The post hoc test showed a significant difference in all comparisons , method III—Percoll with Hank′s balanced salt solution (HBSS) and method IV—Percoll and Ficoll. Then, we assessed the purity of the granulocytes isolated by each method . The higp < 0.001). The post hoc test showed a significant difference in all comparisons  between methods III and IV. In relation to the isolated granulocyte yield, means of (4.99 ± 1.24) × 106 per mL and (3.51 ± 1.40) × 106 per mL were found by methods III and IV, respectively, without a significant difference between methods, as shown by a Student′s t-test (p = 0.996). The isolated lymphocyte yield between methods III and IV were significantly different by the Mann-Whitney test (p = 0.025), with respective means of (0.33 ± 0.07) × 106 per mL and (0.09 ± 0.06) × 106 per mL.The purity analysis of isolated granulocytes and their respective contaminants in methods III and IV showed a proportional inverse relationship, in which method IV had the highest and the lowest contaminant rate, as depicted in the boxplot of The M-SPION optical characterization depicted in To analyze the electrostatic interaction of granulocytes with M-SPIONs, the surface charge was measured using the same pH of 7.4 found in the cell labeling, and a zeta potential of approximately 32 mV was verified.After labeling the isolated granulocytes with M-SPIONs, internalization was verified through fluorescence A,C,E–G aAfter 4 h of labeling, granulocyte viability was evaluated with M-SPION concentrations of 10, 30 and 50 µg Fe/mL, which showed viability (double-negativity for Annexin-FITC and PI-PE) values of 99.3%, 99.6% and 98.6%, respectively H–J.MRI, ICP-MS, and NIRF were used to quantify the iron mass internalized into granulocytes after M-SPION incubation at concentrations of 10, 30 and 50 µg Fe/mL. The quantification results of the iron mass per cell and the number of nanoparticles per granulocyte are shown in −4 ms−1 µg Fe−1 mL for M-SPIONs, as depicted in the inset of The MRI quantification was begun by calculating the r2 value; for this purpose, the T2-weighted image of the phantom was obtained using different echo times (TE) and containing the following concentrations of M-SPIONs dispersed in 1% agarose at: 5, 10, 15, 20, 25, 30, 35, 40 and 50 µg Fe/mL A. The co4 M-SPION/cell, 2.41 ± 0.15 pg/cell or (3.03 ± 0.19) × 104 M-SPION/cell and 6.40 ± 0.18 pg/cell or (8.06 ± 0.22) × 104 M-SPION/cell, respectively , the T2 values of granulocytes labeled with M-SPION concentrations of 10, 30 and 50 µg Fe/mL and of the control granulocytes not labeled with M-SPIONs were calculated. From the curves of the signal intensity of MRI as a function of TE B, the foectively . Figure 8 was obtained, as shown in 4 M-SPIONs/cell; for the M-SPION concentration of 30 µg Fe/mL, it was 3.49 ± 0.19 pg/cell or (4.39 ± 0.23) × 104 M-SPIONs/cell and for the M-SPION concentration of 50 µg Fe/mL, it was 4.64 ± 0.14 pg/cell or (5.84 ± 0.18) × 104 M-SPIONs/cell  × [Fe] + (1.89 ± 0.14)] × 10ONs/cell .4 M-SPIONs for the concentration of 10 µg Fe/mL, 2.56 ± 0.16 pg/cell or (3.21 ± 0.20) × 104 M-SPIONs at 30 µg Fe/mL and 6.83 ± 0.47 pg/cell or (8.56 ± 0.59) × 104 M-SPIONs at 50 µg Fe/mL . However, until now, no method using the combination of Percoll and Ficoll has been reported, only each technique separately. In addition, there is no consensus on the best technique for isolating granulocytes to achieve high purity. The methods applied for granulocyte isolation are diverse ,68,69,706 cells/mL  is the concentration of intracellular iron on agarose gel and r2 is the transverse relaxivity of M-SPIONs.For quantification of M-SPIONs internalized into the cell, a phantom for each well containing 2 × 10The complement of the results was used to calculate the number of nanoparticles internalized into granulocytes using Equation (2):6 cells/mL dispersed in 1 mL of DMEM and 1 mL of nitric acid (37%) for digestion over 4 h at 70 °C. The digested samples were diluted 10 times with Milli-Q® water  and were analyzed with ICP-MS equipment  to determine the iron content of each sample. Sample measurements were performed in quintuplicate, and quantification was based on a calibration curve using certified standard iron (NexION #N8145054) at the following concentrations: 5, 10, 20, 30 and 40 ppb.The quantification of the iron load of the granulocyte labeling with M-SPIONs by ICP-MS used 2 × 106 cells dispersed in 1 mL of DMEM placed in an Eppendorf tube. The NIRF signal of the triplicate samples was acquired by applying an excitation of 750 nm, registered in a range of emission of 810–875 nm, using an exposure time of 2.5 ms, binning of 4 and f/stop of 2. The sample images were acquired using IVIS® Lumina LT Series III equipment , and the fluorescence intensity signals were analyzed in absolute radiation units (photons/s). For the quantification analysis, the calibration curve was constructed using iron concentrations of 0.5, 1, 2, 8, 10 and 15 µg Fe/mL, and the fluorescence intensity signal of the samples was compared to a calibration curve to quantify the iron mass internalized by isolated granulocytes.The quantification of iron load by NIRF was performed after granulocyte labeling with 2 × 10p-value of less than 0.05. Statistical tests were performed using JASP v. 0.10.2.0 .The experimental data are presented as the mean and standard deviation. For comparison of the multiple isolation methods, differences between the means of the groups were tested by a nonparametric test for unpaired samples , following the post hoc analysis corrected by Bonferroni. The difference between two independent sample means was tested by a nonparametric Mann-Whitney test. Significant results were considered a


However, the current recovery process leads to a loss of TiO2-NPs; therefore, there is a need to immobilize TiO2-NPs on the substrate used. Herein, TiO2-coated yttria-stabilized zirconia/silica nanofiber (TiO2-coated YSZ/silica NF) was prepared by coating TiO2 on the surface of YSZ/silica NF using a sol–gel process. The TiO2 coating layer on the nanofiber surface improved the separation ability of the membrane as well as the photocatalytic degradation ability. The pore size of the TiO2-coated YSZ/silica NF membrane was less than that of the pristine YSZ/silica NF membrane, and it rejected over 99.6% of the 0.5 μm polymeric particles. In addition, the TiO2-coated YSZ/silica NF membrane showed excellent adsorption/degradation of humic acid , methylene blue , and tetracycline . Six recycling tests were performed to evaluate the reusability of the TiO2-coated YSZ/silica NF membrane. The adsorption/degradation efficiency for HA, MB, and TC decreased by 3.7%, 2.8%, and 2.2%, respectively. We thus verified the high separation ability, excellent photocatalytic degradation ability, and excellent reusability of the TiO2-coated YSZ/silica NF membranes.Numerous reports have elucidated that TiO For these reasons, electrospun nanofibers have been applied in various environmental fields such as (1) water and air purification membranes (or filters)6, (2) photocatalysts7, (3) adsorption8, and (4) gas sensors9. In addition, electrospun nanofibers have been the focus of various studies aimed at the simple manufacture of polymer materials. However, polymer nanofibers cannot be used under harsh conditions due to their (1) poor thermal stability, (2) low chemical durability. To overcome this problem, numerous researchers have developed inorganic nanofibers (such as ceramics) using electrospinning technology14.Electrospinning technology can continuously produce fibers with diameters that range from several nanometers to several micrometers by using a strong electric field. Electrospinning technology has numerous advantages including the provision of (1) a wide selection of materials, (2) a controllable pore structure, (3) high interconnectivity, and (4) the fabrication of complex structures17. This is because water purification technology using photocatalytic NPs uses light energy, rendering it an eco-friendly technology. Further, photocatalytic degradation processes do not produce harmful substances. In particular, TiO2 is a representative photocatalyst that is widely applied for environmental purification purposes since it has various advantages such as (1) strong optical adsorption, (2) chemical stability, and (3) low cost. Moreover, TiO2 NPs have good photocatalytic degradation capacities due to their high specific surface areas. However, there are several challenges faced including difficulties in separating, recovering, and recycling TiO2-NPs. Moreover, TiO2-NPs easily form agglomerates due to their high surface energy. This problem requires additional processes such as centrifugation and filtering in the recovery process of TiO2-NPs20 wherein some TiO2-NPs are lost. Ultimately, the loss of TiO2-NPs causes secondary water pollution, low reproducibility, and reduced photocatalytic degradation efficiency22. To address these disadvantages, various studies have been conducted where TiO2-NPs are immobilized on various substrates (membranes or filters)28.In recent decades, the use of photocatalytic nanoparticles (NPs) has been considered as an excellent water purification technology2 coating layer on electrospun ceramic nanofibers. Several inorganic materials  were feasible to avoid the risk of polymeric decomposition by OH radicals and hydrogen peroxide (H2O2) that are generated during the photocatalytic reaction31. The immobilization of TiO2 coating layers on ceramic NF membranes not only addresses the disadvantages of water purification technology using TiO2-NPs, but also presents a new direction towards advancing electrospun ceramic NF. Herein, the TiO2-coated YSZ/silica NF membrane was developed for three purposes: (1) simultaneous separation and photocatalysis, (2) improved photocatalysis (by large surface area), and (3) self-cleaning capability . The achievement of these properties requires not only the improvement of both separation and photocatalysis, but also the prevention of TiO2 loss, which is responsible for the reduction of the photocatalytic efficiency. Accordingly, the morphology, mean/largest pore size, and the specific surface area of the TiO2-coated YSZ/silica NF membrane were characterized. Furthermore, the gas and pure water permeabilities as well as the rejection rate (%) were analyzed. Lastly, the efficiency of adsorption/photocatalytic degradation was