The Manganese- Zinc N.S.F.s nanomaterials were deeply characterized, utilizing UV-Vis (reflectance) spectroscopy, X-ray diffraction, N2 adsorption isotherm measurements, and S.E.M., SEM-EDX mapping, and T.E.M. The Kinetic model for the photodegradation of penicillin G (as a model molecule) is investigated using visible light as a source of energy. The kinetic study shows that our results fit well with the modified pseudo-first-order model. The Pen G degradation are 88.73%, 66.65%, 44.70%, 37.62% and 24.68% for x = 0.5, 0.4, 0.3, 0.2 and 0.1, respectively, against 14.68% for the free Cd spinel sample. The pseudo-rate constant is bandgap dependent. From the intra-diffusion rate constant (Kd), we developed an intra-diffusion time (τ) model, which decreases exponentially as a function of (x) and mainly shows the existence of three different domains versus cadmium coordination in spinel ferrite samples. Hence, Cadmium’s presence generates spontaneous polarization with a strong opportunity to monitor the charge separation and then open the route to a new generation of “assisted” photocatalysts under visible light.Penicillin G is an old and widely used antibiotic. Its persistence in the environment started to appear in many environmental samples and food chains. The removal of these emerging pollutants has been a challenging task for scientists in the last decades. The photocatalytic properties of Cd Nowadays, the contamination of water bodies due to the presence of pharmaceutical drugs cause a devastating effect on the environment and is considered to be a serious concern worldwide. Among them, antibiotics are widely used medicines to treat human and veterinary infections . A human3), and super oxide radical (O2-°), which facilitate the oxidation of antibiotics and transform them into harmless by-products O4 (x ≤ 0.05) NSFs onto penicillin G antibiotic utilizing visible light as an energy source. The main objective of this work is to evaluate the photocatalytic properties of Cd2+ doped Manganese- Zinc NSFs with empirical formula (Mn0.5Zn0.5)[CdxFex2−]O4 (x ≤ 0.05) NSFs were produced by the UV irradiation method. A proper ratio of Fe[NO3]3.9H2O (Iron Nitride), Zn[NO3]2 (Zin Nitride), Cd[NO3]2.4H2O (Cadmium Nitride), and MnCl2.4H2O (Manganese Chloride) were dissolved in 100 mL of Deionized water. After stirring the solution continuously for one h, the pH was amended at 11 by drop wisely of NaOH. Subsequently, the final solution was exposed to the U.W. irradiation for 40 min via 70 W and 20 kHz Ultrasonic Homogenizer from UZ SONOPULS HD 2070. The resulting mixture was washed 3 to 5 times with Deionized hot water and then dried overnight at 60 °C.Cd3) containing Penicillin G  as a model contaminant. The reaction was carried out in a double-walled thermostat Pyrex photoreactor (100 cm3) with an optical window area of 12.5 cm2. The concentration of photocatalyst was set at 1.0 g.L−1. The pH of the different solutions was close to 6.0 ± 0.2 and the temperature was set as constant with chiller at 20.0 ± 0.2 °C for all the experiments. HQI-E 400 W/n plus visible lamp coated with UV filter was used for visible irradiation. The rated lamp efficacy in the standard condition is 91 lm/W; the filter coats the lamp for UV radiation removal Typically, in a photocatalytic experiment, 30 mg of (Mn0.5Zn0.5)[CdxFex2-]O4 (x ≤ 0.05) NSFs ultrasonically suspended in 30 mL of 100 ppm aqueous solution of contaminant (PenG). Stirring the mixture for 60 min to reach the adsorption equilibrium in the dark; then, while the mixture was air bubbled and stirred, it was photo-irradiated at 20.0 ± 0.2 °C, utilizing a solar lamp (HQI-E 400 W/n plus visible lamp). After reaching equilibrium, the substrate concentration was measured, and considered the initial concentration (C0) to deduct the dark adsorption. PenG samples were taken at different intervals (Ct) from the reactor’s solution upper part, and then filtered using 0.45 µm nylon syringe filters. Consequently, the samples were analyzed utilizing a Shimadzu High-Performance Liquid Chromatography (HPLC) and (300 mm × 7.8 mm) Hypersil Gold column and a U.V.–Vis detector (λ = 210 nm). A mobile phase of H2SO4 (510−3 mol/L) was used, flowing at 1 mL/min. During all the experiments, three runs for all the photocatalytic tests have been achieved with good reproducibility. Photocatalytic tests were performed using an aqueous solution  XRD system with Cu Kα radiation. The microstructure was analyzed via  scanning electron microscopy (S.E.M.) coupled with E.D.X. T.E.M., SAED patterns, and high-resolution T.E.M. (HR-TEM) analyses were performed using an F.E.I. Titan S.T. microscope (300 keV). Pore structure and surface area measurements were performed using a Micrometrics ASAP 2020 instrument, Norcross, USA. Before the adsorption measurement, 0.05 g of the calcined catalyst was degassed by flowing nitrogen for 3 h at 240 °C. The adsorption isotherms were plotted at 196 °C (liquid nitrogen temperature). Relying on the B.J.H. adsorption calculation method, pore diameter, pore-volume, and pore surface area were measured.0.5Zn0.5)[CdxFex2-]O4 (x ≤ 0.05) NSFs. The characteristic peaks of Manganese- Zinc spinel ferrite structure with space group Fd3m appeared in all samples. There is a miner phase of CCdO3 at x = 0.3 to 0.5. The lattice constant ‘a’ was increased by raising the Cd ratio in the range of 8.312(5)–8.508(0). Further, the average crystal size was evaluated using the Debye-Scherrer formula and was found to be in the range of 6–9 nm. 0.5Zn0.5)[CdxFex2-]O4  NSFs. The intermediate magnification images indicated a cluster of small, homogeneously distributed semi-cubic particles. The efficiency of the preparation method was approved through elemental mapping and EDX spectra of (Mn0.5Zn0.5)[CdxFex2-]O4 (x = 0.2) NSFs, as seen in 2 physisorption measurements. In order to gain more insights into the textural properties of the prepared materials, the surface area analysis was carried out using the TriStar II PLUS surface area analyzer from Micromeritics, Norcross, USA, which was used to obtain measurements of pore size distribution, pore-volume, and surface area of the samples. The materials were degassed at 190 °C for three hours under a vacuum in order to eliminate impurities prior to NThe more complex pore structures resulted in the materials x = 0 and x = 0.1 being close to type H2 where the network effects are significant. According to IUPAC, the other materials show the adsorption hysteresis H3, indicating that the materials’ pores are slit-shaped . H3 hysteresis desorption curves contain slopes associated with a force on the loop of the hysteresis, owing to what is called the tensile strength effect ,21,22. 0.5Zn0.5)[CdxFex2-]O4 (0.0 ≤ x ≤ 0.05) NSFs were investigated using a diffuse reflectance UV-vis spectrometer. The optical properties of (F(R∞Eg) of samples are given by the expression:Eg and (n = 1/2) directly symbolizes the allowed electronic transition. By plotting Eg values can be estimated. A straight line fit to the linear section of the graph intercepts with the energy (y) axis at Eg in eV units. 0.5Zn0.5Fe2O4 has a 1.62 eV Eg value. Cd3+ ion coordinated samples have band gaps 1.67, 1.68, 1.74, 1.84 and 1.87 eV of magnitude, corresponding to increasing Cd3+ ion proportion from x = 0.1 to x = 0.5. Hence, it is observed that the coordination of Cd3+ ion causes significant increments at direct Eg of mixed spinel ferrite sample. However, all estimated Eg data are in the bandgap range of semiconductors. Ashok and Nam’s groups report the direct Eg values of 1.98 and 1.99 eV for mixed Mn0.5Zn0.5Fe2O4 NPs, which were prepared via hydrothermal and sonication assisted microwave irradiation methods, respectively [0.5Zn0.5Fe2O4 and Mn0.5Zn0.5DyxFe2-xO4 (x = 0.01–0.03) NPs were produced using ultrasonic irradiation in a narrow range from 1.61 to 1.67 eV [Eg data for (Mn0.5Zn0.5)[CdxFex2-]O4 NSFs in the literature. Linear correlation between optical absorption and optical energy band gap (Equation :(4)[CdxFex2-]O4 (x ≤ 0.05) NSFs are herein evaluated under visible light radiation. 0.5Cd1.5xFe2-xO4 14.88% (x0), 24.68 %(x0.1), 37.62%(x0.2), 44.70%(x0.3), 66.65%(x0.4) and 88.73% (x0.5).The photodegradation of Pen G onto (Mn0/C as a function of cadmium coordination (x). The first overview of the curvatures’ trend indicates that the degradation is enhanced by increasing Cd coordination in mixed spinel Mn0.5Zn0.5Fe2O4 catalysts  pairs recombination. atalysts . The PenThe photodegradation rate of Pen G can be expressed asIf we consider that the reaction follows pseudo first order kinetics, then a = 1, and thus the equation (Equation (5)) becomes:By rearranging equation (Equation (6)) we obtain:0 is Penicillin G concentration at time zero = C0, and [Pen G]t is the concentration at time (t > 0) C t.By integrating the expression (Equation (7)) from the t = 0 at initial concentration to t > 0 at final concentration, the Equation (7) becomes:Therefore, we can get the rate constant by plotting 0.5Zn0.5Fe2O4 spinel catalysts assigned to pseudo first-order. From 1 (min−1) increases versus Cd loading. Nevertheless, from N2 adsorption-desorption results, the specific surface areas of Mn0.5Zn0.5Fe2O4 spinel catalysts decreased tremendously vs. Cd loading (the SBET is 138 m2·g−1 for Mn0.5Zn0.5Fe2O4 against 43 m2·g−1 for Cd (x = 0.5) Mn0.5Zn0.5Fe2O4). The amount of catalyst was kept constant for all the experiments, and to elucidate the effect of the variation of surface area, the pseudo rate constants can be expressed per m2 . The natural logarithm ln(k1) and ln(k’1) were plotted vs. Cd loading for the different spinel catalysts  and intrinsic rate (lnk’1), showing no significant effect on the surface area. Moreover, the kinetic process seems to be more affected by cadmium loading (x). Three domains are mainly obtained, namely: (I) for x < 0.2, ki increases linearly; (II) for 0.20 < x < 0.30, ki increases slowly and (III) for x > 0.30, ki increases linearly. From n (n > 1 and n ≠ 1) for correlating the present degradation (Equation (10)).n = 1.65). Nevertheless, the few values of (C/C0) and the lack of smoothness in n-value. For this constraint, and to reduce the discrepancy between experimental and estimated values, we thought it better to extend the model of the first pseudo order (Equation (9)) by a small modification in the second number, which becomes a second-degree polynomial by adding a t2-term, and this was expressed as follows:a1) has been put in the common factor (Equation (12)), which becomes equivalent to a kinetic rate constant where the new derived parameter (α) represents an increment and its amount shows how much the kinetic process deviates from the true pseudo first-order.In order to compare the first pseudo-order (Equation (9)) with the proposed modified one (Equation (11)), parameter (R), which exhibit a clear improvement. In addition, the values of (α) decrease with the increase of (x), showing that the nature of the kinetic process approaches the true first