Staphylococcus aureus (MRSA). This study evaluated a novel exolysin containing an SH3b domain for its antibacterial activity against MRSA.Enzybiotics are promising alternatives to conventional antibiotics for drug-resistant infections. Exolysins, as a class of enzybiotics, show antibacterial effects against methicillin-resistant Escherichia coli (E. coli). Finally, the antibacterial effects of LYZ2-SH3b compared with LYZ2 and vancomycin against reference and clinical isolates of MRSA were measured using the disc diffusion method, the minimal inhibitory concentration (MIC), and the minimal bactericidal concentration (MBC) assays.This study designed a chimeric exolysin by fusing the Cell-binding domain (SH3b) from Lysostaphin with the lytic domain (LYZ2) from the gp61 enzyme. Subsequently, LYZ2-SH3b was cloned and expressed in Analysis of bioinformatics showed that LYZ2-SH3b was stable, soluble, and non-allergenic. Protein purification was performed with a 0.8 mg/ml yield for LYZ2-SH3b. The plate lysis assay results indicated that, at the same concentrations, LYZ2-SH3b has a more inhibitory effect than LYZ2. The MICs of LYZ2 were 4 µg/mL  and 8 µg/mL , whereas, for LYZ2-SH3b, they were 2 µg/mL  and 4 µg/mL . This suggests a higher efficiency of LYZ2-SH3b compared to LYZ2. Furthermore, the MBCs of LYZ2 were 4 µg/mL  and 8 µg/mL , whereas, for LYZ2-SH3b, they were 2 µg/mL  and 4 µg/mL , thus confirming the superior lytic activity of LYZ2-SH3b over LYZ2.The study suggests that phage endolysins, such as LYZ2-SH3b, may represent a promising new approach to treating MRSA infections, particularly in cases where antibiotic resistance is a concern. But further studies are needed.The online version contains supplementary material available at 10.1186/s12866-023-03002-9. This problem is even more significant when it comes to bacteria .Staphylococcus aureus (S. aureus) is a gram-positive bacterium found as natural microflora in human and animal bodies. These bacteria can cause skin infections, food poisoning, dangerous shocks, and autoimmune disorders under special conditions such as stress, viral diseases, tissue injury, or any factor weakening the immune system [S. aureus have become resistant to antibiotics, including penicillin, methicillin, and vancomycin. Moreover, like other drugs, antibiotics have side effects, including allergic reactions, toxicity to various organs , and an inhibitory impact on natural flora. Therefore, finding materials with antibacterial properties is one of the basic needs in the battle against antibiotic-resistant S. aureus [e system , 3. Most. aureus .S. aureus phages belong to the Caudovirales order and are strictly lytic [Researchers have focused on phage therapy due to widespread antibiotic use. Most ly lytic . Howeverly lytic , 7. Due ly lytic –14. Throly lytic . The PGHly lytic .Staphylococcus simulans. Structure analyses show this enzyme has a C-terminal CBD named SH3b and its catalytic domain. This domain explicitly identifies the PG layer of S. aureus. Recent research indicates that using the SH3b domain of Lysostaphin to produce engineered chimeric enzybiotics significantly increases their activity and efficiency against the pathogen S. aureus [The enzyme gp61, a type of exolysin, is synthesized by the phage ΦMR11. Gp61 has an N-terminal CHAP  domain and a C-terminal LYZ2 domain, which performs the enzyme’s catalytic activity. Recent structural investigations of this protein indicate that the LYZ2 domain has much greater catalytic activity than the other domains of this enzyme. Furthermore, this enzyme lacks a cell wall binding domain . Bacteri. aureus .S. aureus bacteria [Numerous studies have been conducted on the role and production of chimeric endolysins in fighting bacteria , 9, 18. bacteria .S. aureus. SH3b is the cell-binding domain of the bacteriocin lysostaphin, which specifically targets the peptidoglycan layer of S. aureus [S. aureus.Phage-lytic peptidoglycan hydrolases, such as endolysins and exolysins, are antibacterial against gram-positive pathogens. However, this enzyme is not specific for identifying . aureus . TherefoPhysicochemical Properties, Solubility, and Allergenicity.Table E. coli by upgrading the CAI from 0.50 to 0.96. A CAI of 1.0 is ideal, and a CAI of ≥ 0.8 is considered appropriate for expression in the host organism (Sup 1 A). GC content and unfavorable peaks have been optimized to prolong mRNA half-life. The GC content changed from 44.29 to 54.60. The ideal percentage range of GC content is between 30 and 70% (Sup 1B). FOP of 91–100, 81–90, and 71–80 in the gene after optimization were 84,6 and 8%, respectively. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism (Sup 1 C).A wide variety of factors regulate and influence gene expression levels. The optimumGeneTM algorithm considers as many of them as possible, producing a single gene that can reach the highest possible expression level. In this case, the native gene employs tandem rare codons that can decrease translation efficiency or even disengage from the translational machinery. This algorithm changed the codon usage bias in E. coli DH5α cells. Protein purification was performed using a Ni-NTA column yielding 0.8 mg/ml for LYZ2-SH3b and 0.5 µg/ml for LYZ2. The purity and molecular weight (MW) of the proteins (LYZ2-SH3b: 36 kDa and LYZ2: 26 kDa) were confirmed through SDS-PAGE analysis (Sup 2 A-B). Furthermore, western blot analysis authenticated all the proteins (Sup 2 C).The optimized DNA sequences of LYZ2-SH3b (962 bp) and LYZ2 (695 bp) were correctly cloned into competent We assessed the lytic activities of the LYZ2 and LYZ2-SH3b enzymes against MRSA ATCC 43,300 and clinical isolate ST239 through plate lysis, MIC, and MBC assays. Plate lysis assay results are detailed in Table S. aureus’ PG layer. Following, we evaluated the antibacterial effects of LYZ2-SH3b compared with LYZ2 and vancomycin. This represents the first report on designing and synthesizing this specific chimeric enzyme. The results of this study suggest that LYZ2-SH3b may represent a promising new approach to treating MRSA infections.Antibiotic resistance in clinical settings is a growing concern, and novel therapeutic options are needed beyond conventional antibiotics . AdditioWe predicted its physicochemical properties after designing the chimeric enzyme (LYZ2-SH3b) Table . Our finAfter expressing and purifying LYZ2-SH3b, its antibacterial activity against MRSA was compared with that of LYZ2 using various methods, including disc diffusion, MIC, and MBC. The plate lysis assay showed that at similar concentrations, the diameter of the clear zone was not the same for LYZ2-SH3b and LYZ2   were utilized for plasmid cloning and expressing the chimeric enzymes (LYZ2-SH3b). Mueller-Hinton broth and agar  were utilized for bacterial growth. Bacteria were cultured overnight at 37 °C.Two strains of MRSA were employed to determine antibacterial activity: (i) the reference strain  and (ii) a clinical isolate, ST239. The https://www.uniprot.org/) in FASTA format. Subsequently, these sequences were directly joined without linkers  and SH3b (Accession No: M15686) domains from the UniProt database (https://web.expasy.org/protparam/) was utilized to evaluate the physicochemical properties of LYZ2-SH3b. These properties include the instability index, theoretical PI, amino acid composition, molecular weight (MW), aliphatic index, estimated half-life, and the grand average of hydropathicity (GRAVY). The solubility of LTZ2-SH3b in the E. coli host was predicted using the SOLpro server (https://scratch.proteomics.ics.uci.edu/). Furthermore, allergenicity was checked by the AllerTOP 2.0 server (https://www.ddg-pharmfac.net/AllerTOP/).The ProtParam tool on the ExPASy server (https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/). To attain a significant increase in LYZ2-SH3b expression within E. coli, gene optimization was carried out employing the OptimumGeneTM Optimization algorithm. This process involved the use of the algorithm available at . Various sequence factors were evaluated, such as GC content, codon adaptation index (CAI), frequency of optimal codons (FOP), CIS-acting elements, and repeat sequences.The sequence (LYZ2-SH3b) was back-translated using the EMBOSS Backtranseq server . Chemically competent E. coli DH5α cells  were transformed using a heat shock protocol with the recombinant constructs (pET-LYZ2-SH3b and pET-LYZ2). The plasmids were extracted from transformants using GeneAll Plasmid Miniprep Kit , and the accuracy of the cloning was confirmed by nucleic acid sequencing.DNA fragments for LYZ2-SH3b were synthesized  and inserted into the pET-28a (+) expression vector  at the rs Table  from theE. coli BL21 (DE3)  chemically competent cells were transformed with recombinant plasmids (pET-LYZ2-SH3b and pET-LYZ2) and grown at 37 ºC in 100 mL of Luria Broth (LB) medium containing 100 µg/mL kanamycin. To induce protein expression, after reaching the cell density to an OD600 of 0.5, 1 mM isopropyl-b D-thiogalactopyranoside (IPTG) was added to the cultures, which were incubated for 6 h at 37 ºC (160 rpm). Cells were then harvested by centrifugation at 6000 ×g at 4 °C for 20 min. Bacterial pellets were resuspended and