evaluated using major pollutants which represent natural organic matter (HA), organic dye (MB), and antibiotic (TC).We therefore endeavored to immobilize the TiO2-coated YSZ/silica NF with various TTIP concentrations. The FE-SEM micrographs show the change in the diameter (TiO2 coating layer plus YSZ/silica NF diameter) depending on the TTIP concentration. This indicates that the TiO2 coating layer increased as the TTIP concentration raised. For example, the diameters of the YSZ/silica NF and the 0.1 M and 1.0 M TiO2-coated YSZ/silica NFs were 267 ± 29 nm, 275 ± 31 nm, and 315 ± 28 nm, respectively. The diameter of 1.0 M TiO2-coated YSZ/silica NF increased by approximately 48 nm (thickness of 24 nm) as compared to that of pristine YSZ/silica NF. An increase in diameter of the TiO2-coated YSZ/silica NF is able to affect all subsequent membrane characteristics. For example, a reduction in the membrane pore size leads to an increase in the rejection of pollutants, thereby improving the separation capability. The increased TiO2 coating layer enhances the photocatalytic degradation efficiency. The formation of a TiO2 coating layer on electrospun nanofibers have also been reported using electrospraying32 or the sol–gel method33. The TiO2 coating using electrospraying has a disadvantage in that it resulted in TiO2-NP aggregation which over-coated the electrospun nanofibers.Figure 2 coating on YSZ/silica NF. Figure 2-coated YSZ/silica NF, confirming the co-existence of zirconium, yttrium, silicon, and oxygen. Titanium was only observed in the 1.0 M TiO2-coated YSZ/silica NF, indicating that the TiO2 coating layer was formed. Similarly, Fig. 2-coated YSZ/silica NF, respectively. It shows a uniform Ti distribution in the 1.0 M TiO2-coated YSZ/silica NF, indicating that TiO2 was homogeneously coated over the entire nanofiber bundle without aggregation.High-resolution transmission electron microscope (HR-TEM) micrographs and Energy dispersive spectrometry (EDS) spectra confirmed the formation of the TiO2-coated YSZ/silica NF. The diffractogram of the calcined YSZ/silica NF indicates that the stabilized zirconia had a cubic phase (JCPDS No. 01-081-1550). The peaks assigned to zirconia are 30.0°, 34.7° 50.0°, 59.4°, 62.3°, and 73.4° corresponding to the (111), (200), (220), (311), (222), and (400) crystallographic planes, respectively12. However, new peaks were observed at 25.3°, 37.8°, 53.9°, and 55.1° in TiO2-coated YSZ/silica NF, which show the crystalline anatase TiO2 phase (JCPDS No. 89-4320) and crystallographic planes of (101), (004), (105), and (211), respectively36. In addition, the grain size of the anatase TiO2 phase in the 0.1 M, 0.5 M, and 1.0 M TiO2-coated YSZ/silica NF was calculated using Scherrer’s equation . In general, it is known that the grain size of TiO2 increases with increasing TTIP concentration37. This is because the higher TTIP concentration, the faster the growth rate of nuclei than the nucleation rate39.Figure 2-coated YSZ/silica NF in order to analyze the weight loss and changes in heat capacity. The pyrolysis of the as-coated 1.0 M TiO2-coated YSZ/silica NF can be divided into 4 stages, as shown in Fig. 2-coated YSZ/silica NF in Fig. 41. Stage B showed a weight loss of approximately 16% due to the thermal decomposition of the organic residues that occurred during the oxidation of TTIP that transformed it into an amorphous structure43. In stage C, the weight was reduced by approximately 2% due to the thermal decomposition of organics present inside the 1.0 M TiO2-coated YSZ/silica NF45. Stage D shows complete removal of all organic compounds and residues, and as a result, no weight change was observed. We analyzed the calcined 1.0 M TiO2-coated YSZ/silica NF to identify any remaining organic compounds (for reference). No weight loss was observed for the calcined 1.0 M TiO2-coated YSZ/silica NF in the TGA curve.Thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) were conducted on the as-coated and calcined 1.0 M TiO2-coated YSZ/silica NF, as shown in Fig. 47. As shown in stage C, the exothermic peak (417 ℃) is located in the crystallization zone of the anatase TiO2 phase48. The exothermic peak  is due to the thermal decomposition of organic compounds present in the 1.0 M TiO2-coated YSZ/silica NF. The calcined 1.0 M TiO2-coated YSZ/silica NF showed no change in heat flow as observed in the DSC curve, and this confirmed that no organic compounds were remaining in the sample. If TiO2-coated YSZ/silica NF is calcined above 500 ℃, the anatase phase can be converted to the rutile or brookite phase49. Studies have shown that the anatase phase has better photocatalytic degradation efficiency than the rutile and brookite phases52. Thus, in our present study, we calcined the samples at 500 ℃ to obtain the anatase TiO2 phase, which has less recombination potential than the rutile and brookite phases.The DSC curve shows the heat flow of the as-coated 1.0 M TiO2-coated YSZ/silica NF. We confirmed the change in the specific surface area (SSA) owing to the formation of the TiO2 coating layer on YSZ/silica NF. As shown in Table 2/g, and the SSA of TiO2-coated YSZ/silica NF increased as the TiO2 coating layer raised from 0.1 M (10.2 m2/g) to 0.5 M (11.6 m2/g) and then to 1.0 M (11.7 m2/g). This is because the increased TiO2 coating layer on the YSZ/silica NF contributes to expand the SSA. The SSA is a critical factor in adsorption and photocatalysis. The SSA of electrospun ceramic nanofiber membranes have smaller than NPs, but higher than that of conventional ceramic membranes [especially microfiltration (MF) and ultrafiltration (UF) membranes]54. TiO2-NPs generally have a high SSA, but the problems of aggregation, separation, and recovery hinder their applicability. Although the SSA of TiO2-coated YSZ/silica NF is relatively smaller than that of the TiO2-NPs, our research was able to compensate for the drawbacks of TiO2-NPs.Table 2-coated YSZ/silica NF membranes through the analysis of capillary flow porometry (CFP). The mean pore size of the YSZ/silica NF membrane (0.51 ± 0.05 μm) gradually decreased as the TTIP concentrations increased from 0.1 M (0.48 ± 0.01 μm) to 0.5 M (0.46 ± 0.02 μm) and then to 1.0 M (0.42 ± 0.01 μm)  is 11.8 ± 0.9 L/cm2 min bar. In addition, the pure water permeability of YSZ/silica NF membrane is 46,704 ± 2064 L/m2 h bar and the pure water permeability of 1.0 M TiO2-coated YSZ/silica NF membrane is 31,992 ± 3766 L/m2 h bar, as shown in Fig. 2-coated YSZ/silica NF membranes decrease as the largest pore size reduces. Nevertheless, conventional multi-layer ceramic membranes  have a large number of closed pores, which decreases the gas and pure water permeability relative to electrospun ceramic nanofiber membranes that have an open pore structure57. For example, the gas permeability of ceramic membranes is approximately 0.001 L/cm2 min to 0.004 L/cm2 min, and the pure water permeability is approximately 250 L/m2 h to 600 L/m2 h60. Therefore, the gas and pure water permeability of the TiO2-coated YSZ/silica NF membranes is much higher than that of ceramic membranes.Fig. 2-coated YSZ/silica NF membranes using 0.5 μm polymeric particles. The mean pore size of YSZ/silica NF and TiO2-coated YSZ/silica NF membranes affected the rejection rate (%) because the mean pore size corresponds to the average index of the pores of the membrane can separate particles larger than the mean pore size. The YSZ/silica NF membrane with the largest mean pore size (0.51 μm) showed the lowest rejection rate (99.6% ± 0.1%), and the 1.0 M TiO2-coated YSZ/silica NF membrane with the smallest mean pore size (0.42 μm) showed the highest rejection rate (99.9% ± 0.1%). Overall, we confirmed an excellent rejection rate (more than 99.6%) in all the membranes. The difference in the rejection rates (%) is due to the mean pore size of TiO2-coated YSZ/silica NF membranes, which was mostly less than 0.5 μm as shown in Fig. 14. The variation in the rejection rate (%) is directly correlated to a difference in the mean pore size of the TiO2-coated YSZ/silica NF membranes, which gradually decreased due to the increase in the TiO2 coating layer. In other words, the post-processing for the formation of TiO2 coating layer on YSZ/silica NF is able to govern the pore size and rejection rate (%).Figure 2-coated YSZ/silica NF was characterized by selecting HA, MB, and TC, which represent natural organic matter, organic dyes, and antibiotics, respectively. In particular, the same pollutant concentration (20 mg/L) was adopted to compare the differences in photocatalytic degradation behavior between pollutants. The pollutant concentrations are likely to be varied for research articles, and the variations are very broad. They are mostly distributed in the range of 5 to 10 mg/L, but a relatively high concentration of 20 mg/L was used herein to demonstrate the great photocatalytic degradation performance of TiO2-coated YSZ/silica NF62. That is, we endeavored to accomplish the excellence of our study by using a relatively higher pollutant concentration than the commonly used pollutant concentration. Figure 2-coated YSZ/silica NF membranes. As shown in Fig. 2-coated YSZ/silica NF membranes were 52.4% ± 0.2% , 78.4% ± 0.8% , and 88.2% ± 0.3% , respectively. In Fig. 2-coated YSZ/silica NF membranes using the Langmuir–Hinshelwood rate equation35, which gave the k of YSZ/silica NF membrane for HA as 0.03 h−1, and the k of 0.1 M, 0.5 M, and 1.0 M TiO2-coated YSZ/silica NF membranes were 0.12 h−1, 0.21 h−1, and 0.35 h−1, respectively. The adsorption/degradation efficiency and apparent reaction rate of HA increased with an increase in the TiO2 content. We observed similar results for the adsorption/degradation efficiency and apparent reaction rate of MB and TC, as shown in Figs. (A2), (A3), (B2), and (B3). The difference in adsorption/degradation efficiency and the apparent reaction rate of 1.0 M TiO2-coated NF membrane was HA , MB , and TC . As a result, the order of the adsorption/degradation pattern of 1.0 M TiO2-coated NF membrane is HA < MB < TC. This was caused by the difference in the (1) adsorption rate, (2) bond dissociation energy (BDE), and (3) molecular weight (MW) of each pollutant. In general, the interfacial interaction between pollutants and TiO2 most influences the photocatalytic degradation capability63. That is, the lifetime of OH radicals due to the photocatalytic activity of TiO2 is very short (~ ns), so that only