pseudo order for high values of x. We note that this ascertainment is also confirmed by the R-values increase in k1).a1 (a2 (x). We noticed the presence of three domains with distinct behaviors, which confirms the earlier observation in a1) shows a clear jump for x higher than 0.3. a1 a and a2 a1 (a2 b vs. Cd −3 s−1 (0.02 10−3 min−1). The same authors found that the U.V./peroxodisulfate (P.D.S.) process enhanced the photodegradation rate of Pen G; a pseudo rate constant of 0.5 × 10−3 min−1 is achieved in the presence of 5 mM P.D.S. In the present work, mixed spinel ferrite (x = 0.5) shows a pseudo-rate constant of 12.47 × 10−3 min−1 (~25 times higher) under visible light. Moreover, the drawback of the U.V./peroxodisulfate (P.D.S.) process is sulfate ions formation.Zhou et al.  investig2+ system is effective in the photodegradation of four classical penicillins: ampicillin, amoxicillin, and both G&V penicillins. The pseudo-rate constant for penicillin G is 0.349 × 10−3 min−1. Using the Cd2+/UV system, the pseudo-rate constant for penicillin G reached 0.79 × 10−3 min−1 O4 (x ≤ 0.05) NSFs. From pendence . (Mn0.5Z0.5Zn0.5)[CdxFex2-]O4 (x ≤ 0.05) NSFs on the photodegradation of penicillin G (Pen G) was herein studied. The physical characterization was achieved using X-ray diffraction, Raman, UV-vis (reflectance) spectroscopies, N2 adsorption isotherm measurements, and S.E.M., SEM-EDX mapping, and T.E.M. allow for determining the influence of cadmium coordination on the photocatalytic response of Manganese-ZincNSFs nanostructured materials. Several catalysts were tested with different Cd coordinations  for the photodegradation of penicillin G (Pen G) as a model molecule. The kinetic study shows that our results fit well with the pseudo-first-order model. The rate constant k1 increases versus Cd coordination and clearly highlights bandgap (Eg) dependence. For x = 0.5, the rate constant is enhanced by a factor of 13.5 with respect to the Cd-free spinel ferrite catalyst (x = 0). On the contrary, the rate constant increases slightly between x = 0.2 and x = 0.3 and rapidly above x = 0.3. Hence, Cadmium’s presence probably induces the electric field’s formation, which decreases the recombination rate of electron-hole  pairs. This finding highlights a new generation of photocatalysts with a “tuned bandgap.” Further experiments are ongoing for the treatment of industrial effluents containing penicillin G antibiotic.The effect of Cadmium metal coordination on the structural, textural, and morphological properties of spinel ferrite (Mn


These can effectively reduce the volume change of Si, extend the cycle life, and increase the lithium-ion battery capacity due to the dual protection of MnO2 and rGO. The sea urchin-like Si@MnO2@rGO anode shows a discharge specific capacity of 1282.72 mAh g−1 under a test current of 1 A g−1 after 1000 cycles and excellent chemical performance at different current densities. Moreover, the volume expansion of sea urchin-like Si@MnO2@rGO anode material is ~50% after 150 cycles, which is much less than the volume expansion of Si (300%). This anode material is economical and environmentally friendly and this work made efforts to develop efficient methods to store clean energy and achieve carbon neutrality.Si is a promising material for applications as a high-capacity anode material of lithium-ion batteries. However, volume expansion, poor electrical conductivity, and a short cycle life during the charging/discharging process limit the commercial use. In this paper, new ternary composites of sea urchin-like Si@MnO As a carrier of clean energy, lithium-ion batteries play a pivotal role in the country’s goal of achieving carbon neutrality ,2,3,4. AmAh g−1) ,6. It ismAh g−1) ,8. The smAh g−1) ,10,11,122 (1233 mAh g−1) [2 in lithium-ion intercalation and deintercalation is very small. A reasonable coating structure was used to grow MnO2 evenly outside the Si nanospheres, which can avoid the material crushing and form an unstable solid electrolyte interface (SEI) film due to the volume expansion of Si nanospheres during the circulation process, lowering the capacity. At the same time, rGO has high electrical conductivity and excellent physical properties. The low conductivity of transition metal oxides and Si nanospheres can be compensated by rGO. MnO2 wraps the Si nanosphere to avoid the agglomeration of the Si nanosphere, increase the contact area with the rGO sheet, ensure the interface strength between MnO2 and rGO, and improve the structural stability of the material [The rational design of an anode has been considered as an effective strategy to enhance Si-based lithium-ion batteries’ performances ,14. Some2 13 mAh g−1mAh g−1) ,26,27,28material ,30,31,322@rGO as anodes. In this unique structure, MnO2 and rGO surrounding Si nanospheres formed a strong armor, relieving the mechanical strain generated by the volume expansion of the Si nanospheres and providing enough space to buffer, which helps provide to a long lifetime. In addition, the sea urchin-like Si@MnO2@rGO anode showed an initial discharge capacity of 1378.15 mAh g−1 at a current density of 0.1 A g−1 and a discharge specific capacity was maintained at 1282.72 mAh g−1 under a test current of 1 A g−1 after more than 1000 cycles, showing excellent chemical performance at different current densities. Therefore, this rational design provides a new route for the development of high-performance Si-based anodes; this work made efforts to store clean energy and achieve carbon neutrality [Herein, we propose a simple hydrothermal method to produce sea urchin-like Si@MnOutrality ,34.4, KMnO4, KH550, and NaBH4 were obtained from National Medicines Corporation Ltd, Shanghai, China.All chemicals were analytical reagent grade and used as received. Graphene oxide (GO) was purchased from XFNANO Materials Tech Co., Ltd, Nanjing, China. Si nanospheres, ethanol, MnSO4 were ultrasonically dispersed in 100 mL of deionized water with stirring treatment at 80 °C for 24 h. After cooling to room temperature, the sample was centrifuged to obtain reduced graphene oxide (rGO). The above-prepared rGO of 0.05 g was dispersed in 20 mL of deionized water and sonicated for 30 min, denoted as Solution A.First, 0.5 g of GO and 0.1 of M NaBH4, and 1 g of KMnO4 were sequentially added in a reaction vessel and stirred with magnetic force for 2 h. The reaction temperatures were set as 20, 50, or 160 °C, respectively. After cooling to room temperature, the products were centrifuged with water and ethanol several times to remove the residual reaction products. A freeze-dried treatment was used to remove the water. Finally, the samples were denoted as Si@MnO2@rGO-20°C, Si@MnO2@rGO-50°C, and Si@MnO2@rGO-160°C, respectively.Then, 0.086 g of Si nanospheres (50 nm) were dispersed in a beaker containing 20 mL of water and 20 mL of alcohol, followed by the addition of 50 μL of KH550 silane coupling reagent. The sample was sonicated for 30 min to form a homogeneous solution, denoted as Solution B. Next, Solution A, Solution B, 0.64 g of MnSO2 was similar to the synthesis Si@MnO2@rGO, only missing the rGO, denoted as Si@MnO2-20°C, Si@MnO2-50°C, and Si@MnO2-160°C, respectively.The synthesis of Si@MnO2@rGO, acetylene black, and polyvinylidene fluoride (PVDF) in N-methyl-2-pyrrolidone (NMP) with a mass ratio of 8:1:1. After fully stirring, the prepared slurry was evenly coated on a copper foil by a sputter coater, whose space was 25 μm. The dimeter of electrode was 1.5 cm and the anode material mass load was ~1 mg. The electrolyte of each coin cell was ~45 μL. Then, the electrode sheet was placed into a vacuum drying oven for 12 h at 80 °C. The working electrode was the prepared electrode sheet (Si@MnO2@rGO composites). The electrolyte was a LiPF6 (1.0 mol L−1) in a 1:1:1 v/v/v mixture of ethylene carbonate, dimethyl carbonate, and ethyl methyl carbonate. The separator was Celgard 2400 . The capacity calculation and cycling rate were set by a battery testing system . The galvanostatic charge/discharge (GCD) tests were conducted in the voltage window of 0.1–3.2 V. The cyclic voltammetry curve (CV) was carried out with scan rate of 0.1 mV s−1 between the voltage range of 0.1–3.2 V using an electrochemical workstation . The electrochemical impedance spectroscopy (EIS) was performed over the frequency range from 100 kHz to 0.1 Hz with an alternating current (AC) impedance of 5 mV and was also recorded by an electrochemical workstation .The homogeneous slurry was prepared by mixing Si@MnOField-emission scanning electron microscopy  and field-emission transmission electron microscopy  were used to characterize the morphology and elemental distribution of the electrode materials. X-ray diffraction  was used to characterize the phases, crystallinity, and crystal structures of the samples. The species and chemical composition of the surface elements of the samples were analyzed using X-ray photoelectron spectroscopy .2@rGO composites. First, GO sheets were reduced to rGO by a hydrothermal method with NaBH4. Then, the rGO was collected and dispersed in deionized water (Solution A). Next, Si nanospheres were dispersed in a solution of water, alcohol, and the KH550 silane coupling agent (Solution B). The composites were well grafted by KH550, improving the conductivity. Finally, Solution A, Solution B, KMnO4, and MnSO4 were added in a reaction vessel and stirred with magnetic force for 2 h. The reactions were carried out at different temperatures, resulting in different morphologies. Si nanospheres were coated with MnO2 and rGO, which may have contributed to the KH550. The unique double-layer structure of MnO2 and rGO effectively mitigated the volume expansion of Si nanospheres during the charging/discharging process. In addition, rGO had excellent electrical conductivity, compensating for the low electrical conductivity of Si nanospheres and MnO2. This was the basis of the rational design of the Si-based anode material for enhancing lithium-ion battery performance.2-20°C is shown in 2 formed disordered and entangled filaments on the surface of Si nanospheres. 2-20°C at a high magnification. The surface of the material was rough and the diameter of Si@MnO2-20°C particles was 200 nm. 2@rGO-20°C, showing that the rGO covered the Si@MnO2-20°C. However, some MnO2 nanowires appeared on the surface of rGO, which suggested that the material was not stable. 2-50°C at different magnifications, respectively. As shown in 2 formed a sea urchin-like shell coating on the surface of Si nanospheres. 2-50°C. The stings (MnO2) were 100–300 nm in length. 2@rGO-50°C. The rGO wrapped the Si@MnO2-50°C without obvious damage to the structure. Interestingly, this rGO surface was clean, without any MnO2 nanowires, indicating the sea urchin-like structure was more stable. MnO2 nanowires were vertically oriented on the outer layer of the Si nanospheres, imbedding rGO layers, forming a stable structure, which can buffer the excessive expansion of the Si nanospheres during the charging/discharging process and extend the batteries’ lifetime. 