homogenized in 5 ml lysis buffer  for 45 min at 4 °C. PMSF (Phenylmethylsulfonyl Fluoride) as a protease inhibitor was added in 1 mM concentration for inhibiting proteases. The pellets were sonicated to disrupt bacteria under ice conditions: ten cycles of sonication at 45% amplitude for 10 s and intervals of 10 s. The resulting lysates were then centrifuged at 13,000×g for 30 min at 4 °C. Supernatants were applied to Ni-NTA resin  for 2 h at 4 °C. Subsequently, the resin was washed twice with 5mL wash buffer . The specifically bound protein was washed out of the resin with 5 mL of elution buffer . Finally, the protein concentration was determined using the BCA protein assay kit .The presence of recombinant proteins in the purified proteins (LYZ2-SH3b and LYZ2) was demonstrated through SDS-PAGE and confirmed by western blot analysis . For SDSThe antibacterial activity of the purified enzymes (LYZ2 and LYZ2-SH3b) was measured through the disc diffusion method, minimal inhibitory concentration (MIC), and minimal bactericidal concertation (MBC) against two strains of MRSA (i) the reference strain  and (ii) a clinical isolate ST239. All antibacterial activity assays were measured according to the guidelines of the CLSI  . In addiS. aureus (MRSA) from the MHA plate was resuspended in sterile distilled water to achieve a density of McFarland 0.5 (2 × 108 CFU/ml). Subsequently, the purified enzymes were serially diluted 1:10 in PBS to achieve 50, 5, and 0.5 µg/ml concentrations. Then, 10 µl of each dilution was added to a blank antibiogram disk (6.4 mm) spotted onto a plate of S .aureus. The MHA plate was incubated at 37 °C overnight. The control plate was spotted with an antibiogram disc containing only buffer (PBS). The diameters of the inhibition zones formed by the enzymes were measured and compared.The antimicrobial activity of the purified enzymes (LYZ2 and LYZ2-SH3b) was assessed using the agar disc diffusion method . PickingS. aureus on agar plates after overnight incubation at 37 °C. As identified in the MIC determination experiment, an aliquot of 50 µl from each non-turbid well was spread on agar plates.The MIC of each enzyme (LYZ2 and LYZ2-SH3b) was determined using microdilution in sterile 96-well plates . BrieflyAll tests were carried out in duplicate. The data were represented as the mean ± standard deviation (SD). The experimental results were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for all pairwise comparisons. All statistical analyses were performed using the statistical package for social science version 19 . In all cases, a p-value < 0.05 was established as the threshold for considering a result statistically significant.Below is the link to the electronic supplementary material.Supplementary Material 1


We here conducted a pot experiment to compare the effects of biochar application on rice yield, nitrogen (N) uptake, NH3 and N2O losses in paddy soil with low, medium, and high N inputs at 160 kg/ha, 200 kg/ha and 240 kg/ha, respectively. The results showed that: (1) Biochar significantly increased the rice grain yield at medium (200 kg/ha) and high (240 kg/ha) N inputs by 56.4 and 70.5%, respectively. The way to increase yield was to increase the rice N uptake, rice panicle number per pot and 1,000 grain weight by 78.5–96.5%, 6–16% and 4.4–6.1%, respectively; (2) Under low (160 kg/ha) N input, adding biochar effectively reduced the NH3 volatilization by 31.6% in rice season. The decreases of pH value and NH4+-N content in surface water, and the increases of the abundance of NH4+-N oxidizing archaea and bacteria (AOA and AOB) communities contributed to the reduction of NH3 volatilization following the biochar application; (3) Under same N input levels, the total N2O emission in rice season decreased by 43.3–73.9% after biochar addition. The decreases of nirK and nirS gene abundances but the increases of nosZ gene abundance are the main mechanisms for biochar application to reduce N2O emissions. Based on the results of the current study, adding biochar at medium (200 kg/ha) N level (N200 + BC) is the best treatment to synchronically reduce NH3 and N2O losses, improve grain yield, and reduce fertilizer application in rice production system.Biochar application can improve crop yield, reduce ammonia (NH China rice production accounts for 27% of global rice production and about 35% of China’s total grain production (3) volatilization is one major N loss pathway and account for 10–60% of the total N fertilizer input in the rice season. Moreover, nitrous oxide (N2O) emissions from rice fields can account for 7–11% of N2O emissions from agricultural