the pollutants adsorbed on the TiO2 coating layer can be degraded31. As shown in Fig. 2 has an IEP of 7.0, the IEP of HA and TC were 4.7, 4.8, respectively, and MB is a cationic dye67. The pH of each pollutant solution was adjusted to 6.5, and the surface charge of TiO2 was positive, while HA and TC were negatively charged, and MB was positively charged. As MB has the same surface charge as TiO2, it has the smallest adsorption rate due to electrostatic repulsion. HA and TC, which have similar IEPs, have an opposite surface charge from TiO2, so their adsorption rates are higher than that of MB due to electrostatic attraction. However, the adsorption rate of TC is almost 3 times higher than that of HA, this is because TC is hydrophilic71. In general, since common metal oxide ceramics are hydrophilic, the hydrophilic TC had a higher adsorption rate than the hydrophobic HA and MB72. Secondly, each pollutant has different functional groups that are collapsed by OH radical attack. The chemical structure of the pollutants affects photocatalytic degradation behavior, and in particular, the appropriate photocatalytic materials should be selected according to the functional groups. In previous studies77, the functional group cleaved by OH radicals in HA and TC is C = C (BDE: 618.3 ± 15.4 kJ/mol), while that in MB is C = S (BDE: 548.9 kJ/mol). This order of BDE of MB < HA = TC indicates that MB can be better cleaved by OH radicals than HA and TC. Finally, the MW of each pollutant is different. The MW of HA is over 10,000 g/mol, MB is 319.85 g/mol, and TC is 480.90 g/mol (MB < TC ≪ HA). Taken together, TC has the highest adsorption/degradation efficiency, because TC has the highest adsorption rate (12.1%) and relatively smaller MW (480.90 g/mol). MB has the second highest adsorption/degradation efficiency, because the lowest BDE (548.9 kJ/mol) and MW (319.85 g/mol). HA has the least adsorption/degradation efficiency, because relatively smaller adsorption rate (4.6%) and the largest MW .The photocatalytic degradation of TiO2-coated YSZ/silica NF membrane and TiO2-NPs, we measured adsorption/degradation efficiency and the apparent reaction rate using 5 mg of TiO2-NPs, as shown in Fig. 2-NPs was HA , MB , and TC [98.4% ± 0.1% , k = 0.69 h−1), respectively. It was lower than the adsorption/degradation efficiency and the apparent reaction of 1.0 M TiO2-coated YSZ/silica NF membrane. This is because the dispersion of TiO2-NPs varies depending on the shape of the container, and the distance and position from the UV light source are not constant. Moreover, the TiO2-NPs having high surface energy form agglomerates which can reduce the SSA of the TiO2-NPs and slowed the reaction of TiO2-NPs with UV light. As a result, it was verified that the TiO2-coated YSZ/silica NF membrane had higher adsorption/degradation efficiency than TiO2-NPs and can effectively photodegrade pollutants from different sources such as HA, MB, and TC.To compare the adsorption/degradation efficiency and the apparent reaction rate of 1.0 M TiO2-coated YSZ/silica NF membrane. The adsorption/degradation efficiencies of HA, MB, and TC tend to reduce during a total of six recycling tests. For example, HA decreased by 3.7% , MB by 2.8% , and TC by 2.2% . The reason is that the amount of HA, MB, and TC remaining after adsorption and photocatalytic degradation influenced the subsequent recycling test, as shown in Fig. 78 experimented on the recycling of Fe3O4/SiO2/TiO2 NPs using MB under UV irradiation for 90 min at each cycle, and they observed a decrease of approximately 14% . In addition, Y. Shi et al.19 conducted a study on the recycling of palygorskite-supported Cu2O-TiO2 composite using TC under Xe lamp irradiation for 240 min at each cycle, and they observed a reduction by approximately 7.57% . The main reason for the decrease in adsorption/degradation efficiency in most of the recycling tests are as follows. In each cycle, photocatalytic nanomaterials are more likely to be lost in the recovery process such as filtering and centrifugation, which directly causes the adsorption/degradation efficiency to decrease35. Meanwhile, in order to minimize photocatalyst loss and prevent secondary environmental pollution, we developed a sol–gel process to tightly secure TiO2 on the surface of YSZ/silica NF membrane. Even if the photocatalytic recycling test was carried out 6 times in a row for 7 h each time, the decrease in adsorption/degradation efficiency is minimal. Such an excellent reusability is one of the greatest advantages of our study.Figure 2-coated YSZ/silica NF was successfully developed by forming a TiO2 coating layer on the surface of YSZ/silica NF. This study mainly considered two aspects: (1) the ability to simultaneously perform separation and degradation and (2) improving the efficiency of photocatalytic degradation and imparting self-cleaning functionality. To achieve these goals, the TTIP concentration was controlled accordingly during TiO2 coating process. As the TTIP concentration increased, the TiO2 coating layer of the YSZ/silica NF raised. The increasing TiO2 coating layer reduced the pore size of the membrane, and thus the separation ability of the TiO2-coated YSZ/silica NF membrane was improved. Moreover, the TiO2 coating layer formed was able to efficiently degrade various pollutants with excellent adsorption/photocatalytic degradation. To verify the stability of the formed TiO2 coating layer, recycling tests were carried out, which confirmed the stability and reusability of TiO2-coated YSZ/silica NF membranes. In conclusion, we succeeded in developing a stable TiO2-coated YSZ/silica NF membrane capable of simultaneously separating and photocatalytically degrading HA, MB, and TC, which is expected to play an important role in water purification.In this study, TiO2 coating on the YSZ/silica NF, a TiO2 sol was prepared with 0.1 M, 0.5 M and 1.0 M titanium tetraisopropoxide  as the TiO2 precursor, acetylacetone , 2-propanol , acetic acid , and deionized (DI) water, which were stirred at 60 ℃ for 5 h. The YSZ/silica NF were submerged in the TiO2 sol for 5 min under vacuum and then rinsed with 2-propanol in triplicate. The TiO2-coated YSZ/silica NF were dried overnight in ambient conditions and calcined at 500 ℃ for 1 h at a heating rate of 5 ℃/min in air. Finally, the TiO2-coated YSZ/silica NF were evaluated with respect to their physicochemical properties, membrane performance, and photocatalytic degradation.An electrospinning solution containing 1.6 M zirconium (IV) propoxide solution , 8 mol% yttrium (III) nitrate hexahydrate , 30 mol% tetraethyl orthosilicate , 10 wt% polyvinylpyrrolidone , N,N-dimethylformamide , and acetic acid  was prepared for the fabrication of YSZ/silica NF. The solution was stirred at 30 ℃ until it was clear. Electrospinning of YSZ/silica NF was conducted in a closed chamber system  under a controlled relative humidity (40% ± 5%) and temperature (25 ± 2 ℃). The electrospinning solution was placed in a plastic syringe and then ejected through an electrically conducting stainless-steel nozzle (23 G) at a feed rate of 1 ml/h. The applied voltage was 20 kV. The distance between the nozzle tip and the collector was set to 10 cm. The as-spun YSZ/silica nanofibers were dried in a vacuum oven at 30 ℃ for 5 h and then calcined at 800 ℃ for 2 h at a heating rate of 5 ℃/min in air. For TiO79. The SSA was characterized using the BET  analysis with nitrogen as the adsorbent. TGA and DSC  were conducted within a temperature range of 20 to 900 ℃ at a heating rate of 10 ℃/min in an air atmosphere. The mean pore size and the largest pore size were analyzed using CFP . The mean pore size was mainly associated with the rejection (or separation) capacity of the nanofibrous membranes. The largest pore size is more relevant to the air/water permeability (or transport property) of the nanofibrous membranes80.The surface morphology was characterized using FE-SEM  at an accelerating voltage of 5 kV and a magnification of 20,000×. HR-TEM  was also used for morphological and elemental analysis at an accelerating voltage of 200 kV and a magnification of 150,000× along with EDS . All electron microscope micrographs were analyzed using an image processing software  to calculate the average fiber diameter (from more than 30 randomly selected ceramic nanofibers). The crystalline structure was analyzed with XRD  using Cu kα radiation (λ = 1.5406 Å) at 40 kV and 200 mA under the scanning speed of 4°/min and a scan range (2θ) from 10 to 90°. The grain size was calculated using Scherrer’s equationCf) and that of the permeate concentration (Cp) were measured using ultraviolet–visible (UV–vis) spectroscopy  at a wavelength of 600 nm. The rejection rate (%) was calculated using the following Eq. . The measurement was carried out at a pressure of 1.0 bar with air. The pure water permeability was determined using a cross-flow membrane testing system . The measurement was conducted at a transmembrane pressure (TMP) of 0.15 bar with a fluid velocity of 2.5 L/min. The rejection rate (%) was analyzed using 0.5 μm polymeric particles . The solution containing polymeric particles was prepared by diluting it with DI water to a concentration of 250 ppm. The contents of feeding concentration  HA , (2) MB  and (3) TC . The schematic diagram of adsorption/photocatalytic degradation process is described in Fig. 2-coated YSZ/silica NF membranes (50 mm in diameter) were completely submerged in the pollutant solutions  and left in a dark condition for 1 h to reach the adsorption equilibrium. The photocatalytic degradation was initiated using UV irradiation  for 6 h while the pollutant solutions were constantly shaken to ensure proper mixing. Aliquots were taken at regular time intervals (1 h) to measure the absorbance of pollutant solutions using UV–vis spectroscopy . The specific wavelengths of 254 nm, 663 nm, and 356 nm were adopted to determine the HA, MB, and TC contents, respectively. The rate constant for the photodegradation reaction was determined using Eq. . Furthermore, a recycling test of 1.0 M TiO2-coated YSZ/silica NF membrane was conducted to determine its reusability following the same way as the adsorption/degradation test described above. It was carried out 6 times in a row and reused after washing (with DI water for 1 h) and drying (in a 50 ℃ oven for 4 h) every cycle.The adsorption/photocatalytic degradation of the TiOSupplementary Information.