2-160°C. The MnO2 nanowires grew longer and coated the surface of the Si nanospheres. 2 nanowires were interleaved. 2@rGO-160°C. It can be clearly seen that the MnO2 nanowires were broken and dispersed on the surface of rGO, which indicates that the Si@MnO2@rGO-160°C was not stable enough. The stability of the material is one of the important factors of the lithium-ion batteries’ performance. The high stability of the anode material may suggest the long life-time. Therefore, the Si@MnO2@rGO-50°C was chosen for the following test.2@rGO-50°C contained ~48% Si, ~21% rGO, and ~31% MnO2. 2, ~35.6%; and rGO, ~1.1%). The existence of MnO2 can not only resist the volume expansion of Si, but also provide the capacity contribution. 2@rGO-50°C composite at different magnifications. Si nanospheres were coated with rGO and MnO2. The lattice fringes corresponded to the (111) plane of Si, having a separation of 0.31 nm [2 [2@rGO-50°C composite is shown in 2; Si element contributed to Si nanospheres. C element dispersed throughout the image may have contributed to both the substrate and rGO. 0.31 nm . In addi31 nm [2 ,37,38. T2-50°C, and Si@MnO2@rGO-50°C, respectively. In the XRD pattern of Si@MnO2-50°C, the pronounced peaks at 28.5°, 47.4°, 56.2°,58.9°, 69.2°, 76.5°, and 88.2° corresponded to the (111), (220), (311), (222), (400), (331), and (422) planes of Si (PDF#77-2108). In addition, the characteristic peaks at 12.7°, 18.0°, 28.7°, 37.6°, 41.1°, 49.8°, 59.5°, 65.5°, 68.5°, and 72.5° corresponded to the (110), (200), (310), (121), (420), (411), (260), (002), (202), and (631) planes of MnO2 (PDF#72-1982). The XRD pattern of the Si@MnO2@rGO-50°C composites contained peaks corresponding to Si and MnO2, as well as the broad peak ranges from 20° to 30°, which were indexed to the standard peaks of rGO [2@rGO-50°C composites, which revealed the presence of Si, Mn, O, and C, corresponding to element mapping (3/2 (642.5 eV) and Mn 2p1/2 (654.1 eV) of MnO2, whose separation between these peaks was 11.6 eV.s of rGO ,40,41,42 mapping d–g. Figu2@rGO-50°C as the independent anodes of lithium-ion batteries for the first four cycles at a scan rate of 0.1 mV s−1 between 0.1 V and 3.2 V. In the first cycle, a clear cathodic peak at 0.16 V corresponded to the lithium alloying process of crystalline Si and the formation of an amorphous LixSi phase; a clear cathodic peak at 0.10 V corresponded to the formation of the Li15Si4 phase. An anodic peak at 0.24 V was related to the delithiation of Li15Si4; an anodic peak at 0.50 V was related to the transition from the LixSi phase to amorphous Si, according to Equations (1) and (2).A CV experiment carried out to further evaluate the lithium storage behavior is shown in 2, according to Equation (3). When the cathodic peak was 0.36 V, Li was inserted into the anode to form LiO2 and MnO2 was reduced to Mn. An anodic peak at 1.23 V was related to the charging process of the lithium ion battery; Mn can facilitate the decomposition of LiO2.The redox peaks of Si coincided with those reported previously ,44,45. I2@rGO-50°C, indicating the good electrochemical reversibility for lithium-ion batteries.In the subsequent scans, the redox peaks were largely coincident, which was attributed to the stability of the Si@MnO2@rGO-50°C at 0.1 A g−1 over the range between 0.1 V and 3.2 V. In the first cycle, the initial discharge capacity was 1378.15 mAh g−1. In the second, third, fifth, and 10th cycles, the discharge specific capacities were 1279.21, 1208.02, 1150.74, and 1093.70 mAh g−1, respectively. In the subsequent cycles, the charge and discharge curves basically overlapped, which indicated good capacity retention. In addition, the plateau of Si at 0.5 V in the figure was consistent with the CV results.2-50°C, Si@MnO2@rGO-20°C, Si@MnO2@rGO-50°C, and Si@MnO2@rGO-160°C) at a current density of 0.1 A g−1. Of these samples, Si@MnO2@rGO-50°C showed the best cycling performance. The initial discharge specific capacity of Si was 1855.62 mAh g−1. However, after 20 cycles, the discharge specific capacity decayed to 330.03 mAh g−1; after 150 cycles, the discharge specific capacity was almost 0. This is because, during the cycling process, the slurry of the active material became dislodged from the collector due to the serious volume expansion of Si nanospheres caused by lithium-ion intercalation and deintercalation. When the Si nanospheres underwent volume changes during the charging/discharging process, the formed SEI film was broken, resulting in new surfaces being exposed in the electrolyte. The exposed surfaces needed external lithium ions to form a stable SEI film, which led to a dramatic decrease in capacity. Although Si@MnO2-50°C had a higher discharge specific capacity than Si nanospheres alone after 150 cycles, it still did not meet current demands for battery energy storage. The initial specific capacity of Si@rGO-50°C was 1180.41 mAh g−1; after 150 cycles, the capacity was maintained at 543.84 mAh g−1  before cycling, which was the lowest among Si, Si@MnO2@rGO-50°C, and Si@MnO2-50°C. This result was attributed to the rGO networks, prominently improving the electronic conductivity of the electrodes and lowering the charge transfer impedance. 2@rGO-50°C before and after 150 cycles at 0.1 A g−1; cycled Si@MnO2@rGO-50°C was fitted with the same equivalent circuit model in CT (72.7 Ω) was lower after cycling, which implies the charge transfer impedance decreased substantially, demonstrating the stable SEI film and good structure stability during cycling of Si@MnO2@rGO-50°C.EIS measurements were carried out to further investigate the electrochemical mechanism shown in material ,57. The −1. After cycles, the volume expansion was ~323% (from 5.3 μm to 22.41 μm). The huge change in volume expansion of Si may have contributed to the intercalation and deintercalation of lithium ions during cycles and the exfoliation of the active materials. As shown in 2-50°C electrode after 150 cycles at 0.1 A g−1 was ~198% (from 4.29 μm to 12.8 μm). Interestingly, the volume expansion of Si@MnO2@rGO-50°C electrode was ~59% (8.13 μm to 12.9 μm) after 150 cycles at 0.1 A g−1. Such a small volume change in the active materials guarantees long-term cycling stability. 2@rGO-50°C after 150 cycles at 0.1 A g−1. The electrode sheet was relatively intact without signs of rupture. xSi) (0.16 V). Then, the amorphous LixSi was transformed to crystalline Li15Si4 (0.1 V). The delithiation process involved the transformation from crystalline Li15Si4 to amorphous LixSi (0.24 V) and, finally, to amorphous Si (0.5 V) [ (0.5 V) ,59,60.2@rGO-50°C as an anode for lithium-ion batteries was presented. The reversible capacity, cyclability, and rate capability were very high, which was attributed to the dual protection of rGO and MnO2. The discharge specific capacity was maintained at 1282.72 mAh g−1 under a test current of 1 A g−1 after more than 1000 cycles. Such high cyclability may be attributed to the sea urchin-like structure reducing the volume expansion of anodes during the charging/discharging process. The present results indicate that the sea urchin-like Si@MnO2@rGO-50°C is a good candidate for high performance anodes of lithium-ion batteries. This work made efforts to develop efficient methods to store clean energy and achieve carbon neutrality.In summary, sea urchin-like Si@MnO


Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete  In addition to these structural components, a solvent-soluble fraction, commonly referred to as extractives, constitute up to 10 w/w% of the total plant biomass, though the chemical compounds vary with tree species, age and position, i.e. bark, heartwood or sapwood 5. Extractives are considered to be a factor impeding or excluding fungal colonization of the freshly harvested conifers6. The ligninolytic basidiomycete Phlebiopsis gigantea can rapidly invade such softwood7 but little is known about the mechanism(s) of how P. gigantea could contend with the extractives from the plant9. A measure of this efficient softwood colonization has been the use of P. gigantea inoculum to reduce the spread of Heterobasidion root rot on conifer stumps11. Thus, P. gigantea has evolved a unique system to efficiently transform and/or utilize conifer extractives.The most abundant source of terrestrial carbon is woody plant biomass. The cell wall largely consists of cellulose, hemicellulose and lignin, the proportions of which differ between hardwood and softwood speciesP. gigantea possesses hundreds of genes encoding carbohydrate active enzymes (CAZy) for plant cell wall depolymerization and utilization12. Our recent proteomic analyses demonstrated that P. gigantea employs a suite of classical cellulases, hemicellulases, and lignin-degrading enzymes when colonizing freshly cut pine in the presence of various extractives13. The transcriptome profiles further suggest that P. gigantea processes coniferous extracts by using ATP-binding cassette (ABC) transporter and a glutathione S-transferase similar to other fungi. For example, a set of genes encoding an efflux ABC transporter in Grosmannia clavigera is thought to confer monoterpene-tolerance14, and a glutathione S-transferase in Phanerochaete chrysosporium provides antioxidant activity15. Other genes are suggested to function in lipid metabolism by P. gigantea but our knowledge of specific genes involved in the metabolism(s) of coniferous extractives by this fungus remains uncertain in large part due to the complexity of the woody substrate13. In short, it is not clear whether the coniferous extractives specifically induced the observed metabolic pathways in P. gigantea.Consistent with the observed efficient utilization of lignocellulosic materials, genome analyses shows that 13. However, how this group of fungi regulates the genes involved in utilizing lipids in the wood extractives remains unknown. Here, we attempt to elucidate the extractive-induced mechanism(s) of P. gigantea. We cultivated the fungus on a defined culture medium containing microcrystalline cellulose as a carbon source and supplemented with different amounts of the Pinus taeda sapwood extracts, which contained high levels of the lipids. Metabolites of lipophilic extractives in the culture supernatant were determined by GC–MS. Transcriptomic analyses of the grown mycelia and proteomic analyses of the supernatants were performed by RNA-seq and LC–MS/MS, respectively, to identify genes encoding enzymes related to transformation or degradation of the extractives by P. gigantea. A putative extracellular lipase was highly expressed and regulated in response to the extractives. This enzyme was produced in the heterologous expression system of Pichia pastoris, and functionally characterized as a novel basidiomycete lipase.Free and esterified fatty acids together with resin acids are major components of softwood extractives, and basidiomycetes are known to secrete lipases that hydrolyze glycerides to release fatty acidsP. taeda