fields in China , soil NH4+-N, and nitrate (NO3−-N) contents and functional microbial gene abundance by a pot experiment. The results and conclusions of the study can provide technical support and theoretical basis for the mutual interaction of N fertilizer and biochar to achieve N fertilizer reduction and biochar resource conservation, crop yield stabilization and environmental protection in rice production.The integrated evaluation of crop yield, soil NHion rate . However2.2.1.3) as in the field. About 20 kg soil was filled into each soil column according to the volume of soil column and the soil bulk density. The selected properties of 0–20 cm topsoil was as follows: pH 6.38 (soil: water ratio 1: 5), soil organic matter 29.2 g/kg, total N 1.72 g/kg, available P 23.1 mg/kg, available K 159.3 mg/kg, and CEC 22.6 cmol/kg. In this experiment, biochar was produced using wheat straw that had been heated to 500°C. The reactor was heated by a stepwise procedure under oxygen-limited conditions. For pyrolysis, the temperature was raised to 500°C at a rate of 5°C/min and held constant for 8 h. The measured properties of the biochar were pH 9.80, total N 0.81%, total C 37.5%, and BET surface area 32.0 m2/g. At the initiation of the experiment, at the same time of repacking soil into column/pot, biochar was mixed with the top layer (0–20 cm) soil.Test soil for this study was collected at depth of 0–20 cm from an approximately 30 ha paddy field in Zhoutie town of Yixing city , Jiangsu Province, which is located at the Taihu Lake region of China. This region has a subtropical monsoon climate, with an annual mean air temperature and rainfall of 15.7°C and 1,177 mm, respectively. The soil was mixed, air-dried for approximately 10 days, ground and sieved through a 2-mm nylon sieve, and repacked layer-wise (0–10 and 10–20 cm) into soil columns  at similar bulk density  seedlings (28 days old) were transplanted into the soil columns (three holes in each column and two plants per hole) on July 1, 2021. The total fertilizer N application was split into a basal dressing at transplanting, and two top-dressing during the season in the ratio of 40–30–30%, respectively. Application periods were July 1, July 16, and August 14 in 20,218. Calcium superphosphate and potassium chloride were applied to all treatments including the CK at the rates of 90 kg/ha (P2O5) and 150 kg/ha (K2O), respectively, at the time of transplanting as basal fertilizer. The floodwater was drained at a mid-season drainage period from July 31 to August 7, 2021 to control invalid tillering and improve the rice root development. During other times, a 3–5 cm depth floodwater level was maintained with tap water. Weeds and pests were controlled according to the local farmers’ traditional practices. The rice shoots (including straw and grain) were manually harvested on November 8, 2021.Rice  were also recorded. Rice straw and grain were oven-dried at 105°C for 30 min, and then dried at 80°C until constant weight. The dried plant samples were ground into powder using a high-speed crusher (DS-YM-001), passed through a 0.2 mm nylon sieve. Ground plant samples were kept in sealed containers until digestion. Sub-sample (0.25 g) of ground plant samples were digested in a mixture of HWhere To reflect the leaf chlorophyll content, SPAD values of rice leaves were measured using the chlorophyll content meter  at the tillering, earing, and maturation stage, respectively. This was done by selecting three rice plants from each soil column and three leaves of each rice plant were measured. Therefore, the SPAD values presented in this study represented average SPAD value of the three plants in each replicate .2.3.2.3 volatilization rates were measured at three N fertilizer applications, using the sponge absorption method . The NH3 volatilization was calculated using Equation 2 outlined below:During sampling, the lower sponge was taken out (8: 00 am), immediately sealed in a bag, and replaced in the gas-capturing device with a new sponge also beforehand dipped in phosphoglycerol. The upper sponge was replaced once every 2 days. The sampled sponge was placed into a 500 mL plastic bottle, submerged in 300 mL of 1 M KCl, and shook at 180 r per minute for 1 h. The NHω =−3(2)ω: NH3 content in a single collection device (mg); 4+-N concentration (mg/L); 3 emission factor and yield-scale NH3 volatilization were calculated according to that introduced in our previous work  at 350°C equipped with an electron capture detector (ECD). Total N2O emission was calculated from the individual fluxes and the interval times . A sub-sample of 50 