Candida parapsilosis, Candida albicans, Candida tropicalis, Aspergillus niger, and Trichophyton rubrum, via a 3--2,5-diphenyltetrazolium bromide (MTT) microdilution assay. Compounds GKV10, GKV11, and GKV15 emerged as promising antifungal agents against all the fungal strains used in the current study. One of the highly active antifungal compounds, GKV10, was selected for a single-crystal X-ray diffraction analysis to unequivocally establish its molecular structure, conformation, and to understand the presence of different intermolecular interactions in its crystal lattice. A cooperative synergy of the C-H···O, C-H···N, C-H···S, C-H···π, and π···π intermolecular interactions was present in the crystal structure, which contributed towards the overall stabilization of the lattice. A molecular docking study was conducted for all the test compounds against Candida albicans lanosterol-14α-demethylase (pdb = 5 tzl). The binding stability of the highly promising antifungal test compound, GKV15, from the series was then evaluated by molecular dynamics studies.A series of 2,4,5 trisubstituted-1,2,3-triazole analogues have been screened for their antifungal activity against five fungal strains,  Fungi are organisms with similar properties to plants . Many fu1H and 13C), High-resolution mass spectrometry (HRMS), and selected compounds using the single-crystal X-ray method, as per an earlier study [Synthesis of the title compounds GKV1–15  was achier study . The yieer study .The molecular structure of the molecule GKV10 in the crystal shows that it is conformationally flexible . The magCandida (C.) tropicalis (0.49 μg/mL). They showed similar activity against C. parapsilosis and C. albicans, as well (0.98 μg/mL). Compound GKV15 had higher activity against Aspergillus (A.) niger and Trichophyton (T.) rubrum (0.98 μg/mL). The promising results of these antifungal activities may require the optimization of highly active compounds in the future.The in vitro antifungal minimum inhibitory concentration (MIC) was determined by using the two-fold dilution method  and the results are presented in The molecular docking study was performed to evaluate the binding orientation of the tested compounds within the active site of lanosterol-14-α-demethylase. We computed scores for the free energy of binding, affinity, clash score, and the root of the mean square deviation (RMSD). Compound GKV15 showed the top-ranked ΔG value , affinity (30.01), and was the lowest in RMSD (0.81 Ǻ) and clash score (2.25) . CompounN-atom of the triazole ring affected the binding. Presence of methyl group in compounds like GKV1-3, GKV6-11, and 15 had an electron-donating effect that enhanced the electron cloud around the nitrogen atom of the triazole ring and potentiated its coordination with the heme. However, the branching as well as increase in the carbon chain length resulted in the increase in steric hindrance, thereby effecting the coordination with the heme which probably could be the reason for detrimental effect in the activity as evident in case of the compounds 4, 5, 12, 13 and 14  to evaluate the strength of their binding. The oscillations of the tested compound started with RMSD 1.5 Ǻ during the first 3 ns. Then, from 3 ns to 8 ns, there were oscillations suspended between 1.25 Ǻ and 2.00 Ǻ. Equilibrium was reached at 2.00 Ǻ at exactly 8 ns, which was then maintained . Interesα radiation (λ = 0.71073 Å). Data collection was carried out at 173(2) K using an Oxford Cryostream cooling system featuring the Bruker Apex II software. The in vitro antifungal test was performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.The synthesis of the test compounds was achieved by two-step chemical synthesis and is depicted in α radiation (λ = 0.71073 Å). Unit cell measurement, data collection, integration, scaling, and absorption corrections for the crystal were carried out using the Bruker Apex II software [H-1,2,3-triazol-4-yl)(thiophen-2-yl)methanone (GKV10) are summarised in Single-crystal data were collected on a Bruker APEX II diffractometer equipped with a CCD detector, using monochromated Mo Ksoftware . Data resoftware . The crysoftware  and refisoftware , which isoftware . Absorptsoftware . All nonsoftware . Geometrsoftware  and PLATsoftware . The sinCandida parapsilosis (ATCC RCMB 05064); Candida albicans (ATCC RCMB 05064); Candida tropicalis (ATCC RCMB 05064); Aspergillus niger (ATCC RCMB 05064); Trichophyton rubrum (ATCC RCMB 05064). Microdilution assay was used to evaluate the tested compounds (GKV1-15) for their antifungal activity [The calculation of the minimum inhibitory concentrations (MICs) for the tested compounds was done against activity . DiffereThe Molecular Operating Environment (MOE) program was used for performing the docking studies. The chain A was selected as the main chain, which was complexed with heme–porphyrin and a reported azole inhibitor. The amino acids within a radius of 6.5 Å were selected in the binding site. The triangle method was the main placement method. London dG was used as a rescoring method. The Merck molecular force field 94x (MMFF94x) was selected as a force field .3. Assisted Model Building with Energy Refinement 10:Extended Hückel Theory (Amber 10:EHT) was selected as a force field. The heat was adjusted in order to increase the temperature of the system from 0–300 K, which was followed by equilibration and production for 300 ps; cooling was then initiated until 0 K was reached. The simulation was conducted over a 20 ns period of time .The docking of GKV15 revealed a stable pose that was kept in the active site. The protein geometries, electron density and temperature-related factors were tested. All hydrogens were added, and energy minimization was calculated. Any foreign solvent molecules in the system were deleted; salt atoms were then added to the system to surround the biomolecular protein–ligand complex in a spherical shape. Sodium chloride was added in a 0.1 mol/L concentration. The cell dimensions were of 81.9 × 81.9 × 81.9 Å. The total number of molecules within the system was 18,295, 1.01 g/cmC. parapsilosis, C. albicans, C. tropicalis, A. niger, and T. rubrum at an MIC of 0.98, 0.98, 0.49, 0.98, and 0.98 µg/mL, respectively. This result was combined with the molecular docking results of free energy, affinity, clash score, and RMSD at −24.75, 30.01, 2.25, and 0.81, respectively. The activity of the test compound GKV15 was similar in magnitude to the standard drug. Thus, the test compound GKV15, reported herein, is a promising starting point for the development of an antifungal drug candidate.A series of 2,4,5-trisubstituted-1,2,3-triazole derivatives (GKV1-15) bearing a substituted phenyl moiety at the fourth position of the 1,2,3-triazole nucleus was synthesized, and the derivatives were evaluated for their antifungal activity. The single-crystal X-ray diffraction studies of compound GKV10 established that C-H···O, C-H···N, C-H···S, C-H···π, and π···π intermolecular interactions primarily stabilized the supramolecular packing arrangement of the molecules in the solid-state. The antifungal results indicate that all the test compounds exhibited promising antifungal effects against the tested ATCC strains. The title compound GKV15 was highly promising, as it exhibited the best antifungal activity against


On-Hep1) and the synthetic FITC-labelled mature peptides sMatOn-Hep1 and sMatOn-Hep2 were analyzed. Moreover, sMatOn-Hep1 and sMatOn-Hep2 were mildly detected in the lymphocytes of peripheral blood mononuclear cells (PBMCs) and strongly detected in head kidney macrophages. The in vitro binding and antibacterial activities of these peptides were slightly effective against several pathogenic bacteria. Immune regulation by sMatOn-Hep1 was also analyzed, and only sMatOn-Hep1 significantly enhanced the phagocytic index in vitro (p < 0.05). Interestingly, intraperitoneal injection of sMatOn-Hep1 (10 or 100 µg) significantly elevated the phagocytic activity, phagocytic index, and lysozyme activity and clearly decreased the iron ion levels in the livers of the treated fish (p < 0.05). Additionally, sMatOn-Hep1 enhanced the expression levels of CC and CXC chemokines, transferrin and both On-Hep genes in the liver, spleen and head kidney, for 1–96 h after injection, but did not properly protect the experimental fish from S. agalactiae infection after 7 days of treatment. However, the injection of S. agalactiae and On-Heps indicated that 100 μg of sMatOn-Hep1 was very effective, while 100 μg of rProOn-Hep1 and sMatOn-Hep2 demonstrated moderate protection. Therefore, On-Hep is a crucial iron-regulating molecule and a key immune regulator of disease resistance in Nile tilapia.In this study, the functions of a recombinant propeptide (rPro Oreochromis niloticus) is the most important freshwater fish cultured in the world. The global production of farmed tilapia was approximately 6.5 million metric tons in 2019, and Thailand is among the most important producers, with an annual production of approximately 2 × 105 tons [Aeromonas hydrophila, Flavobacterium columnare and Streptococcus agalactiae [Nile tilapia (Morone chrysops × M. saxatilis) in 2002 [Ictalurus punctatus) [Oreochromis mossambicus) [Oryzias melastigma) [Scophthalmus maximus) [Oncorhynchus mykiss) [Gobiocypris rarus) [Scatophagus argus) [Trachidermus fasciatus) [Larimichthys crocea) [Carassius auratus) [An evaluation of fish immunology is required to overcome these problems, for the prevention and treatment of infectious diseases. Based on current information, vertebrates possess both innate and adaptive immunity. Innate immunity is the first line of defense against pathogens ,4. It is in 2002 . Since t in 2002 , zebrafi in 2002 ,9, channnctatus) , tilapiaambicus) ,12, mariastigma) , turbot maximus) ,15, rains rarus) , spotteds argus) , roughsksciatus) , large y crocea)  and goldOn-Hep), as both a propeptide and a mature peptide, and to determine their antibacterial activities against pathogenic bacteria. Transcriptional level expression of On-Hep was quantitatively analyzed under pathogenic bacterial infection. The regulation of important immune-related gene transcription and iron levels in response to On-Hep application was also demonstrated. The antimicrobial activity of On-Hep1 was investigated in both in vitro and in vivo experiments. Importantly, the efficacy of On-Hep1 has been proven to act as an effective safeguard against harmful S. agalactiae. The information obtained from the current study is crucial for understanding the immune mechanisms and effective functional roles of On-Hep1, which is useful for generating prophylactic and therapeutic strategies to improve disease resistance against deadly pathogenic bacteria in the Nile tilapia aquaculture industry.Little information is known about the immune function and regulation of hepcidins in Nile tilapia. Therefore, the purposes of this research were to overexpress and structurally analyze Nile tilapia hepcidin (https://www.ncbi.nlm.nih.gov) using blastN and blastX programs. The 5′ UTR, ORF and 3′ UTR were analyzed by Genetyx 7.0.  