sapwood was analyzed by GC–MS , the equivalent amount of extractives present in the P. taeda wood (AV1X), twofold (AV2X), and fourfold P. taeda extractives (AV4X), which were also used as the culture medium of P. gigantea. The 20 representative compounds  including extractives. All transcript data, along with manual annotations, gene ontology terms (GO), and genome locations, are summarized in Supplemental data file P gigantea, 11,437, 11,434, 11,431, 11,423 and 11,394 transcripts were identified in the cells grown on AV0X, AV1X, AV2X, AV4X and LPAS cultures, respectively. The average expression values (RPKMs)/standard deviation were 75.5/257, 73.8/236, 74.1/243, 77.6/243 and 80.5/345 for the datasets of AV0X, AV1X, AV2X, AV4X, and LPAS, respectively. With notable exceptions, transcript profiles of P gigantea genes were generally conserved in microcrystalline cellulose-based media AV0X, AV1X, AV2X, and AV4X  but diverged substantially from loblolly pine medium (LPAS) as described in Fig. P < 0.02, > twofold, RPKM ≥ 10)  , 16 and 10 transcripts were up-, and down-regulated, respectively, relative to the AV0X , compared to that of no extractives (AV0X), respectively  in response to supplementation with pine extractive , which converts oxaloacetate in the TCA cycle into oxalate, were not up-regulated in the presence of extractives. This observation was different from the comparative transcriptome results of pine wood powder relative to glucose13. Considered together with the limited CAZy gene regulation, use of extractive-coated microcrystalline cellulose seems well suited for investigating genetic regulation of degradative processes without the complexities inherent in woody substrates.An integrated pathway of lipid metabolism was proposed in a previous studyP. gigantea in response to increasing amounts of extractives, the time course of lipase activity was measured in culture supernatants from AV0X, AV1X, AV2X, and AV4X, using p-nitrophenol-dodecanoate (pNPD) as the C12 substrate which is one of model compounds of lipase activity. Significantly increased lipase activities were observed in the culture supernatant of AV1X, AV2X, and AV4X media during time course, compared to AV0X   Fig. . A secreP. pastoris culture supernatants , which is one of the most common conifer species and heavily used as lumber and pulpwood. P. gigantea is known to spread fast and deeply on softwood stumps preventing growth of softwood pathogens. Previous work on P. gigantea revealed a distinctive repertoire of genes involved in a unique and efficient system for degrading all components of softwood. Transcriptome analyses had identified hundreds of potential CAZymes, many of which were upregulated on P. taeda wood relative to media containing glucose as the sole carbon source13. Extending transcript analyses to pine devoid of acetone soluble extractives, comparative analyses suggested that unknown extractives in P. taeda influenced the regulation of a diverse array of P gigantea genes13. In this study, P. gigantea was shown to utilize microcrystalline cellulose as a sole carbon source and induced various CAZymes like other white-rot fungi38. To more precisely identify the genes involved in the response to the extractives, we examined the P. gigantea transcriptome and proteome when grown on defined microcrystalline cellulose media supplemented differing amounts of P. taeda sapwood extractives. Transcripts encoding protein Phlgi_19028 accumulated sevenfold in non-extracted wood (LPAS) relative to microcrystalline cellulose (AV0X) in this study  possibly involved in tolerance to terpenes, glutathione S-transferase (Phlgi_101998), aldehyde dehydrogenase (Phlgi_121047), and aryl-alcohol dehydrogenase (Phigi_89048) in the transformation and detoxification of extractives , flavonoids (e.g. flavone) and coumarins (e.g. 7-ethoxycoumarin)29. Likewise, induced P450 genes CYP512B (CYP503 family), and CYP5035A (CYP67 family) were shown to catalyze oxidation of these plant defense chemicals. CYP53 family represented is known to hydroxylate benzoate and its derivatives30. Beyond these simpler aromatic compounds, P450 monooxygenases CYP512, CYP5141, and CYP5150 have also been shown to oxidize more complex aromatic compounds including polyaromatics and heteroaromatics31. Considering their regulation and catalytic potential, P. gigantea P450s likely play a role in the metabolism of P. taeda extractives.e Phlgi_11047, and33. Especially, the optimal pH 4.5 is apparently lower than other lipases, but it seems consistent with P. gigantea’s external environment, which is usually acidic due to secreted organic acids such as oxalic acid34. Additionally, from our biochemical characterization, this novel lipase releases various unsaturated fatty acids from Pinus extractives at ambient temperature under acidic conditions  Fig. A. The reons Fig. . Hence, http://www.led.uni-stuttgart.de/). PgLip19028 is categorized in abH23 subfamily, which includes lipases reported from various pathogens, i.e. Aspergillus flavus and Fusarium graminearum, and some of these lipases are involved in penetration to the host organisms24. In conifers, extractives occur in resin canals and the surrounding parenchyma cells in sapwood, which structurally prevent fungal developments within cell lumens35. In addition, it has been reported that larger amounts of triglycerides were extracted from sapwood, compared to heartwood in Pinus species9. The growth rate of P. gigantea greatly varies depending on softwood species, i.e., it grows well on Pinus species but not on spruce, larch and fir36. Therefore, PgLip19028 is thought to play important roles in the penetration into Pinus sapwood by P. gigantea, and there would be value in examining P. gigantea responses to different extractives from various softwood species in future studies. Considering the wide sequence conservation , are classified into 38 abH subfamilies based on amino acid sequences and 3D structures  in Macon County, Alabama and sequenced (https://mycocosm.jgi.doe.gov/Phlgi1/Phlgi1.home.html) as described13. For each treatment, a single-basidiospore derivative of 11061-1 strain 5–6 was cultivated in triplicate on 250 mL basal salts medium38 containing: (i.) 1.25 g ground (1 mm mesh) debarked loblolly pine sapwood that had been suspended in 10 mL acetone and subsequently dried in a roto-evaporator (LPAS); or (ii.) 1.25 g microcrystalline cellulose  that had been suspended in 10 mL acetone and subsequently dried in a roto-evaporator (AV0X); or (iii.) 1.25 g microcrystalline cellulose that had been suspended in 10 mL of an acetone soxhlet extract of 1.25 g, 2.5 g or 5 g loblolly pine and subsequently dried to reconstitute the extractive content at the same (AV1X), two times (AV2X) and four times (AV4X) the native loblolly pine extract concentration, respectively. All loblolly pine was ground from the same 22 cm cross sectional bolt containing 16 annual rings at waist height. After debarking, the material was primarily composed of sapwood. The use of plant parts in the present study complies with international, national and/or institutional guidelines. The composition of basal salts medium contained, per liter, 2 g of NH4NO3, 2 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.1 g of CaCl2·2H2O, 1 mg of thiamine hydrochloride, and 10 mL of mineral solution. Mineral solution contained, per liter, 1.5 g of nitrilotriacetic acid, 3 g of MgSO4·7H2O, 0.5 g of MnSO4·H2O, 1 g of NaCl, 0.1 g of FeSO4·H2O, 0.1 g of CoSO4, 0.1 g of CaCl2, 0.1 g of ZnSO4·7H2O, 0.01 g of CuSO4, 0.01 g of AlK(SO4)2·12H2O, 0.01 g of H3BO3, and 0.01 g of NaMoO4·2H2O39. Cultures with solid materials sinking to the bottom were inoculated with mycelial plugs and incubated at 22 °C in a rotary shaker (150 rpm) for 5 days. The culture filtrate and mycelia were separated after the cultivation, and total RNA were purified from the mycelia by RNeasy Mini Kit (Qiagen) with DNase treatment (Qiagen) as previously described41. For GCMS-based metabolome analyses, the harvested entire cultures including the substrates and fungal mycelium from all the treatments were frozen in liquid nitrogen and lyophilized.The P. gigantea isolate 11061-1 strain CR5-6 was cultivated in triplicates in the culture medium of AV0X, AV1X, AV2X and AV4X, the culture supernatants were collected after 3, 5 and 7 days of cultivation, and protein concentration in the culture supernatants was measured by Protein Assay (Bio-Rad). To determine lipase activities in the secretomes, p-nitrophenol-dodecanoate  was used as substrate. Two microliter of 75 mM pNPD in dimethylsulfoxide (DMSO) was mixed with 50 µL of culture supernatant and 25 µL of 100 mM acetic acid buffer (pH 5.0) in a total volume of 100 µL. After the incubation at 37 °C for 30 min, 25µL of 100 mM Na2CO3 was added to stop the reaction. The released pNP amount was measured at 405 nm using the standard curve for pNP (Sigma-Aldrich). One unit of lipase activity was defined as the amount of enzyme releasing one micromole of pNP per a minute.https://support.illumina.com/sequencing/sequencing_kits/truseq-stranded-mrna.html). Total RNA starting material was 100 ng per sample and 10 cycles of PCR was used for library amplification. Total RNA qualities were examined by using Fragment Analyzer (Agilent Technologies), and RNA quality numbers (RQN) scores were 7.2 ± 0.3, 7.2 ± 0.8, 8.0 ± 1.7, 6.4 ± 0.2, 6.9 ± 2.7 for AV0X, AV1X, AV2X, AV4X and LPAS, respectively. The prepared libraries were quantified using KAPA Biosystem’s next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. The libraries were then multiplexed with other libraries and sequencing was performed on the Illumina NovaSeq sequencer using NovaSeq XP V1 reagent kits, S4 flow cell, following a 2x150 indexed run recipe. Sequence data for the libraries were deposited in SRA and assigned accession numbers SRP245000 through SRP245010 and 245022 through 245025.The transcriptomes were analyzed using Illumina RNA-Seq. Plate-based RNA sample prep was performed on the PerkinElmer Sciclone NGS robotic liquid handling system using Illumina's TruSeq Stranded mRNA HT sample prep kit utilizing poly-A selected mRNA as outlined by Illumina  was used to visualize the normalized reads and compute differential expression in pairwise comparisons. Unless otherwise indicated, parameters used to call genes differentially expressed between culture conditions were P value < 0.02 or P value < 0.05 when the stringency is relaxed, with > twofold change and RPKM values > 10. Differential expression of all genes were illustrated in volcano plots in Fig. Illumina reads were filtered and trimmed using for artifacts, RNA spike-in reads, PhiX reads and reads containing any Ns. Quality trimming was performed using the phred trimming method set at Q6. Finally, following trimming, reads under the length threshold were removed . Filtered reads from each library were aligned to the reference genome using HISAT2 version 2.1.047. Filtrates from microcrystalline cellulose cultures, with  or without (AV0X) addition of loblolly pine acetone extract, were filtered after 5 days and analyzed. Filtered proteins were precipitated with 10% (wt/vol) trichloroacetic acid and washed three times in cold acetone before air drying. Total proteins from the pellets were further purified via methanol/chloroform/water partitioning, where chloroform and methanol were added to pellets first, followed by water and allowed to partition with a protein interphase formed between polar and non-polar fraction. After multiple methanol washes, these purified protein preps where ultimately resolubilized in 8 M urea/50 mM NH4HCO3 (pH8.5)/1 mM TrisHCl.With minor modification, NanoLC-MS/MS analysis identified extracellular proteins in culture filtrates as describedP. gigantea protein database via the JGI portal  with a list of common lab contaminants (available at ftp://ftp.thegpm.org/fasta/cRAP) to establish False Discovery Rate  using in-house Mascot search engine 2.2.07 [Matrix Science] with variable methionine oxidation, asparagine and glutamine deamidation, plus fixed cysteine carbamidomethylation. Scaffold  was used for spectral based quantification and to validate MS/MS peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm48. Each detected protein abundance was calculated as exponentially modified protein abundances (emPAI)19. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Sequence data was deposited in PRIDE (PRoteomics IDEntifications Database) and assigned identifier PXD021332 (https://www.ebi.ac.uk/pride/archive/projects/PXD021332).For NanoLC-MS/MS protein identification of samples, equal amounts of total protein per sample were trypsin/LysC digested, OMIX C18 SPE purified (Agilent Technologies), and finally 2 µg loaded for nanoLC-MS/MS analysis using an Agilent 1100 nanoflow system (Agilent Technologies) connected to a hybrid linear ion trap-orbitrap mass spectrometer  equipped with an EASY-SPRAY electrospray source. Chromatography of peptides prior to mass spectral analysis was accomplished using a capillary emitter column  onto which 2 µL of purified peptides was automatically loaded. The nanoHPLC system delivered solvents A: 0.1% (v/v) formic acid , and B: 99.9% (v/v) acetonitrile, 0.1% (v/v) formic acid at 0.50 µL/min to load the peptides (over a 30 min period) and 0.3 µL/min to elute peptides directly into the nano-electrospray with gradual gradient from 3% (v/v) B to 20% (v/v) B over 154 min and concluded with 12 min fast gradient from 20% (v/v) B to 50% (v/v) B at which time a 5 min flash-out from 50 to 95% (v/v) B took place. As peptides eluted from the HPLC-column/electrospray source, survey MS scans were acquired in the Orbitrap with a resolution of 120,000, followed by MS2 fragmentation of 20 most intense peptides detected in the MS1 scan from 380 to 1800 m/z; redundancy was limited by dynamic exclusion. Raw MS/MS data were converted to mgf file format using MSConvert (ProteoWizard: Open Source Software for Rapid Proteomics Tools Development) for downstream analysis. Resulting mgf files were used to search against forward and decoyed-reversed 4. The temperature program started at 100 °C (1 min), raised to 380 °C at 10 °C min−1, and held for 5 min. The transfer line was kept at 300 °C, the injector was programmed from 120 °C (0.1 min) to 380 °C at 200 °C min−1, and helium was used as carrier gas at a rate of 2 mL min−1. Compounds were identified by mass fragmentography, and by comparing their mass spectra with those of the Wiley and NIST libraries and standards. Quantification was obtained from total-ion peak area, using response factors of the same or similar compounds . N, O-Bis(trimethylsilyl)trifuluoroacetamide (BSTFA) in the presence of pyridine was used to prepare trimethylsilyl derivatives.About 0.4 g of the lyophilized culture samples (n = 1) of microcrystalline cellulose coated with acetone extracts of loblolly pine were Soxhlet-extracted with acetone for 6 h, dried and redissolved in chloroform for GC–MS analyses. GC–MS analyses were performed with a Varian 3800 chromatograph coupled to an ion-trap detector (Varian 4000) using a medium-length fused-silica DB-5HT capillary column  from J&W Scientific, enabling simultaneous elution of the different lipid classes49, PgiLip19028F_PIPE_Pic, TCTCTCGAGAAAAGACTCCCATCGCCTGTCCAC, PgiLip19028R_PIPE_Pic, AGTTTTTGTTCTAGATTAAGTACAATAAATAGTTCCG, PicVector_PIPE_F, TCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCG, PicVector_PIPE_R, TCTTTTCTCGAGAGATACCCCTTCTTCTTTAGCAGCAATGCTG (underlined sequences were overlapped). The recombinant lipase was produced by transformed Pichia pastoris overexpressing Lip19028 following the manufacturer’s instructions (Invitrogen). The culture supernatant of Pichia pastoris was applied to saturated ammonium sulfate precipitation, and the resulting protein precipitate was dissolved in 100 mM acetate buffer (pH 5.0). Ten micrograms of crude protein were denatured and digested by Endoglycosidase H  for protein deglycosylation, then protein samples were applied to SDS-PAGE analysis (Bio-Rad), and the gel was stained by Coomassie Brilliant Blue (CBB) R-250 .The DNA encoding the class 3 lipase (PgLip19028), minus the native secretion signal, was synthesized by JGI service Fig. . The codhttps://www.expasy.org) as follows. Secretion signal peptides were predicted by SignalP ver 5.0 . N-glycosylation and O-glycosylation sites were predicted by NetNGlyc ver 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc ver 4.0 (http://www.cbs.dtu.dk/services/NetOGlyc/), respectively. The theoretical molecular weight was calculated by compute pI/Mw (https://web.expasy.org/compute_pi/). Secondary structure prediction was performed by Phyre 2 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). The multiple alignments of nine lipase amino acid sequences from P. gigantea and other reported lipases were performed by ClustalW, and a phylogenetic tree was executed by RaxML with bootstrap, and it was drawn by Figtree software.Sequences were analyzed by web-based tool Expasy (tert-butyl methyl ether (tBE), 5% DMSO and 25 mM buffer in total volume of 160 µL. The final concentration of tBE was kept 15%, which showed the highest activity compared to 5%, 10%, 15%, 20%, 25%, and 30% tBE. After the reaction mixture was incubated at the appropriate temperature for 10 min, 40 µL of 100 mM Na2CO3 was added to stop the reaction. One unit was calculated as described above. For optimal pH determination, tartaric buffer ranging from pH 3.0 to pH 3.5, sodium acetate buffer ranging from pH 3.5 to pH 5.5 and potassium phosphate buffer ranging from pH 6.0 to pH 6.5 were assessed. Lipase activity of PgLip19028 was corrected by subtracting activity in the supernatant of a vector-only control culture. Sensitivity of lipase activity to metals was determined with 1 mM of ZnCl2, MnCl2·4H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·2H2O, CdCl2, LiCl, CaCl2·2H2O, MgCl2·6H2O, or ZnCl2. For glycerides degradation assay, 1 µg/mL of crude protein was mixed with 10 mg/mL triolein or Pinus extractives in tBE, 5% DMSO and 25 mM acetate buffer (pH 4.5) in total volume of 1000 µL. The reaction mixture was incubated at 25 °C for 17 h with shaking at 120 rpm, lyophilized and the residue dissolved in chloroform. The sample was applied to TLC plates32 and the remaining sample analyzed by GCMS after trimethylsilylation by BSTFA.Prior to lipase activity determinations, the protein concentration of crude proteins was diluted to 10 µg/mL and then 1 µg/mL of crude protein was mixed with 1.5 mM pNPD in Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Marinobacterium litorale was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density fermentation, 35 gDCW/L of biomass with overexpressed SMO was produced. SMO exhibited excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess (˃95–99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, -1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-cell SMO.Styrene monooxygenases are a group of highly selective enzymes able to catalyse the epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds containing oxirane ring or products of their hydrolysis represent key building blocks and precursors in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial scale. Two-component recombinant styrene monooxygenase (SMO) from  Pseudomonas and Rhodococcus species, SMO is comprised of two subunits—FAD-dependent monooxygenase (StyA) and NADH-dependent flavin-reductase (StyB)—encoded by styA and styB of styABDE gene cluster responsible for the degradation of styrene [styA and styB are possible candidates for biocatalysis. Initially, wild-type SMOs of Pseudomonas were widely employed for biotransformation of styrene, but, to date, many other bacterial species that share styrene degradation pathways have been described [Escherichia coli [Pseudomonas sp. and, in 2002, produced 388 g of enantiopure (S)-styrene oxide in a 30 L scale by the whole-cell SMO expressed in E. coli JM101, which is the highest (S)-styrene oxide yield achieved so far. However, only a low cell concentration (12 g/L) was reached during fermentation, despite being operated on a pilot-scale [Styrene monooxygenase (SMO) is a flavoprotein monooxygenase that incorporates a single oxygen atom into a styrene molecule to form styrene oxide . Origina styrene ,3. Sinceescribed . Such dihia coli ,4,5,6,7.hia coli ,9,10,11.ntration  g/L was ot-scale ,14. Few ot-scale ,16.Chiral epoxides comprise a group of important building blocks and intermediates of biologically active pharmaceuticals . To dateMarinobacterium litorale was cloned to a pET28b(+) vector and expressed in Escherichia coli BL21(DE3) under the control of isopropyl-β-D-thiogalactopyranoside (IPTG) inducible lacT7 promoter. Recombinant SMO originating from M. litorale has been expressed and characterised in [styA and styB was fused by the linker L2 designed in [In this work, the gene of SMO from igned in   fermentation is a well-established method for recombinant enzyme production. However, the process performance and productivity highly depend on many factors  and require optimisation ,23,24.E. coli generates organic acids such as acetate, which significantly decreases biomass growth at a concentration above 2 g/L [DCW/L, but under these conditions, the cell growth was inhibited immediately. The concentration of IPTG is one of the crucial factors that affect the growth of E. coli, the level of expression, and also the activity of a recombinant enzyme [DCW/L was too low for the induction. Expression of SMO was further induced at 15 gDCW/L (OD600 = 50) for 5.5 h  expressing recombinant SMO was as follows: M9 inoculation medium and induction with 0.25 mM IPTG at 30 °C and 15 gDCW/L in a batch mode. Following the protocol, HCD fermentation with 1.5 L provided 35 gDCW/L of overexpressed SMO with a specific activity of 9.6 U/gDCW.The final, optimised HCD fermentation protocol for 2+ affinity chromatography according to [SMO per gDCW). Since this is not the first report of SMO isolation