ml filtered water was stored in clean plastic bottles at −20°C for further analysis. The concentrations of NH4+-N and NO3−-N in overlying water were determined by an autoanalyzer .Overlying water samples were collected using a syringe on the same day and time as NH2.3.5.4+-N and NO3−-N determination. Soil extracts were filtered through a 0.45 μm membrane filter and the NH4+-N and NO3−-N concentrations were determined using an autoanalyzer . The gene copy numbers of AOA and AOB amoA, nirK, nirS, and nosZ of soil samples were determined by Shanghai Majorbio Biomedical Co., Ltd. according to the procedures detailed in At the end of the experimental period after rice harvest, three soil cores in each pot were randomly sampled at 0–20 cm depth using a soil drill (50 mm in diameter), top layer soil was sampled at selected points. Soil samples were composited, mixed manually, placed in self-sealing bags, and brought back to the laboratory in collar box with ice. Soil samples were divided into two parts: one was stored at −80°C for molecular analysis and another at −20°C for analysis of other properties. The soil pH was measured in the 1:2.5 (w/v) soil: water suspension using a combined reference electrode (Ф255 pH/temp/mV meter). Soil samples were extracted with 2.0 M KCl  for NH2.4.p < 0.05).A statistical analysis was performed using SPSS 22.0, and one-way analysis of variance (ANOVA) was used to determine the significance of the difference between treatments. The level of significance was measured using Duncan’s multiple-comparison test (3.3.1.3.1.1.p < 0.05) increased the straw biomass by 54.0 and 77.0% relative to the control treatment. Both N160 and N160 + BC had no difference in straw biomass compared to the control. The combinations of BC with N200 and N240 significantly (p < 0.05) increased rice straw biomass by 79.5 and 42.3% compared to N200 and N240 alone, respectively. However, there was no difference in straw biomass between N160 + BC and N160. Interestingly, the grain yield in N added treatments (70.53–145.50 g/pot) were significantly (p < 0.05) higher than the no N added control treatment (42 g/pot). Moreover, N200 + BC and N240 + BC treatments significantly (p < 0.05) promoted the rice grain yield by 31.9 and 70.6% relative to N200 and N240 treatments, respectively. Nevertheless, amendment of biochar induced no difference in rice grain yield at low N input level (160 kg/ha).The results in p < 0.05) 16 panicles/pot and 6.1% higher than N200, respectively. Compared with N240 treatment, however, N240 + BC treatment significantly (p < 0.05) increased the number of panicles, grains per panicle and 1,000 grain weight of rice by 6 panicles/pot, 35.9, and 4.4%, respectively  compared with N240 treatment (p < 0.05) increased by 5.0–11.0% and 7.1–19.5%, and rice leaf SPAD value was significantly (p < 0.05) increased by 9.9–13.4% and 17.6–29.9% (p < 0.05) when biochar was applied at medium (200 kg/ha) and high N (240 kg/ha) application levels. Among the different treatments at both early and maturing stage, the N200 + BC treatment had the highest values in terms of rice plant height and leaf SPAD value  increase of 9.7 and 40.7% in seed N content and uptake, compared with N160 treatment, respectively. Biochar addition significantly (p < 0.05) increased rice straw N content by 39.8–75.7% and N uptake by 148–152% at medium and high N supply. Overall, biochar addition exerted no effect on seed N content of rice receiving same N fertilizer, while biochar addition significantly (p < 0.05) increased seed N uptake by 54.7–73.0% at middle (200 kg/hm2) and high (240 kg/hm2) N supply. Meanwhile, the total N uptake (straw + seeds) in N200 + BC and N240 + BC treatments was 1.8 and 2.0 times higher compared to N200 and N240 treatments, respectively (No significant difference in N content and uptake by rice straw among the treatments with N fertilizer but without biochar . Howeverectively .3.2.3 volatilization rate (24.95–67.03 mg/pot/d) was observed on the third days after BF, while the peak NH3 volatilization rate (29.48–110.63 mg/pot/d) was observed on the first day after SF2. Except for the N240 treatment, NH3 volatilization could reach its peak (41.33–74.84 mg/pot/d) within the first 3 days after SF1. The NH3 volatilization rate in all N fertilizer applied treatments dropped to a low level as that in the control treatment within 7 days after N each fertilizer application conducted.As shown in 3 volatilizations in the rice season under N applied treatments were 0.20–0.53 g/pot, accounting for 10.5–28.7% of the fertilizer N input into the rice paddy. The NH3 losses