The signal peptide was predicted by the DAS transmembrane prediction server (http://www.sbc.su.se/~miklos/DAS/maindas.html). A homology analysis of On-Hep1 and other reported hepcidin gene sequences (http://www.angelfire.com/nj2/arabidopsis/MatGAT.html) was conducted. Full-length On-Hep1 was aligned with other known tilapia hepcidin proteins using Genetyx 7.0.In this experiment, cDNA encoding uncharacterized On-Hep1 was retrieved from the cDNA library of Nile tilapia spleen deposited in the GenBank database (FF280957). The obtained cDNA sequence was reblasted against information available in the GenBank database , and a neighbor-joining evolutionary tree was analyzed with 1000 bootstrapping values using MEGA version 6.0 (www.megasoftware.net).A phylogenetic tree of equences  were aliFive hundred healthy Nile tilapia (84 ± 3.6 g) were obtained from Manit Genetics, Ltd., Phetchaburi Province, Thailand. The fish were acclimatized in a 500-L glass tank containing clean dechlorinated freshwater with an aeration system, for 7 days. During acclimatization, the fish were fed three times daily with commercial feed at 5% body weight. All experiments were conducted in accordance with the Ethical Principles and Guidelines for the Use of Animals recommended by the National Research Council of Thailand, for the care and use of animals for scientific purposes. The protocol was approved by the Animal Ethics Committee, Kasetsart University, Thailand, with the ethic ID of ACKU61-FIS-004.Three fish were randomly collected and anesthetized with 80 mg/L clove oil , and whole blood was withdrawn from the caudal vein using a 2-mL heparinized syringe with a 21-G needle. PBLs were isolated with the method previously described by Chung and Secombes [On-Hep1 in each tissue was performed. One microliter of first-strand cDNA from each tissue was subjected to a Mx3005P real-time PCR system  equipped with analytical software version 4.0  and Brilliant II SYBR Green qPCR Master Mix , according to the manufacturer’s recommended protocol, using the specific primer pairs On-Hep1 F and On-Hep1 R  of the t method . The thrFlavobacterium columnare (AQFC001) and S. agalactiae (AQSA001), were obtained from the Laboratory of Aquatic Animal Health Management, Faculty of Fisheries. A single colony of S. agalactiae was cultured in 10 mL of trypticase soy broth (TSB) and incubated in a shaking incubator, at 30 °C for 24 h. The bacterial cells were centrifuged at 2500 rpm for 10 min, washed and resuspended in PBS (pH 7.4), adjusted to reach an absorbance of 0.669 at 600 nm, to obtain a concentration of 1 × 109 CFU/mL, and further tenfold diluted with PBS to obtain a final concentration of 1 × 107 CFU/mL. A single colony of F. columnare was inoculated with 10 mL of Shieh medium broth. Then, this bacterium was incubated and grown under the above conditions. The pellet was re-suspended in sterile water. The concentrations of F. columnare were adjusted to 1 × 107 CFU/mL with optical densities of 0.27 at 525 nm. This bacterial suspension was used for other experiments below.Two severely pathogenic bacteria, 7 and 1 × 109 CFU/mL of S. agalactiae and F. columnare, respectively. These concentrations were selected based on a preliminary test that they could induce moderate and severe responses, days 5–7 after injection. At 0 h, 6 h, and 12 h and days 1, 2, 3 and 7, the liver, spleen and head kidney of 4 fish in each tank were collected for total RNA extraction, 1st-strand cDNA synthesis and qRT-PCR of On-Hep1 with the methods described above. The relative expression of On-Hep1 at h 0 was used as a calibrator for the transcription level analysis of each pathogenic induction.One hundred and fifty Nile tilapia in the above section were selected, placed in 5 250-L fiberglass tanks (30 fish each) and acclimatized as described above for 7 days. After that, all fish in tank 1 were intraperitoneally injected with 100 μL of PBS (pH 7.4), while fish in tanks 2-3 and 4-5 were injected with 100 μL of PBS (pH 7.4), containing 1 × 10On-Hep1 peptide  competent cells, using the heat-cold shock method. Blue-white colony screening was conducted on Luria–Bertani (LB) agar containing ampicillin (100 mg/mL), isopropyl-β-d-1-thiogalactopyranoside (IPTG) (1 mM) and X-gal (50 mg/mL). The plasmids were extracted from selected positive clones using the FavorPrep Plasmid Extraction Mini Kit  following the manufacturer’s protocol. Double restriction enzyme digestion with Xho I and Nde I was conducted following the recommendation of the company’s protocol , and the products were run on a 1% agarose gel. The desired digested DNA fragment was purified using the FavorPrep Gel/PCR Purification Mini Kit .Specific primers were designed to amplify the sequence encoding the ProXho I/Nde I-cut pET28b expression vector and transformed into JM109 competent cells as previously described. The transformed bacteria were selected and grown in LB broth (supplemented with 100 µg/mL kanamycin) at 37 °C. The positive clones were isolated, kept and extracted for plasmid DNA as described above. The obtained plasmid was sequenced with a similar method as described above, to check for the correct ProOn-Hep1 sequence. The rProOn-Hep1 plasmid was transformed into BL21 host cells on LB agar supplemented with 100 µg/mL kanamycin. The positive cells containing the rProOn-Hep1 plasmid were subcultured and kept in LB agar supplemented with 100 µg/mL kanamycin for further experiments.The purified DNA was ligated into a On-Hep1 plasmid grown on LB agar plus 100 µg/mL kanamycin was inoculated into 3 mL of LB broth containing kanamycin (LBk) in a 15 mL test tube, and incubated at 30 °C overnight. Four hundred microliters of prepared bacterial preculture was transferred into 40 mL of LBk in a 50 mL conical PE tube incubated in a shaking incubator at 37 °C, for approximately 3 h. When the absorbance of the culture reached 0.6 at 600 nm, IPTG was added to a final concentration of up to 1 mM. The bacterial culture was further grown, and 1 mL of bacterial suspension was collected and put into 1.5 mL Eppendorf tubes at 1 h, 2 h, 3 h, 4 h and 5 h, and then centrifuged at 5000 rpm for 5 min. rProOn-Hep1 expression was determined using 12% SDS-PAGE, as described by Nakharuthai et al. (2017) [A single colony of BL21 cells containing the rPro. (2017) .On-Hep1 were grown and harvested in 500 mL Erlenmeyer flasks containing 250 mL of LBk under the previously described conditions. The target protein was purified by the Ni-NTA Purification System as described by the manufacturer with a modified protocol for an inclusion body protein . Finally, the recombinant protein was eluted by elution buffer (pH 8.0), and various protein fractions were collected into new Eppendorf tubes and characterized by SDS-PAGE techniques as described above. The obtained recombinant protein was dialyzed, and protein concentrations were determined by using the Bradford protein assay, compared with standard serial two-fold protein dilutions of 2 mg/mL albumin. All set concentrations were quantified for absorbance at 595 nm using an iMark™ Microplate Absorbance Reader . Total protein was also determined by SDS-PAGE using a Coomassie Protein Assay Kit (Thermo Scientific).Based on the optimal induction times in the previous section, bacterial cells producing rProOn-Hep1 protein. The recombinant ProOn-Hep1 protein was separated on a 12% SDS-PAGE gel and further electrotransferred to a nitrocellulose membrane using a Mini Trans-Blot® cell  at 0.11 A for 1 h and 10 min. The membrane was blocked with 5% skimmed milk at room temperature for 1 h, incubated with a 1:6000 dilution of a mouse anti-His tag antibody for 1 h, washed with 1X TBS + 0.1% Tween and incubated with a 1:5000 dilution of goat anti-mouse IgG-alkaline phosphatase (GAM-AP) for 1 h. The hybridized protein was detected using the Vectastain® ABC kit . The membrane was incubated with nitro blue tetrazolium (NBT)/ 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrate and incubated with detection buffer  for 30 min until color appeared.Western blot analysis was used to confirm the existence and molecular weights of the obtained rProOn-Hep1 and sMatOn-Hep2) produced by China Peptides Co., Ltd. (China) and our own rProOn-Hep1 were used to evaluate MICs by liquid growth inhibition methods [A. hydrophila (AQAH009), S. agalactiae (AQSA001), Vibrio parahaemolyticus (AQVP001) and V. vulnificus (AQVV001). A. hydrophila and S. agalactiae were cultured in 10 mL of TSB medium and incubated in a shaking incubator at 30 °C for 24 h, while V. parahaemolyticus and V. vulnificus were grown in 10 mL of TSB supplemented with 1.5% NaCl, under conditions similar to those of the first two bacteria. The bacterial cells were centrifuged at 2500 rpm for 10 min, washed and resuspended in PBS (pH 7.4), adjusted to an absorbance of 0.669 at 600 nm to obtain a concentration of 1 × 109 CFU/mL, and further tenfold diluted with PBS to obtain a final concentration of 1 × 108 CFU/mL. Mueller–Hinton (MH) broth was used as an assay medium and supplemented with 1.5% NaCl for V. parahaemolyticus and V. vulnificus in a 96-well microtiter plate. The final concentrations of each peptide (128 to 0.25 μg/mL) in 200 μL of tested medium (in triplicate) were obtained by serial dilution, and bacterial concentrations of 1 × 105 CFU were applied. Wells that contained only MH broth and bacteria were provided as a negative control. Plates were incubated at 30 °C, and the growth of bacteria in each well was followed by monitoring the absorbance at 600 nm at 0 h and then every 3 h until 24 h, using an iMark™ Microplate Absorbance Reader (Bio-Rad). After a 24 h incubation period, the lowest concentration that caused complete inhibition, as indicated by clear characteristics, was defined as the MIC value.Two commercially synthesized FITC-labeled mature peptides  and then centrifuged at 200× g for 10 min, twice. The obtained cells were numerated using a hemocytometer and further diluted to reach a final concentration of 5 × 106 cells/mL.A healthy Nile tilapia (160.5 g) was used to isolate PBMCs using a heparinized syringe with a 23-G needle. One milliliter of whole blood was withdrawn from the caudal vein and put into a 15 mL PE tube containing 2 mL of RPMI medium . Diluted blood was mixed gently, loaded into a new 15 mL PE tube