causing an obstacle [Due to the presence of the His tag, recombinant SMO was isolated by immobilised Nirding to  , unconjugated aromatic alkenes (2), unsaturated aldehydes and ketones (3), unsaturated carboxylic acid and esters (4), linear alkenes (5), and unsaturated amines and imines (6–8).The affinity of SMO towards a wide range of alkenes  was exam1b–e alkenes were converted to epoxide. Both double bond and aromatic ring substitution presumably acted as a steric barrier to the active site of the enzyme. On the other hand, the bioconversion of 4-Cl-substituted styrene derivative (1f) and unconjugated allylbenzene (2a) was high. In both cases, a higher activity of SMO compared to styrene was achieved. Both 4-chlorostyrene oxide and allylbenzene oxide were formed in the upscaled process, with excellent conversion (more than 99%), ee, and yield . SMO showed no affinity towards 5a–c, e, and f, but was highly active in the epoxidation of 5d, implying a trend of rising activity with increasing distance between the double bond and the hydroxyl group. A similar trend was confirmed compared to the epoxidation of bromo-alkenes 5i and 5j. On the contrary, the activity of SMO decreased with increasing distance of the oxirane ring from the double bond . Eventually, both epoxides were converted to diepoxides, but the conversion of longer 5h and ee of the corresponding diepoxide was lower compared to 5g.2-hydroxyl substitution of the aromatic ring also seemed unfavourable (3b was transformed to epoxide with excellent enantiopurity (ee ˃ 99%). Interestingly, 3b only differs from 3a by one methyl- substitution on C2 carbon, which supports the previous finding of SMO activity enhancement by double bond methylation [4c was observed, suggesting that the combination of methyl- and carbonyl- substituents was crucial. Unfortunately, SMO showed no affinity to any tested unsaturated ketone, carboxylic acid, ester, amine, or imine.From the group of unsaturated aldehydes and ketones, only hylation . On the S) configuration, similar to previous reports [R,5R)-1,2:5,6-diepoxyhexane and 2-(3-bromopropyl)oxirane enzymatic production by SMO in such a configuration and/or enantiopurity.To evaluate the scope of SMO applicability, upscaled biotransformations were performed. It was found that the crude extract of SMO was not suitable for upscale epoxidations, as many undesired compounds were repeatedly isolated from crude reaction mixtures besides epoxides (data not shown), since additional purification steps provided only trace final amounts of epoxides. Therefore, whole-cell SMO was employed, providing the same enantioselectivity and even higher conversion compared to crude extracts . As Tabl reports ,29,32. AR)-1f epoxide is a possible intermediate in the synthesis of neuroprotective agent Eliprodil. Similarly, (R)-3-1f epoxide serves as an intermediate of antidiabetic and antiobesity agents and is produced on an industrial scale (100–4000 L) [2a epoxide is an intermediate for the synthesis of (R)-(−)-mevalonolactone, a key intermediate of cellular biological processes and regulation [The application of chiral epoxides prepared in this work has not been reported so far. However, structurally similar (–4000 L) ,33. 2-megulation . The posDespite the rising trend of biocatalysis or chemo-biocatalysis involvement in industrial production, chiral epoxides are still preferably produced by the manner of chemical catalysis. The ability of SMOs to operate at mild reaction conditions, together with their tremendous enantioselectivity, thereby emphasise their potential for industrial applications.® . NAD+ was purchased from Prozomix . N-hexadecane was purchased from Alfa Aesar . Ethyl acetate was purchased from Merck . Styrene, styrene oxide, and 4-chlorostyrene oxide were purchased from Sigma Aldrich . 4-chlorostyrene was purchased from Carbosynth . Other alkenes and standards of epoxides were provided by the Department of Organic Chemistry of the Slovak University of Technology.Kanamycin was purchased from Gibco4)2SO4, 3.64 g/L NaH2PO4.2H2O, 4.53 g/L K2HPO4, 4 g/L citric acid, 1% (v/v) trace element solution, 1 g/L MgSO4, 7H2O. Mineral M9 medium contained 5 g/L glycerol, 0.5 g/L MgSO4.7H2O, 0.11 g/L CaCl2, 0.01 g/L thiamine-HCl, and 5% (v/v) M9 salts . All media were supplemented with kanamycin to a final concentration of 30 µg/mL.Lysogeny broth (LB) was prepared according to . SemidefstyA and styB [Marinobacterium litorale were linked with linker WYHHHH according to [Escherichia coli BL21(DE3).Codon-optimised genes and styB  encodingrding to  and wereE. coli BL21(DE3) with pET28b(+)SMO was transferred to 3 mL of LB/M9 medium supplemented with kanamycin (30 µg/mL) and cultivated at 37 °C and 200 rpm overnight. A total of 100 mL of LB/M9 medium in a 500 mL shaking flask were inoculated with 1% (v/v) of overnight culture and cultivated at 37 °C and 200 rpm until OD600 reached 0.5–0.7. The temperature was reduced to 20/30 °C and the expression of SMO was induced by the addition of 0.25/0.5/1 mM of IPTG. To determine OD600 and level of SMO expression performed by denaturing polyacrylamide gel (SDS-PAGE) electrophoresis , 1 mL samples were taken regularly after induction, which took 22 h. The cells were then harvested by centrifugation  and applied for biotransformation.A single colony of Fermentations were performed in 1.3/3 L New Brunswick BioFlo Bioreactors  with an initial fermentation medium volume of 500 mL/1.5 L.v/v) of the preinoculum culture and cultivation at 37 °C, 200 rpm until OD600 reached 2.5. Fermentation was started by the inoculation of a bioreactor containing the semidefined medium with 2% (v/v) of inoculum culture. Temperature and pH were set to 37 °C and 7, maintained by the addition of 26% (v/v) ammonia and 3.1 M phosphoric acid. Air saturation of fermentation medium was maintained at dissolved oxygen (DO) = 30% by aeration (1 vvm) and agitation speed cascade (200–1200 rpm). When agitation reached 1200 rpm, the temperature was reduced to 30 °C and the expression of SMO was induced by the addition of 0.25 mM IPTG. To determine OD600, glycerol and acetic acid concentration, and specific activity and level of SMO expression, samples of fermentation medium were withdrawn regularly. Samples were processed as described above, except the supernatant was transferred to a fresh tube, diluted, and analysed by high-performance liquid chromatography (HPLC). Biomass for SMO activity determination was sampled separately and kept at 4 °C until the activity assay. Fermentation was terminated after complete substrate utilisation. Cells were harvested by centrifugation  and stored at −80 °C.Preinoculum was prepared in a glass tube as described above. The inoculum was prepared by inoculation of 100 mL of M9 medium in a 500 mL shaking flask with 1%  . Crude cell extract (CE) was clarified from cell debris by centrifugation . The reaction mixture (1 mL) containing CE, glucose (100 mM), GDH (0.1 mg/mL), and NAD+ (1 mM) was prepared into 4 mL screw-neck vials and tempered in a shaking incubator at 30 °C and 200 rpm for 3 min. Biotransformation was started by the addition of 10 µL of styrene from 150 mM stock in methanol. To obtain a styrene oxide linear concentration increase, samples were withdrawn regularly and analysed by gas chromatography (GC). Each biotransformation was performed in two parallel runs, and given values represent mean values. One unit (U) was defined as the amount of SMO that catalyses the formation of 1 µM of styrene oxide in 1 min. The specific SMO activity was calculated as U per g of dry cell weight (U/gDCW).Harvested cells were resuspended in 100 mM potassium phosphate buffer (pH 8) to OD600 = 50) were prepared as described above. CE was filtered by vacuum filtration  or microfiltration  performed on an ÄKTA flux filtration unit . Recombinant SMO was purified by fast protein liquid chromatography (FPLC) performed on an ÄKTA purifier/ÄKTA pilot unit equipped with 5 mL His Trap FF /50 mL Ni-NTA  affinity column according to [® ultracentrifuge membrane  or ultrafiltration cassette . The concentration of purified SMO was measured by an Eppendorf µCuvette® . The molar extinction coefficient of SMO was determined using ProtParam tool.CE of SMO (biomass diluted to ODrding to . In brie+ (1 mM), and potassium phosphate buffer  were prepared. Biotransformations were performed as described above. To determine the optimal temperature, the same biotransformations were performed in a water bath  instead of a shaking incubator.To determine the optimal pH, purified SMO was employed in the biotransformation of styrene. Reaction mixtures (1 mL) containing glucose (100 mM), GDH (0.3 mg/mL), SMO (1 mg/mL), NAD600 = 50) were stored at 4, −20, and −80 °C. Reaction mixtures contained glucose (100 mM), GDH (0.3 mg/mL), CE (OD600 = 25), NAD+ (1 mM), and potassium phosphate buffer  or glucose (100 mM) and cell suspension (OD600 = 25) in potassium phosphate buffer  were prepared. Biotransformations were performed as described previously.The storage stability of CE of SMO was determined, when 1 mL aliquots  as described above. Additional biotransformations of 5, 10, and 20 mM 1f, 2a, 3b, 5d, 5g, 5h, 5j alkenes were performed and allowed to proceed for 2–5 h. Specific SMO activity was expressed in U/gDCW. One unit (U) was defined as the amount of SMO that catalyses the formation /depletion (for 3b) of 1 µmol of epoxide/alkene in 1 min.Small-scale (1 mL) biotransformations were performed in 4 mL screw-neck vials employing CE of SMO  and glucose (100 mM) was tempered for 5 min in a shaking incubator at 30 °C and 200 rpm, then 1 mL of 1f, 2a, 5d, 5g, 5j from a 2 M stock solution in methanol was added to start the biotransformation. Biotransformations were allowed to proceed for 9 h. To determine the conversion of alkenes and ee of corresponding epoxides, 100 µL samples were withdrawn for GC analysis. The rest of the reaction mixture was used for isolation and purification of corresponding epoxides performed immediately after biotransformation termination. All small-scale and upscale biotransformations were performed in two parallel runs.Upscale , an Agilent Infinity 1260 RI detector , and 9 mM H2SO4 as a mobile phase under the following conditions: injection volume 20 µL, temperature 50 °C, and flow 0.8 mL/min.To determine glycerol and acetic acid concentrations, samples of cell-free fermentation medium were diluted with deionised water into a glass vial and analysed by an Agilent Infinity 1220 LC System  equipped with a WATREX polymer IEX Hn-hexadecane as an internal standard by 30 s vortexing. The organic phase, containing the substrate and product, was separated from the water phase by centrifugation  and transferred to a glass vial. To determine the conversion of biotransformation, samples were analysed by an Agilent 6890N gas chromatograph  equipped with a DB5 capillary column , a flame-ionisation detector (FID), and H2 as a carrier gas under the following conditions: injection volume 1 µL, split 50:1, temperature