after BF, SF1 and SF2 were 0.06–0.18 g/pot, 0.09–0.23 g/pot, and 0.05–0.20 g/pot, respectively (3 volatilization at both SF1 (38.9%) and SF2 (37.2%) observations, compared with N160 treatment, and this effect resulted in an overall significant (p < 0.05) reduction in cumulative amount of NH3 volatilization by 31.6%. Moreover, the application of biochar at N160 level significantly (p < 0.05) reduced the NH3 emission factor by 37.2%. Nevertheless, the total NH3 losses and emission factor in the rice season were not influenced by biochar at 200 kg/ha and 240 kg/ha applications. Interestingly, biochar addition significantly (p < 0.05) reduced yield-scale NH3 volatilization at all three N application levels by 29.7, 52.7, and 46.3% relative to N160, N200, and N240 kg/ha supply, respectively (The cumulative NHectively . N160 + ectively .3.3.2O emissions in the rice season increased significantly (p < 0.05) with the increasing N application (p < 0.05) reduced the cumulative N2O emissions in rice season by 54.1, 43.3, and 73.9% relative to N160, N200, and N240, respectively. Meanwhile, results in 2O emissions of N200 + BC and N240 + BC treatments in rice season can be reduced to the N160 level, and the N2O emissions of N160 + BC treatment can be reduced to the control treatment level.Under no biochar additions, Nlication . Biochar3.4.3.4.1.4+-N concentrations in the overlying water of each treatment during the BF occurred on the 2–5 days (16.1–29.0 mg/L) after N fertilizer application  and high (240 kg/ha) N supplied treatments increased the average NH4+-N concentration of overlying water by 4.4 and 33.9%, respectively. After the SF1 and SF2 applied, the addition of biochar at low (160 kg/ha) N supply level increased the average NH4+-N concentration of overlying water by 10.0 and 11.8%, respectively. Nevertheless, biochar addition at medium (200 kg/ha) and high (240 kg/ha) N supply levels reduced the mean overlying water NH4+-N concentrations by 20.3–54.5% and 31.0–53.7%, respectively, during the same periods.At the BF observation, compared with N200, the average NH3.4.2.3−-N concentration in the overlying water by 16.2, 3.8, and 11.0%, respectively  N level it decreased the mean NO3−-N concentration in the overlying water by 41.3%. During the SF2, the average NO3−-N concentration in the overlying water of N200 + BC and N240 + BC treatments was 3.5 and 1.2 times higher than that of N200 and N240 treatments, respectively  and high (240 kg/ha) N fertilizer application conditions. The soil NH4+-N content in N160 + BC treatment was significantly (p < 0.05) higher than N160 treatment by 9.8%, while that in N240 + BC was significantly (p < 0.05) lower than N240 treatment by 15.0% (4+-N contents in the N240 + BC treatment were significantly (p < 0.05) 48.8 and 79.7% higher than N240 treatment during the SF1 and SF2, respectively.During the BF, biochar had varied effects on soil NHby 15.0% . The soi3−-N content was not remarkably affected by N fertilizer application and biochar addition during the BF and SF1 (p < 0.05) increased the soil NO3−-N content, and the N200 + BC treatment had 3.3 times more soil NO3−-N content than the N200 treatment  increased by 37 and 146%, relative to their counterparts N160 and N240 treatments, respectively. However, soil AOB abundance under medium (200 kg/ha) N supply conditions was significantly (p < 0.05) increased by 1.5 times with the addition of biochar.n levels . Howeverp < 0.05) lower number of nirS, nirK, and nosZ functional genes by 78.0, 25.3, and 73.1%, respectively compared to N160. In contrast, the copies of nirS, nirK, and nosZ in N240 + BC soil were 10.8, 3.7, and 7.4 times significantly (p < 0.05) higher than N240 soil. No significant effect of biochar on the copies of all three denitrifying bacterial functional genes was found at medium (200 kg/ha) N supply level.N160 + BC had significantly  N level  N condition in this experiment were mainly found the SF1 (39%) and SF2 (36%). As shown in the 4+-N and pH decreased by 4.07 mg/kg and 0.07 units, respectively, during the SF1  N levels can increase the community abundance of AOB   . Nguyen 60 kg/ha ,B, which biochar , especia biochar . In this biochar , which anirS, nirK, and nosZ genes play key roles in regulating N2O emissions  N levels increased soil nosZ gene copy numbers (2O emissions as biochar was linked to the decreased nirS and nirK genes copies but the increased nosZ gene copies or a decrease in the ratio of (nirK + nirS)/nosZ in other word  N level in this study inhibited soil autotrophic nitrification and denitrification processes by reducing soil nirS and nirK gene