containing 3 mL of LymphoprepTM , and centrifuged in a swing rotor centrifuge at 400× 2 cover glasses and allowed to adhere on the glass surface for 2 h. Monolayer cells were washed 3 times with PBS (pH 7.4) to remove unattached cells. Previously, 200 µL of PBS (pH 7.4) containing 1 × 107 latex beads  supplemented with 100 µg of each rProOn-Hep1, sMatOn-Hep1 and sMatOn-Hep2 was prepared for 1 h. Two hundred microliters of three latex bead types, each incubated with a different peptide, were added onto the cover glass with each duplicate. After 1.5 h, the unattached cells and excess beads were washed 3 times with PBS. The other two cover glasses with only 200 µL of PBS containing latex beads were provided as a control group. All attached cells in each treatment were stained with a Dip-Quick Staining Kit according to the manufacturer’s recommendations . PA was observed under a microscope by counting 100 cells in each cover glass, and percentages of PA and PI were calculated as described in a previously modified method [Two hundred microliters of the above-prepared leukocytes containing phagocytes were loaded onto eight 22 × 22 mmd method , followiA. hydrophila, Flavobacterium columnare, S. agalactiae and the nonpathogenic species Bacillus pumilus were used. All bacteria were cultured and prepared as described above to obtain a final concentration of 1 × 108 CFU/mL in a bacterial suspension. One hundred microliters of each bacterial suspension were separately mixed and incubated with 100 µL of PBS, containing 100 µg of the FITC-labeled peptides sMatOn-Hep1 and sMatOn-Hep2 for 2 h at ambient room temperature (25 °C).In this experiment, the three pathogenic bacteria g for 10 min three times. After the cells were washed, 10 μL of the cell-peptide combinations were dropped onto a glass slide, and the binding activities of each peptide were observed under a confocal microscope  using 100× magnification.The cells were washed with PBS and centrifuged at 400× 8 cells/mL.A healthy Nile tilapia (160.5 g) was isolated for PBMCs based on the above description, and leukocytes from head kidney were isolated using the modified method described by Ortuno et al. (2001) . Cells wOn-Hep1 and sMatOn-Hep2 (100 µg for each), for 2 h at ambient room temperature (25 °C). The cells were washed and centrifuged, and cell binding activity was observed with the same practices as described in the section above.One hundred microliters of diluted cell suspension were mixed with 100 µL of each FITC-labeled peptide, i.e., sMatOne hundred and forty-eight healthy Nile tilapia (70.6 ± 4.7 g) were acclimatized in a 1000-L fiberglass tank containing dechlorinated freshwater, with full aeration conditions, for 7 days. Then, each set of 48 fish was placed into separate 500-L fiberglass tanks containing 400 L of freshwater. During this time, fish were fed twice a day with commercial feed at 5% body weight, and 20% of the water was changed daily.S. agalactiae strains were cultured and prepared as described above to obtain a bacterial suspension at a final concentration of 1 × 108 CFU/mL using PBS (pH 7.4). PBS was used to dilute rProOn-Hep1 solution from a previous section to the 3 concentrations 10, 50 and 100 µg rProOn-Hep1/100 mL.Virulent On-Hep1, was performed as described in a previous section. The fourth group of 12 fish in each tank were similarly injected with 100 µL PBS to serve as controls. Fish continued rearing, and mortality was recorded daily for 10 days. Moribund fish found in each tank were collected and diagnosed for bacterial infection using the liver and spleen by the loop isolation method in TSA.The experiment was conducted and run with 3 replicates under a cohabitation culture system. Each of four groups of twelve fish in each tank from a previous section were differently tagged with one of four colored threads at the first spine of the dorsal fin. Each experimental group was intraperitoneally injected with 100 µL of bacterial suspension. One hour later, a second injection of 100 µL, containing 1 of 3 different concentrations of rProOne hundred healthy Nile tilapia (100.5 ± 9.3 g) were acclimatized in the same environment in a 1000-L fiberglass tank as described above, with slight modification. Then, 50 fish each were brought into a 500-L fiberglass tank containing 400 L of freshwater.S. agalactiae strains were cultured and prepared as described above. Two commercially synthesized FITC-labeled sMatOn-Hep1 and sMatOn-Hep2 peptides from the above section were aliquoted at the 2 concentrations (diluted with PBS) 10 µg and 100 µg peptides/100 μL. The bacterial suspension was diluted with PBS to provide 1 × 108 CFU of S. agalactiae.The virulent On-Hep1 and sMatOn-Hep2 prepared as described above were injected under the same conditions. The other 10 fish in each tank served as controls and were injected with 100 µL of a bacterial suspension. Fish were observed and recorded for mortality until day 10, with a similar procedure, as described above.This experiment was also conducted in cohabitation culture, and 10 fish in each tank were tagged by 5 differently colored threads. Ten fish from each group were intraperitoneally injected with 100 µL of a bacterial suspension. One hour later, 2 different concentrations of sMatOn-Hep1 peptide from the above section was aliquoted and mixed with PBS (pH 7.4) to provide the 2 concentrations 10 and 100 µg sMatOn-Hep1/100 μL.Commercially synthesized FITC-labeled sMatNinety healthy Nile tilapia (130.3 ± 6.5 g) were acclimatized in a 1000-L fiberglass tank containing dechlorinated freshwater with full aeration conditions for 7 days. Then, each group of 30 fish was placed into separate 500-L fiberglass tanks containing 400 L of freshwater to set up 3 experimental groups. Each group of ten fish in each tank was tagged with 1 of 3 different colors of thread. After acclimatization, 2 groups of ten fish each in each tank were intraperitoneally injected with 100 µL of sMatOn-Hep1 solution prepared in the previous section. The other 10 fish in each tank served as the control and were injected with 100 µL of PBS (pH 7.4). At the beginning of the experiment and prior to peptide injection, whole blood and liver of each fish were collected from each tank, to investigate innate immune parameters and heavy metal concentrations, including iron, zinc and copper. PBMCs of blood samples were isolated as described above. After injection, the same investigations of a fish in each replicate of each treatment were conducted at h 0, 1, 6, 12, 24, 48 and 96.8 beads/mL and 5 × 106 cells/mL, respectively, with RPMI medium. The phagocytic assay was conducted with the same protocol as described in a previous section.PBMCs were isolated from 3 fish in each treatment at 0, 1, 6, 12, 24, 48 and 96 h, as described in a previous section. The densities of the latex beads  and phagocytic cells were adjusted to 1 × 10Micrococcus lysodeikticus (200 µg/mL) in PBS (pH 6.2) was prepared next. Ten microliters of fish serum were added to 3 wells of a 96-well flat-bottom microtiter plate containing 250 µL of prepared M. lysodeikticus. The other 3 wells received 10 µL of PBS (pH 6.2) to serve as a control. The absorbance of the reaction wells was measured at 540 nm after 1 and 5 min using an iMark™ Microplate Absorbance Reader (Bio-Rad). Lysozyme activity was calculated with the method described by Sarder et al. [In this experiment, another fish from each treatment at different time points similar to those in the above section was withdrawn, and its whole blood was collected with the same method as described above, transferred to a new Eppendorf tube and rested at ambient temperature for 2 h. Serum was separated from the blood corpuscles by centrifugation at 3500 rpm for 15 min. Approximately 500 µL was transferred and kept in a new tube. A suspension of 8 cells/mL were loaded into every well. The reaction mixtures were incubated for 1 h at 25 °C and centrifuged at 1500× g for 5 min at 25 °C. Finally, 200 μL of supernatant in each well was transferred to a new 96-well flat-bottom microplate, and the absorbance was measured at 540 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad). Complement activity in serum samples in terms of ACH50 was analyzed based on the modified method of Ortuno et al. [One hundred microliters of PBS buffer (pH 7.4) was placed into 10 wells of a 96-well U-bottom microplate. Serial two-fold dilutions were conducted by adding 100 µL of each fish serum from the first to the last well. Unlysed and completely lysed controls were set by using 100 µL of PBS buffer (pH 7.4) and distilled water, respectively. Then, 100 µL of sheep red blood cells  in PBS buffer (pH 7.4) at a concentration of 2 × 10o et al. .3 . The digested sample in acid solution was transferred to a Teflon PFA pressure decomposition vessel  and heated in a microwave digester  at 200 °C for 15 min. In the case of blood serum, 0.5 mL of serum was used and put in a Teflon beaker, containing 1.25 and 0.5 mL of concentrated HNO3 and hydrogen peroxide, respectively. Mixed components were heated under the same conditions as in the abovementioned methods. The resulting transparent liquid of the digested liver, blood corpuscles and serum was moved to a Teflon beaker. Fe, Zn and Cu elements were analyzed by atomic absorption spectroscopy . Standard solutions of each element were prepared by following the methods described by PanReac AppliChem ITW , and used to obtain calibration curves. Each sample solution was analyzed three times with AAS to determine the 3 target elements.Fish livers and blood corpuscles (1 g) were cut into small pieces and predigested in a Teflon beaker with 10.0 mL of HNOCC chemokine-1, CC chemokine-2, interleukin-8 (On-CXC1 and On-CXC2), interleukin-1β, hepcidin1 (On-Hep1), hepcidin2 (On-Hep2) and transferrin  was prepared with the same system as described in the previous section to reach a final concentration of 1 × 108 CFU/mL.Pathogenic On-Hep1 application for 7 days, the remaining fish in each treatment from different tanks in the above section were intraperitoneally injected with 100 µL of bacterial suspension prepared in the previous part. The fish in each tank received good care for 10 days. The mortality assessments and disease diagnoses of the moribund fish were conducted daily, as described above, until day 10 of injection.After sMatOn-Hep1 in qRT-PCR analyses, immune parameters and concentrations of 3 heavy metals were statistically analyzed using analysis of variance (ANOVA), and the means of these parameters were compared by Duncan’s new multiple range test (DMRT) using SPSS software version 20.0 (IBM). A survival analysis in the challenge experiments in each group was performed using the Kaplan–Meier