gradient 90 °C (3.5 min), 30 °C/min to 280 °C; H2 flow 1.2 mL/min. Temperature gradients were adjusted according to the molecular weight of alkenes during substrate specificity screening. Conversion of biotransformation was established according to (1).During all biotransformations, 100 µL of the reaction mixture was sampled regularly and extracted to 200 µL of ethyl acetate containing 0.25 mM of 2 as a carrier gas under the following conditions: injection volume 1 µL; split 50:1; temperature gradient 100 °C (0 min), 2 °C/min to 120 °C (1 min), 30 °C/min to 280 °C (1 min); H2 flow 1.2 mL/min.To determine the ee of the products, samples were analysed by an Agilent 7890N gas chromatograph  equipped with a CP-Chirasil-Dex CB capillary column , FID detector, and H254 , and plates were visualised with UV light or by treatment with permanganate solution followed by heating. Optical rotations were measured with a JASCO P-2000 digital polarimeter with a Na-D lamp (10 cm cell length). Concentrations (c) are given in grams per 100 mL. NMR spectra were recorded with a Varian INOVA-300 spectrometer  in CDCl3 using tetramethylsilane as the internal standard. Data are presented as follows: chemical shift (in ppm), multiplicity , integration and coupling constants (J/Hz). All solvents used were dried and distilled according to conventional methods.Flash column chromatography (FCC) was carried out with a Büchi system (Pump Manager C-615 and Fraction Collector C-660) using Normasil 60 silica gel . Thin layer chromatography (TLC) analysis was carried out using TLC silica gel 60 F4, and the solvent was evaporated under reduced pressure . The crude product was purified by FCC using hexanes as the eluent to give (S)-4-chlorostyrene  oxide as a colourless liquid with spectroscopic data in good agreement with those reported in the literature [The crude biotransformation mixture (100 mL) was vigorously stirred with ethyl acetate (150 mL) for 20 min. Then, the slurry was filtered through a pad of Celite, the organic phase was separated, and an aqueous layer was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were dried over anhydrous MgSOterature ,37.S)-4-Chorostyrene Oxide: δ = 2.75 , 3.14 , 3.83 , 7.18–7.23 , 7.29–7.34 .c 1.35, CHCl3).Rf = 0.15 .S)-4-chlorostyrene oxide c 1.29, CHCl3) for 99% ee [c 1.2, CHCl3) for 99% ee [Reported rotation for -Allylbenzene Oxide, colourless liquid with spectroscopic data in good agreement with those reported in the literature .1H-NMR : δ = 2.55 , 2.78–2.85 , 2.93 , 3.13–3.19 , 7.21–7.34 .c 1.3, EtOH).Rf= 0.30 .S)-allylbenzene oxide c 2.01, EtOH) for 99% ee [c 1.0, EtOH) for 98% ee [Reported rotation for -1,2:5,6-Diepoxyhexane, colourless liquid with spectroscopic data in good agreement with those reported in the literature .1H-NMR : δ = 1.57–1.85 , 2.51 , 2.78 , 2.91–3.01 .c 1.2, CHCl3).Rf= 0.18 .R,5R)-1,2:5,6-diepoxyhexane c 5.03, CHCl3) [c 1.3, CHCl3) [Reported rotation for  , αD20 = , CHCl3) .(S)-4-(Oxiran-2-yl)butan-1-olYield: 76 mg , colourless liquid with spectroscopic data in good agreement with those reported in the literature .1H-NMR : δ = 1.51–1.68 , 2.49 , 2.76 , 2.90–2.96 , 3.67 .c 1.0, CHCl3).Rf= 0.15 .S)-4-(oxiran-2-yl)butan-1-ol c 1.0, CHCl3) for >99.9% ee [Reported rotation for (99.9% ee .2-(3-Bromopropyl)oxiraneYield: 173 mg  of the crude product (could not be purified by FCC because of its instability on silica gel), orange liquid with spectroscopic data in good agreement with those reported in the literature ,44.1H-NMR : δ = 1.53–1.66 , 1.76–1.88 , 1.98–2.08 , 2.51 , 2.77 , 2.92–2.98 , 3.41–3.53 .Rf = 0.19 .DCW/L was optimised. It was found that SMO loses stability in the isolation process and was not suitable for biotransformations in a purified form. The highest specific activity, stability, enantioselectivity, and purity of desired products was achieved by application of whole-cell SMO, which was proven to be the most suitable form for epoxidation of alkenes. In its crude form, SMO exhibited the highest activity at pH 8 and 35 °C and retained 70% of its initial activity within 16 months of storage. The enzyme transformed 7 out of 34 tested alkenes to corresponding epoxides, many of them with higher specific activity than styrene. Eventually, (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, -1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were produced with excellent enantiopurity .Herein, the production of recombinant SMO by HCD fermentation that yielded 35 g


N-acylethanolamine derivatives. As a result, we found that both N-docosahexaenoylethanolamine (synaptamide) and N-eicosapentaenoylethanolamine (EPEA) prevents an LPS-mediated increase in the proinflammatory cytokines TNF-α and IL-6 production in the SIM-A9 microglia culture. In an in vivo experiment, synaptamide reversed an increase in LPS-mediated hippocampal TNF-α and IL-1β, but EPEA did not. However, both compounds contributed to the microglia polarization towards the M2-phenotype. Synaptamide, rather than EPEA, inhibited the Iba-1-positive microglia staining area increase. However, both synaptamide and EPEA prevented the LPS-mediated astrogliosis. A study of BDNF immunoreactivity showed that synaptamide, but not EPEA, reversed an LPS-mediated decrease in BDNF production. Despite the more pronounced anti-inflammatory activity of synaptamide, both compounds were effective in maintaining a normal level of hippocampal long-term potentiation in neuroinflammation. The results indicate a high therapeutic potential for both compounds. However, some tests have shown higher activity of synaptamide compared to EPEA.The search for methods of cognitive impairment treatment and prevention in neurological and neurodegenerative diseases is an urgent task of modern neurobiology. It is now known that various diseases, accompanied by dementia, exhibit a pronounced neuroinflammation. Considering the significant docosahexaenoic and eicosapentaenoic polyunsaturated fatty acids’ therapeutic potential, we decided to investigate and compare anti-inflammatory activity of their  According to the World Health Organization, around 50 million people worldwide suffer from dementia. Moreover, among the population over 60 years of age, the prevalence of dementia is 5–8%. Among the diseases that cause dementia, in addition to neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, and Lewy body disease, there is also a mild cognitive impairment (MCI). The prevalence of MCI among the elderly is 5.13–29.9% , chemokiN- docosahexaenoylethanolamine (synaptamide) activity aimed at neurogenesis, axonal growth, and synaptogenesis is well characterized [The annual increase in the prevalence of neurodegenerative diseases accompanied by neuroinflammation requires the search for their prevention and effective approaches for treatment. There is an increasing interest in drugs that regulate the activity of microglia and control the neuroinflammatory process. Some of these promising molecules include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and polyunsaturated fatty acids (PUFAs) which perform important structural and metabolic functions in the brain. Some studies indicate that the biological activity of DHA and EPA derivatives is significantly higher than that of the PUFAs themselves. For example, several works are devoted to the anti-inflammatory activity of natural DHA derivatives–protectins , resolvicterized ,21. A nucterized ,23,24. Acterized . Endogencterized ,26. In acterized ,21. Thiscterized ,28. It icterized . Howevercterized . In addicterized . Given tp < 0.001) (p < 0.001) (p < 0.01) (p < 0.001) (p < 0.001)   a, IL-1β < 0.001) b, and IL < 0.01) c. EPEA a< 0.001) a and IL-< 0.001) d. Howeve< 0.001) b. In add< 0.001) d.p < 0.001). A similar effect was observed in the study of TNF-α production within the hippocampus. Synaptamide reversed an LPS-mediated TNF-α increase . EPEA in a similar dose was unable to prevent the LPS-mediated increase in TNF-α production. Interestingly, TNF-α in the “LPS + Syn” group was lower than in the “Veh” group  (p < 0.001) (We found that LPS causes an increase in IL-1β production a. The adh” group b. LPS tr < 0.05) c. In add< 0.001) d.p < 0.05). In the “Syn” and “EPEA” groups, there was an even more pronounced decrease in CD86 immunoreactivity compared to the control group  . In the “Syn” and “EPEA” groups, MHCII immunoreactivity was lower than in the “Veh” group (100 ± 3.58%—“Veh” vs. 57.30 ± 2.70%—“Syn” and 51.82 ± 2.20%—“EPEA”)  than in the “LPS” group  e. At the—“EPEA”) f.p < 0.01 and 106.47 ± 2.48%, p < 0.05)  , which converts arginine to polyamines , and CD2 < 0.05) g. The si< 0.001) h.p < 0.001—CA1; 7.93 ± 0.43%—“LPS” vs. 5.29 ± 0.29%—“LPS + Syn”, p < 0.001—CA3; 5.44 ± 0.55%—“LPS” vs. 3.05 ± 0.17%—LPS + Syn”, p < 0.001—dentate gyrus (DG) (p < 0.05). In the CA1 (p < 0.05)  a. At therus (DG) b. It is  the CA1 c and the the CA1 d areas,  < 0.05) e. Such sp < 0.001—“LPS + Syn” and 2.39 ± 0.22%, p < 0.001—“LPS + EPEA”; CA3: 7.74 ± 0.50%—“LPS” vs. 4.55 ± 0.36%, p < 0.001—“LPS + Syn” and 4.73 ± 0.25%, p < 0.001—“LPS + EPEA”; DG: 4.43 ± 0.57%—“LPS” vs. 2.36 ± 0.32%, p < 0.01—“LPS + Syn” and 2.39 ± 0.22%, p < 0.001) . In the stratum radiatum layer, a decrease in GFAP immunoreactivity is also observed in synaptamide and EPEA-treated animals without neuroinflammation   production within the hippocampus demonstrated a significant increase in the CA1, CA3, and DG regions a. At the< 0.001) b. Even t < 0.05) c. In the < 0.05) d. In the < 0.05) e.p < 0.001—“LPS + Syn” and 0.51 ± 0.04%, p < 0.001—“LPS + EPEA”) and the dentate gyrus . It is interesting that, in the CA3 region, both synaptamide and EPEA, administered separately from LPS, reduces S100β immunoreactivity compared to the “Veh” . In the DG, EPEA, administered separately from the LPS, downregulated the immunoreactivity level below the “Veh” group (p < 0.05) (p < 0.001). In the CA3 region, while a significant increase in S100β expression was observed in the str. oriens and the str. luciderm layers, but only in str. luciderm, we observed a significant effect of both synaptamide and EPEA on S100β immunoreactivity . In the stratum lacunosum–moleculare, both synaptamide and EPEA reduced the S100β level in animals untreated with LPS below the “Veh” group level (p < 0.01 for synaptamide and p < 0.001 for EPEA)  b. In the < 0.05) c. Moreovor EPEA) d. In theor EPEA) e.p < 0.05), CA3 (p < 0.001), and DG (p < 0.01) regions (p < 0.05) (p < 0.001 (p < 0.01), which may indicate a stimulating effect of synaptamide on BDNF accumulation (p < 0.05) (p < 0.001) and in the hilus (p < 0.01) (p < 0.05) (Brain-derived neurotrophic factor (BDNF) is one of the most significant regulators of brain synaptic and neurotransmitter processes . As a ru regions a. At the < 0.05) b. This e < 0.001 d. Synaptmulation b. Simila < 0.05) b. In thi < 0.01) e. Within < 0.05) c. At the < 0.05) b. Immuno < 0.05) .p < 0.05) and 172.65 ± 22.04% (p < 0.05) of baseline value, respectively, immediately after tetanic stimulation (p < 0.001) and 