abundance, and at medium (200 kg/ha) N level by reducing soil nirS gene abundance. In contrast, the addition of biochar at high (240 kg/ha) N levels is effective in reducing N2O emissions by increasing the abundance of soil nosZ genes, increasing N2O reductase activity, and promoting the catalytic process of N2O reduction to N2.Functional soil microbial communities represented by missions . In this numbers . Accordiher word . Duan et5.3 and N2O losses in paddy soil and the underlying mechanisms were evaluated by a soil column experiment. The main conclusions were:The combined effects of biochar addition and N reduction on rice yield and N uptake, NH(1) The yield increasing effect of biochar can be realized at medium (200 kg/ha) to high (240 kg/ha) N inputs, attributing to the improving N uptake and increasing panicle number, grain number per panicle and 1,000 seed weight. In particular, the highest yield was achieved by combination of biochar and N fertilizer at medium (200 kg/ha) level.3 volatilization in rice season with low (160 kg/ha) N supply condition. Interestingly, however, at equal N input level, yield-scale NH3 volatilizations were reduced by biochar addition.(2) Biochar addition only effectively reduced the NH2O emissions from rice paddy receiving inorganic N fertilizer from low (160 kg/ha) to high (240 kg/ha) inputs. Varied changes in the functional genes, including nirS, nirK, and nosZ, explained the inhibiting effects of biochar on the N2O emission at different N inputs.(3) Biochar addition was effective in reducing the N2) N levels, which can archive the synergistic benefits of reducing inorganic N fertilizer, promoting crop yield, and decreasing N environmental losses.(4) We recommend the addition of biochar at medium , the Jiangsu Agricultural Science and Technology Independent Innovation Fund [CX(21)3066], the National Natural Science Foundation of China (31972518), and the Qing Lan Project of Jiangsu Province of China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1174805/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using  Antimicrobial peptides (AMPs), a diverse family of short peptides that are important for plants and animals, are powerful weapons in host defense mechanisms and are present in all life domains . DefensiE. coli under aerobic conditions than under anaerobic conditions through multiple actions, such as membrane perturbation, induction of strong oxidative stress, and intracellular effects -Crp4 (Novagen) vector was used in the expression of crp4 as the inclusion body. E. coli cells were crushed by an ultrasonic crusher  at 180 W for 30 min in sonication buffer , and the inclusion body was collected by centrifugation at 4300× g for 20 min at 4 °C. The inclusion body was then solubilized in buffer  at 37 °C, 180 rpm, and refolded into crp4oxi by air oxidization. The supernatant, which contains crp4oxi, was purified by cation exchange chromatography of SP Sepharose FAST FLOW (Cytiva), equilibrated with 50 mM Glycine-NaOH pH 8.5, 3 mM EDTA. Adsorbed crp4oxi was eluted using a linear gradient of 0–1 M NaCl buffer. After overnight dialysis in 0.1% acetic acid, the crude crp4oxi sample was purified by reversed phase HPLC (RP-HPLC) using column COSMOSIL Protein-R . Elution was performed using a linear gradient of 0–50% acetonitrile with 0.1% TFA. Finally, the purified recombinant crp4oxi was lyophilized and stored at −30 °C before use. Crp4oxi was produced by the co-expression system, and purified as described previously . BrieflyTo obtain crp4red, 500 mM dithiothreitol (DTT) was added to 2–5 mg/mL crp4oxi solution. The solution was then incubated at room temperature. After 16 h, the reaction solution was adjusted to pH 2.0~3.0 and crp4red was purified by RP-HPLC with the same elution program, then lyophilized and stored at −30 °C before use.Crp4oxi and crp4red were used in the full range from 0.3125 μg/mL (0.083 μM) to 10,000 μg/mL (2660 μM), and from 1.25 μg/mL (0.332 μM) to 500 μg/mL (133 μM), respectively.E. coli K12 (ATCC 39425) was used throughout this study, which was cultured in 3% TSB (Tryptone soy broth) at 37 °C, with 180 rpm under aerobic cultivation and 50 rpm under the anaerobic condition. It took 3–3.5 h and 4.5–5 h to reach to the exponential phase (absorbance at 600 nm was 0.4) under aerobic and anaerobic cultivation conditions, respectively. An Anaero Pack system  was used to maintain anaerobic growth conditions during the cultivation, incubation, and plating processes. The bacterial solution was collected by centrifugation , washed, and resuspended in an assay buffer containing 10 mM sodium phosphate