method with the same software. p < 0.05 was considered statistically significant.The relative expression ratios of On-Hep1. It comprised 672 bp, including a 114 bp 5′ untranslated region (UTR), a 273 bp open reading frame (ORF) and a 285 bp 3′ UTR with a polyadenylation site (AATAAA). The predicted amino acid sequence of the propeptide (proOn-Hep1) contained 90 amino acid residues. A signal prediction program showed that proOn-Hep1 had a signal peptide and a propeptide, ranging from amino acids 1–20 and 21–90, respectively (On-Hep1), containing 26 amino acids, was found at the C terminus, with eight cysteine residues that are highly conserved among the other Cichlid hepcidin proteins  hepcidin TH 2-3. In comparison, the On-Hep2 similarity and identity were 68.1 and 67.4%, respectively   A. At dayectively B.S. agalactiae was significantly effective, increasing On-Hep1 expression at 6 h and 12 h after injection, by 45.9 ± 6.1- and 438.1 ± 2.2-fold, respectively (p < 0.05). In an F. columnare injection experiment, the highest concentration elevated upregulation of the target gene at 6 h by only 21.5 ± 1.8-fold . No obvious upregulation of the target gene was observed after this period , but no MIC values were clearly observed based on the criteria. However, based on their detected absorbance, some concentrations of rProOn-Hep1 showed mild antibacterial activity against A. hydrophila, S. agalactiae and V. vulnificus  of Nile tilapia phagocytes were examined. It was shown that 100 µg/mL of these proteins could not enhance the PA of Nile tilapia in vitro compared with that of the control  than those of the control and other peptides, which resulted in indices of 1.84 ± 0.03, 2.08 ± 0.04 and 2.01 ± 0.12, respectively (p < 0.05) (The effects of rPro control A. Howeve < 0.05) B.On-Hep1 and sMatOn-Hep2 were investigated   than the control and other treated groups, which exhibited mortality rates of 91.7 ± 8.3%, 77.8 ± 9.6% and 80.6 ± 9.6% (p < 0.05)  A.A survival analysis of the challenge test in each group was performed using the Kaplan–Meier method.On-Hep1 and sMatOn-Hep2 in controlling S. agalactiae was tested in cohabitating conditions. The results in On-Hep1 demonstrated significantly lower mortality (10.0 ± 0.0%) than the control and other fish (p < 0.05). During these periods, fish injected with sMatOn-Hep1 (10 µg) or sMatOn-Hep2 (100 µg) also showed significantly lower mortality (30.0 ± 0.0% and 25.00 ± 7.07%) than fish injected with sMatOn-Hep2 (10 µg) or the control, which had mortality rates of 50.0 ± 0.0% and 75.0 ± 7.1%, respectively (p < 0.05). The mortality of the control group was significantly higher than those of the other groups (p < 0.05).The efficacy of sMatOn-Hep1 on the PA of Nile tilapia phagocytes, an in vitro experiment was designed. At 48 h, just 100 µg of this peptide could significantly enhance (78.0 ± 2.0%) the PA of Nile tilapia compared with those of the control and 10 µg sMatOn-Hep1-injected group  (p < 0.05) (On-Hep1 enhanced the phagocytic index (PI), which was significantly higher for these fish than for the control at 12 h and 24 h (p < 0.05), with values of 1.79 ± 0.04, 1.77 ± 0.14, 1.43 ± 0.03, 1.94 ± 0.31, 2.22 ± 0.05 and 1.53 ± 0.07 (On-Hep1 demonstrated a PI (2.79 ± 0.40) significantly higher than that in the control and other treated groups by 1.43 ± 0.10 and 1.93 ± 0.32, respectively (p < 0.05).To examine the effect of sMat < 0.05) A. Howeve3 ± 0.07 B. FurtheOn-Hep1 significantly induced serum lysozyme activity in Nile tilapia (p < 0.05). The highest induction was observed at h 48, and fish stimulated with 10 and 100 µg of sMatOn-Hep1 expressed lysozyme activities of 310.00 ± 10.00 and 240.00 ± 17.32 units/mL, respectively, which were significantly higher than that of the control (136.67 ± 32.15 units/mL) (p < 0.05)  C.50 in fish serum was investigated. However, no significant difference between fish groups was observed in any period of time (p > 0.05), with the concentration ranging from 91.37 ± 38.35-215.08 ± 60.67 units/mL (The ACHunits/mL D.On-Hep1 on the regulation of iron (Fe), zinc (Zn) and copper (Cu), the concentrations of these metal elements were quantified in the liver, blood corpuscle and serum of stimulated fish.To study the effects of sMatOn-Hep1 used significantly increased Fe concentrations (68.5 ± 3.0 and 73.2 ± 10.5 µg/g) in the liver, higher than those of the control (39.7 ± 2.7 µg/g). Moreover, at 48 h and 96 h, only 100 µg of peptides resulted in significantly higher Fe (79.3 ± 15.5 and 81.7 ± 14.4 µg/g) than the control and other treated group (p < 0.05), with consistent levels of 54.9 ± 6.6 and 45.1 ± 6.6, and 59.9 ± 9.1 and 38.4 ± 10.0 µg/g at the two respective times (p > 0.05) (On-Hep1 (10.2 ± 0.2 and 11.0 ± 1.0 µg/g) could significantly maintain Zn levels in fish liver at 96 h after injection, compared with the control (8.5 ± 0.3 µg/g) (p < 0.05) (On-Hep1 was found to rapidly decrease the Cu levels at 6 h in the liver by 35.5 ± 3.6 µg/g compared to the control and 100 µg of sMatOn-Hep1 treatment (52.2 ± 3.5 and 59.3 ± 11.9 µg/g) (On-Hep1 at 100 µg significantly increased the Cu levels by 1.6 ± 0.4 µg/g compared to the control and 10 µg of sMatOn-Hep1 treatment (0.4 ± 0.0 and 0.2 ± 0.0 µg/g) (p < 0.05)  C. For Zn > 0.05) D,E. Howe < 0.05) F. Regard.9 µg/g) G. sMatOn < 0.05) H. The coOn-Hep1 on the expression of eight immune-related genes, including CC chemokine1 (On-CC1), CC chemokine2 (On-CC2), CXC chemokine1 (On-CXC1), CXC chemikine2 (On-CXC2), interleukin 1b (On-IL 1b), hepcidin1 , hepcidin2 (On-Hep2) and transferrin (On-Trans), in three different organs were investigated by qRT-PCR techniques.In this experiment, the effects of sMatOn-Hep1 significantly enhanced the upregulation of these genes (p < 0.05) compared to their levels in the control fish by 75.4 ± 20.4-, 13.9 ± 4.7-, 2.6 ± 0.8-, 27.7 ± 13.4-, 2.7 ± 1.7-, 5.4 ± 3.3- and 22.2 ± 6.4-fold. At 6 h, this concentration of sMatOn-Hep1 further significantly upregulated On-CC1, On-CC2, On-Hep1 and On-Hep2, by 33.3 ± 6.5-, 11.8 ± 6.7-, 13.6 ± 8.1- and 11.4 ± 2.6-fold, respectively. Additionally, both 10 and 100 µg of sMatOn-Hep1 significantly enhanced the upregulation of On-CC1 at 12 h, On-CC2 at 48 h and On-CXC1 at 24 h, compared to their levels in the control group . However, the downregulated expression of these genes caused by the two concentrations of sMatOn-Hep1 was observed at 96 h after injection.In the head kidney A–H, veryOn-CC1 and On-Hep1 upregulation caused by injection with only 100 µg of sMatOn-Hep1 was demonstrated at 48 h, increasing their levels by 7.1 ± 4.3- and 400.3 ± 184.0-fold (p < 0.05), respectively. Ten micrograms of sMatOn-Hep1 strongly enhanced the expression of On-CXC1 and On-Hep1 at only 12 h, by 6799.3 ± 559.2- and 100.8 ± 31.1-fold, respectively. This concentration also significantly upregulated the expression of On-CC2, On-CXC2 and On-Trans at 12 h and 96 h after injection, resulting in values of 26.4 ± 8.3 and 6.2 ± 0.6, 9.2 ± 3.6 and 16.3 ± 6.8, and 45.3 ± 9.5 and 81.2 ± 26.7, respectively. Both injected concentrations (10 µg and 100 µg) of sMatOn-Hep1 effectively increased the expression fold of On-CC2, On-CXC2, On-IL1b, On-Hep2 and On-Trans expression (p < 0.05) at 48 h .In the liver A–H, cleaOn-Hep1 were found to have significantly downregulated On-CC2, On-CXC1, On-IL1b, On-Hep1 and On-Trans 1–96 h after injection (p < 0.05). In addition, 100 µg of sMatOn-Hep1 significantly increased the expression level of On-CC1 at h 6 by 16.8 ± 3.5-fold, and 10 and 100 µg upregulated On-CC1 expression at h 24 by 6.5 ± 1.4- and 6.5 ± 2.9-fold, respectively, while the control was expressed at approximately 1.2 ± 0.8. Interestingly, it was clear that the application of 10 and 100 µg of sMatOn-Hep1 strongly upregulated the expression of On-CXC2 compared to that of control fish, at 12 h, 24 h, 48 h and 96 h after injection by 0.18 ± 0.3, 295.2 ± 40.3 and 487.5 ± 182.8; 22.9 ± 9.3, 86.8 ± 41.2 and 631.3 ± 84.0; 35.6 ± 21.6, 320.9 ± 226.4 and 833.9 ± 217.7; and 11.46 ± 6.4, 356.4 ± 134.9 and 456.6 ± 174.6. In addition, 100 µg of sMatOn-Hep1 clearly significantly upregulated On-Hep2 expression, compared with that of other tested groups (p < 0.05) at 1 h, 24 h and 96 h, resulting in values of 28.7 ± 15.3, 31.3 ± 12.3 and 94.2 ± 7.8, respectively.In the spleen A–H, fishOn-Hep1 for 7 days; at days 1 and 2 after S. agalactiae injection, only the fish group that were given 100 µg of peptide showed high resistance to infection with no mortality, while the other groups exhibited rapid increases in mortality in these periods (p < 0.05) (On-Hep1-injected groups (p < 0.05). However, at days 6–10, the mortality rate of each group reached the highest points, at 86.7 ± 11.6, 73.3 ± 11.6 and 66.7 ± 11.6%, which were not significantly different until the end of the experiments (p > 0.05).After fish were injected with sMat < 0.05) . HoweverOn-Hep1) was successfully characterized. The full-length cDNA sequence encoding On-Hep1 contains 90 and 70 amino acids for immature and propeptide molecules, respectively, similar to the case for hepcidin molecules found in other vertebrates [On-Hep1 mature peptide consisted of 26 amino acids adjacent to the motif “RQ/H/KR/H” upstream, which is highly conserved in teleosts or cichlid hepcidins [On-Hep1 mature peptide. The theoretical isoelectric point (pI) value of the On-Hep1 mature peptide is 8.78, similar to those of previously reported hepcidins in other teleosts, which have net cationicity for facilitated interaction with negatively charged microbial surfaces [In this study, cDNA encoding the hepcidin gene of Nile tilapia  and hepcidin antimicrobial peptides 2 (HAMP2), based on their expression patterns and functional roles , suggestOn-Hep1 gene in a normal fish showed ubiquitous expression in all tested tissues, with the highest expression in the liver, and moderate expression in the spleen, head kidney, heart and PBLs. These results were similar to those of previous studies of HAMP1 in vertebrates, which were mainly expressed in the liver and moderately in other tissues, while HAMP2 were expressed in only the liver, suggesting that HAMP1 might play a crucial role in innate immune defense in fish in different manners [Tissue distribution analysis of the  manners ,32,33,34On-Hep1 after challenge with S. agalactiae and F. columnare in three tissues, the liver, spleen and head kidney, was clearly demonstrated. The challenge with the highest dose of S. agalactiae found that On-Hep1 expression concisely reached its peak at 12 h after challenge in all three organs. However, F. columnare challenge showed that On-Hep1 reached its highest peak at 6 h. This suggests that On-Hep1 is an acute-phase protein and prone to