136.71 ± 5.53% (p < 0.001), respectively  and incubated for an additional 1 h at 37 °C with 5% CO2. Next, an LPS solution  was added to the wells so that the final concentration was 1 μg/mL and cultured for 24 h at 37 °C with 5% CO2. Cells incubated in a normal culture medium without synaptamide, EPEA, and LPS were used as negative controls. As control of LPS activity, we used cells incubated in a normal culture medium without synaptamide or EPEA, but with LPS.SIM-A9 mouse microglia was seeded in 24-well microplates, cultured in complete DMEM/F12 medium, and incubated at 37 °C with 5% COMale C57BL/6 mice (3-month-old) were obtained from the National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia. The mice were housed 3–4 per cage with a 12-h light/dark cycle. The animals had ad lib access to chow and water. The temperature (23 ± 2 °C) and humidity (55 ± 15%) were constant. All experimental procedures were approved by the Animal Ethics Committee at the National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences (No 1/2021) according to the Laboratory Animal Welfare guidelines and the European Communities Council Directive 2010/63/EU.E. coli O111:B4, Sigma-Aldrich, Bellefonte, PA, USA). Synaptamide was injected subcutaneously (s.q.) in a dose of 10 mg/kg. The mice (n = 80) were divided into the following treatment groups: “Veh” (n = 20)—i.p. saline and s.q. water injection; “LPS” (n = 20)—i.p. LPS and s.q. water; “LPS+Syn” (n = 20)—i.p. LPS and s.q. synaptamide; and “Syn” (n = 20)—i.p. saline and s.q. synaptamide. The i.p. saline or LPS (750 mg/kg) injections were administered for seven consecutive days. The volume of injected substances was 100 μL. The emulsion of synaptamide was prepared by mixing synaptamide with water to obtain a final concentration of 25 mg/mL with constant shaking using a Multi-Vortex shaker . To increase the stability of the emulsion in the process of stepwise dissolution, ethanol was added at a low concentration. For cell culture, the final concentration of ethanol did not exceed 0.1%. For in vivo administration, the amount of ethanol was 1.5% of the injected amount. A similar amount of ethanol was added to water or culture medium administered to control groups or cells.Neuroinflammation was induced by intraperitoneal (i.p.) injections of bacterial lipopolysaccharides . The elution rate was 50 mL/min. Fractions containing resulting N-acylethanolamines were collected, evaporated in vacuo, and analyzed by GC and GC-MS. The resulting N-docosahexaenoylethanolamine and N-eicosapentanoylethanolamine looked like a light-yellow oily liquids with a mild odor at room temperature. The purity of ethanolamides was 99.4%.nolamine  were obtN-docosahexaenoylethanolamine and N-eicosapentanoylethanolamine obtained by GC-MS are given in the To determine the composition of ethanolamides, conversion to trimethylsilyl derivatives (TMS-NAE) was used . For thiTo determine the concentration of the cytokines and glial markers in the cell culture and mouse hippocampus, the enzyme-linked immunosorbent assay (ELISA) was used. For analysis, we used SIM-A9 mouse microglia cell lysate after incubation with LPS, synaptamide, and EPEA. After the cells were collected in the centrifuge tube, 0.5 mL of buffer  was added to each sample. The samples (cells with buffer) were vortexed briefly and incubated on ice for 15–30 min. Then centrifuged at 13,000 rpm for 10 min at 4 °C to pellet insoluble contents. The supernatant was aliquoted to clean tubes on ice and stored at—80 °C.The mice were anesthetized with isoflurane using rodent anesthesia vaporizer  and the hippocampus was quickly extracted, frozen in liquid nitrogen, and stored at a temperature of −80 °C. For analysis, we used both right and left hippocampi. The hippocampi were homogenized using a homogenization buffer consisting of 100 mM of Tris, pH 7.4, 150 mM of NaCl, 1 mM of EGTA, and 1 mM of EDTA; 1% Triton X-100; 0.5% sodium deoxycholate; and protease inhibitors cocktail  incubated on ice for 15 min, centrifuged , and the supernatants were collected. ELISA kits were used for the detection of TNF-α (ab208348), IL-1β (ab197742), IL-6 (ab46100), IL-4 (ab100710) and IL-10 (ab100697), all from Abcam, Cambridge, UK. A BCA Protein Assay Kit  was used for quantitation of protein concentration.2CO3, 6.0 g of NaHCO3, 1000 mL of distilled water, pH 9.6) to obtain a 20-μg/mL concentration. Then, 100 μL of samples (extracts from cells or tissue dissolved with coating buffer) were added to each well of PVC microtiter plate  and incubated at 4 °C overnight. After this, the coating solution was removed, and the plate was washed three times by filling the wells with 200 µL of PBS. To block the remaining protein-binding sites in the coated wells, the 5% non-fat dry milk  was used (2 h at room temperature). After washing, 100 µL of diluted primary antibody was added to each well. In this study, we used the following primary antibodies: rabbit polyclonal anti-CD86 antibody , rabbit polyclonal anti-MHC class II antibody , rabbit polyclonal anti-mannose receptor antibody , rabbit polyclonal anti-liver arginase antibody , and rabbit monoclonal anti-BDNF antibodies , all from Abcam, Cambridge, UK. The plate was covered with an adhesive plastic and incubated for 2 h at room temperature. After washing, 100 µL of peroxidase secondary antibody  was added to each well, and the plate was incubated for 2 h at room temperature. After washing, 50 µL of TMB  was added to each well, and the plate was incubated for 30 min at room temperature before color appears. After sufficient color was developed, 50 µL of stop solution (1N hydrochloric acid) was added to the wells.To determine CD86, MHCII, CD206, and Arg1 antigens, the samples (supernatants of cells or tissue lysates) were diluted with bicarbonate–carbonate coating buffer  at a wavelength of 450 nm. Each sample was analyzed twice, and the results were averaged.2O2 solution for 5 min; (2) blocking non-specific antibody binding: 5% BSA in PBS for 1 h; (3) primary antibodies ; (4) secondary antibodies conjugated to horseradish peroxidase: anti-rabbit, 1:200, PI-1000-1; anti-mouse 1:200, PI-2000-1 ; (5) ImmPACT™ DAB Peroxidase Substrate chromogen ; and (6) washing with 0.1 M PBS (pH 7.2), dehydration and mounting in VectaMount Permanent Mounting Medium . The following primary polyclonal rabbit antibodies were used: anti-Iba-1 rabbit polyclonal antibodies , anti-GFAP antibodies , anti-S100β rabbit monoclonal antibodies , and anti-BDNF rabbit monoclonal antibodies .Immunohistochemical studies were performed on the 7th day after the start of treatment. The animals were deeply anesthetized with isoflurane  using a rodent anesthesia vaporizer  equipped with a rodent mask. Mice were transcardially perfused with 5 mL of PBS (~4 °C), pH 7.2. Then, the brain was rapidly removed from the skull, divided into 2 hemispheres, and placed in 4% paraformaldehyde for 12 h. We used both hemispheres for immunohistochemical study. After paraformaldehyde fixation, the material was washed with PBS (pH 7.2) and embedded in paraffin blocks. After embedding in paraffin, the samples were sectioned to obtain 10-µm slices, using a Leica rotary microtome RM 2245 . The immunohistochemical method used in the study consisted of the following steps: (1) blocking endogenous peroxidase activity: 0.3% HA Zeiss Axio Imager microscope equipped with an AxioCam 503 color and AxioVision software  was used to obtain images. The images were processed and analyzed using ImageJ software . Processing of each micrograph included the following steps: conversion to an 8-bit image; subtracting the background ; and contrast enhancement. To measure the area of marker staining, the necessary area was selected, and the percentage of the colored area was calculated. All measurements were performed by an operator who was blinded to the identity of the sections. For calculations, five sections were used from each animal. For statistical processing, the values obtained for each animal were averaged.2, 0.25 mM of CaCl2, 26 mM of NaHCO3, 1 mM of NaH2PO4, 10 mM of D-glucose, pH 7.4, oxygenated with carbogen 95% O2, 5% CO2. The hippocampus was removed and parasagittal sections with a thickness of 350 μm were prepared using a vibratome. The slices were allowed to recover within 1 h at 33 °C. The recordings were performed in a submersion recording chamber perfused with aCSF . To hold the slices in place in the recording chamber, we used a nylon mesh while aCSF perfusing. Acute hippocampal slices were visualized using an upright microscope (Olympus BX50). The parameters of recording extracellular electrode were: a 1.5-mm outer diameter, a length of 10 cm, and borosilicate glass . The monopolar stimulating electrode consisted of Pl-Ir Teflon wire . The stimuli were triggered using National Instruments Labview 2019 software  with an isolating stimulator (Constant Current Stimulus Isolator WPI). An intracellular amplifier in the bridge circuit mode , with a sampling rate of 15 Hz, was used. The signal was digitized , analyzed, and filtered using the National Instruments Labview 2019 software.Mice were deeply anesthetized using isoflurane  and decapitated; theur brains were removed and transferred to ice-cold aCSF composed of 119 mM of NaCl, 2.5 mM of KCl, 2 mM of MgClThe stimulating electrode was placed into the Schaffer collateral fiber tract between the CA2 and CA1 regions. For extracellular population excitatory postsynaptic potentials (EPSP) recording, an electrode was placed in the stratum radiatum subfield of the CA1 area at a distance of no more than 1500 μm, but not less than 300 μm from the stimulating electrode in order to avoid direct stimulation of cells located near the recording sites. To check if the slice is suitable for recording, an extra-synaptic potential was observed during stimulation of 0.5 mA, and the classic graph of input/output stimulation currents (IO) was obtained. We used a stimulation with a frequency of 1 Hz, 0.4 mA for 30 min, to stabilize the responses. For long-term post-tetanic potentiation development, the amplitude of the testing stimulus was 70% of the maximum extrasynaptic potential amplitude. Long-term potentiation (LTP) was obtained using a 100-Hz stimulation for 1 s.p < 0.05 was considered to indicate a statistically significant difference. For all studies, one animal was used as the analysis unit. All statistical tests were performed using Microsoft Excel software  and GraphPad Prism 4 .Data are presented as the means ± SEM. All data were tested for normal distribution using the Shapiro–Wilk test. Since the data obtained by the ELISA in in vivo experiments and immunohistochemistry were normally distributed, they were subjected to statistical analysis using one-way ANOVA followed by a post-hoc Tukey multiple comparison test. The data obtained by the electrophysiological recording and the ELISA in in vitro experiments were subjected to the Kruskal–Wallis test followed by Dunn’s multiple comparisons tests. A value of