greater sensitivity in response to S. agalactiae than F. columnare. This finding reflects the fact that S. agalactiae is more systemic in inducing immune defenses than F. columnare, which prefers one to externally colonize the external surface areas of the host, such as the gills and skin, during the early stage of infection [Dicentrarchus labrax infected with 1 × 105 CFU of P. damselae spp. piscicida PP3 was strongly upregulated in the liver, spleen and head kidney, at days 1 and 2 after injection. These reports mostly supported many studies conducted in various fish species challenged with different pathogens [Streptococcus species and lipopolysaccharide, strongly induce hepcidin expression in the liver via the induction of IL-6, resulting in a rapid reduction in iron levels in serum [The expression of nfection . The resnfection , who fouathogens ,37,38,39athogens ,41. In vin serum ,43,44,45On-Hep1, sMatOn-Hep1 and sMatOn-Hep2 in controlling pathogenic bacteria, MIC and MBC assays were employed. However, no peptide inactivated those target bacteria, even though a maximal concentration of 128 mg/mL was used. A very mild inhibitory effect was observed in only rProOn-Hep1 against V. vulnificus. The results of this study are similar to the results of previous reports by Cai et al. [Omhep1 showed even better results than the synthetic mature peptide Omhep1. However, these results appear to differ from those of Gui et al. [Staphylococcus aureus, Vibrio anguillarum and Vibrio alginolyticus. Xu et al. [Brachymystax lenok) strongly exhibited antimicrobial activity against all bacterial strains tested for both Gram-negative and Gram-positive bacteria and fungi, such as Acinetobacter baumannii, Aeromonas salmonicida, A. hydrophila, Staphylococcus aureus, Enterococcus faecium and Candida glabrata, by destroying the cell membranes of these microorganisms. Liu et al. [To determine the efficacy of rProi et al. , who foui et al. , who fouu et al.  reportedu et al.  found thOn-Heps with various fish pathogens was first demonstrated in the present study. Moreover, sMatOn-Hep1 and sMatOn-Hep2 did not completely and strongly bind to all tested bacteria, since they bound only some portions in some cells. Additionally, sMatOn-Hep1 and sMatOn-Hep2 coincidentally bound nonpathogenic B. pumilus and adhered to pathogenic bacteria in different manners. Furthermore, sMatOn-Hep1 showed much stronger binding with F. columnare and S. agalactiae than sMatOn-Hep2 did, while sMatOn-Hep2 bound more strongly to A. hydrophila. The study of Sow et al. (2007) [The binding activity of FITC-. (2007)  clearly On-Hep1 significantly increased the PI over that of the control and rProOn-Hep1- and sMatOn-Hep2-treated groups, suggesting that sMatOn-Hep1 is vital for being more efficient at improving antigen engulfment than at enhancing phagocytosed cell numbers. Functionally, sMatOn-Hep1 may refold into a structure similar to the native structure, and act more effectively than rProOn-Hep1. Srinivasulu et al. [The efficacy of these peptides in enhancing phagocytosis in vitro was also analyzed. All peptides did not elevate PA; however, sMatu et al.  reportedOn-Hep1 and sMatOn-Hep2 on PBMCs and head kidney leukocytes was clearly observed in the cytoplasm of very few cells for PBMCs and many more for head kidney-derived cells. In PBMCs, the positive cells clearly exhibited round shapes, such as lymphocytes, which were also small. However, strong reactions of these two peptides were observed in leukocytes from the head kidney, which appeared similar to phagocytic macrophages with several large vacuoles. This suggests that both sMatOn-Hep1 and sMatOn-Hep2 function with only some specific lymphocytes in the blood circulation system and mainly work with mature leukocytes, especially macrophages. The mechanisms of hepcidin molecule ingestion in these cells are unknown. However, based on current information, ferroportin is known as an iron exporter that specifically binds hepcidin and presents on the surface of absorptive enterocytes, hepatocytes, placental cells and macrophages [Very little is known about the localization of hepcidin molecules in immune-related cells. Only Sow et al. (2007)  demonstrrophages . It has rophages , resultiOn-Hep1 on Nile tilapia immune responses were examined by intraperitoneal injection. This study demonstrated the crucial functional roles of sMatOn-Hep1 in enhancing phagocytosis and lysozyme activity, but not complement activity. The results clearly showed that both 10 µg and 100 µg of sMatOn-Hep1 did not enhance the PA of Nile tilapia PBMCs, but significantly enhanced the PI compared with that of the control group. These results suggest that sMatOn-Hep1 possesses a crucial ability to stimulate phagocytes, by enhancing their engulfment efficacy during acute period responses at 12–48 h. These two concentrations also strongly elevated serum lysozyme activity concurrently with the PI during 24–48 h, suggesting that increasing MatOn-Hep1 strongly affected not only the engulfment of antigens, but also lysozyme activity, which is important for innate immune responses to invading pathogens in Nile tilapia.The effects of sMatOn-Hep1 may possess vital activity to modulate macrophages and lymphocytes in Nile tilapia immune responses, similar to some other AMPs.Recently, Zhang et al. 2017  demonstrOn-Hep1 additionally enhanced or suppressed the expression of some immune-related genes in different target organs with different patterns. The application of sMatOn-Hep1 clearly elevated the upregulation of On-CC1 and On-CC2 in head kidney from 1–24 h, and those of On-Hep1 itself or On-Hep2 from 1–6 h or 1–12 h, respectively. Almost all immune-related genes were significantly upregulated at h 12 and 48, especially iron transport-related genes, including On-Hep1, On-Hep2 and On-Trans. On the other hand, in the spleen, all immune-related genes were strongly suppressed, except On-CXC2 and On-Hep2, which maintained their significant expression levels during the experimental period. In agreement with much previous research, hepcidin regularly increased the expression levels of many immune-related genes [hepcidins, ferroportin and ferritin), cytokines  and specific immune responsive genes  [On-Hep1 may be a strong regulator that is a vital part of inducing these key immune responsive genes (like other vertebrate hepcidins), which normally function in both innate and acquired immunity.Furthermore, sMated genes ,52,53,54 MHC II) . This suOn-Heps, enhancing resistance against S. agalactiae, was clearly implicated. Survival analyses of these On-Heps demonstrated that all of them are truly effective at protecting Nile tilapia from this harmful pathogen. The greatest protection was observed following the sMatOn-Hep1 application. The mechanisms by which protection is conferred by sMatOn-Hep1 could be described and supported by its ability to improve the antigen engulfment efficiency and innate immune responses, especially lysozyme activity, binding to pathogens and upregulation of important immune-related genes.The most important ability of Streptococcus species have developed various mechanisms to uptake iron from environments with limited available iron. They can directly extract iron from host iron-containing proteins, such as ferritin, transferrin, lactoferrin and hemoproteins, or indirectly extract it by relying on specialized secreted hemophores (heme chelators) and small siderophore molecules (high-affinity ferric chelators) [S. agalactiae homeostasis [On-Hep1 reduced the Fe and Cu concentrations in the liver and blood corpuscles of treated fish, but not in serum. This finding suggests that the target peptide clearly functions to modulate metals other than Fe and that the highest concentration of this peptide is not enough to affect the reduction in Fe levels in serum.Previously, it was shown that continuously high levels of hepcidin strongly decreased serum iron levels, presumably reducing bacterial growth , and lowelators) . Accordieostasis . In our Dicentrarchus labrax) against V. anguillarum. In addition, Wei et al. (2018) [Ctenopharyngodon idellus challenged with F. columnare. The fish that were fed a diet containing recombinant CiHep had a higher survival rate than other groups. The study also showed that recombinant CiHep effectively regulated iron metabolism, causing iron redistribution and decreasing the levels of serum iron at the early stage of infection, indicating that the host responded by withholding iron from pathogens to slow down their rapid growth. Information from Michels et al. [These results are similar to the results of previous reports by Alvarez et al. , who fou. (2018)  reporteds et al.  stronglyF. columnare. This could be the same function as that shown by the experiments in Nile tilapia in this study.Basically, hepcidin is enormously enhanced during pathogenic infections such as fungal, viral and bacterial infections ,61. Low On-Hep1 could decrease levels of Fe or other metals in fish tissues, to not only inhibit bacterial growth, but also reduce the virulence of bacteria, increasing fish resistance to invading pathogens. To our knowledge, this is the first report of in vivo hepcidin application that provides crucial information for application in streptococcosis resistance in Nile tilapia. Appropriate field trials are needed to prove this indispensable ability of MatOn-Hep1 in Nile tilapia aquaculture.This information indicates that application of sMatOn-Heps were successfully characterized for their structure and evolutionary relationships at the molecular level and compared with various hepcidins reported in vertebrates. qRT-PCR analyses clearly implied significant responses of On-Hep1 to two severely pathogenic bacteria, S. agalactiae and F. columnare, as an acute-phase factor. The results also clearly showed that rProOn-Hep1, sMatOn-Hep1 and sMatOn-Hep2 were effective against pathogenic bacteria, especially sMatOn-Hep1, even though the in vitro antimicrobial activity was unclear. sMatOn-Hep1 was obviously detected in lymphocyte-like cells from PBMCs and macrophage-like cells isolated from the head kidney. Additionally, it crucially functions as an immunoregulator important for elevating immune responses, especially phagocytic and lysozyme activities, the expression of crucial immune-related genes and the regulation of iron ion levels in the liver. Importantly, among all tested peptides, sMatOn-Hep1 perfectly proved its ability to protect Nile tilapia from streptococcosis. Information obtained from this study is important for properly implementing an effective strategy to prevent harmful diseases caused by S. agalactiae in the Nile tilapia aquaculture industry.In the present study,