buffer (pH 7.4) at different concentrations in each experiment. The concentration of bacteria was calculated by colony counting.Facultative anaerobe E. coli solution, prepared using the above protocol, and crp4 solution dissolved in 10 mM sodium phosphate buffer (pH 7.4) were mixed and incubated at 37 °C for 1 h. The concentration of bacteria at this time was 1 × 107 CFU/mL. Serial dilutions of crp4oxi or crp4red at a range of 20 to 0.625 μg/mL were used against E. coli cultivated under the aerobic condition. For cells under anaerobic cultivation treatment, 100, 500, 1000, 2500, 5000, 7500, and 10,000 μg/mL of crp4oxi and 25, 50, 100, and 500 μg/mL of crp4red were used. The final bacterial concentration was 1 × 107 CFU/mL. The mixture was then diluted 1000 times and 50 μL dilution cell suspension was added to a 3% TSB medium with 1.5% agar. After culturing overnight at 37 °C, the number of colonies was compared to the control without antimicrobial peptide under aerobic and anaerobic conditions, respectively, and survival rates were calculated.The minimum bactericidal concentration (MBC) was determined using the broth microdilution method as described previously . BrieflyMembrane damages on the outer membrane (OM) and inner membrane (IM) were confirmed using methods basically as described previously ,22.E. coli cells were washed and diluted to OD600 at 0.2, with 5 mM HEPES buffer (pH 7.2) containing 20 mM glucose, by centrifugation at 2770× g, 25 °C, for 5 min. Afterward, peptides with the final concentration of 80 μg/mL and NPN with the final concentration of 10 μM were added 60 s after starting the measurement. The final bacterial concentration was 4 × 107 CFU/mL. Changes in fluorescence were monitored continuously using a fluorescence spectrometer  at excitation and emission wavelengths of 355 and 408 nm, respectively. For the OM damage assay, cultured E. coli suspension was prepared as the just mentioned method above. Subsequently, bacteria suspensions were incubated with the final concentration of 1 μM DiSC3(5) containing 0.15 mM EDTA at 37 °C, 50 rpm, for 30 min in the dark. Afterward, peptides with the final concentration of 80 μg/mL were added 60 s after starting the measurements. The final bacterial concentration was 4 × 107 CFU/mL. Changes in fluorescence were monitored continuously using a fluorescence spectrometer at excitation and emission wavelengths of 622 and 670 nm, respectively.For the IM damage assay, E. coli suspension prepared by the above protocol was treated for 1 h, at 37 °C with peptides. Two peptide concentrations 0.5×  and 1× MBC  were used. The final bacterial concentration was 4 × 107 CFU/mL. Then, normal centrifugation at 4 °C, 9300× g, for 5 min was performed to precipitate intact or less-damaged cells as pellets (CP). Then, the supernatant was ultra-centrifugated at 4 °C, 252,000× g, for 30 min for obtaining precipitate as membrane fraction-containing membrane vesicles (MV).Membrane vesicles were isolated as described before . BrieflyAcid-urea polyacrylamide gel electrophoresis (AU-PAGE) was performed as previously described . SamplesThe DNA-binding abilities of the peptides were examined using a gel retardation assay. Plasmid DNA (10 μg/mL of the pET16 plasmid) was incubated with peptides at a range concentration of 5, 10, 20, 40, and 50 μg/mL in Tris-EDTA buffer  for 30 min at room temperature. After incubation, the peptide-DNA mixtures were subjected to electrophoresis on a 0.8% agarose gel containing ethidium bromide . The gelE. coli cells were collected as the method mentioned above and washed with 10 mM phosphate buffer (pH 7.4). The final OD600 of bacteria suspension was adjusted to 0.2. Peptides with the final concentration of 80 μg/mL were added 60 s after starting the measurements. The final bacterial concentration was 4 × 107 CFU/mL. Changes in fluorescence were monitored continuously using a fluorescence spectrometer  at excitation and emission wavelengths of 428 and 457 nm, respectively. Bacteria treated with 1% TritonX-100 were used as controls.The cultured E. coli cultivation step, until the exponential growth phase (absorbance at 600 nm was 0.4~0.6). The final concentration of GSH in TSB was 15 mM. The cultivated E. coli solution was washed and diluted using the above protocol, and crp4, dissolved in 10 mM sodium phosphate buffer (pH 7.4), was mixed and incubated at 37 °C for 1 h. For cells treatment, 0.5×, 0.375×, 0.25×, 0.125×, and 0.0625× MBC of crp4oxi (MBC = 5 μg/mL) and crp4red (MBC = 10 μg/mL) were used. The mixture was then diluted and added to a 3% TSB medium with 1.5% agar. After culturing overnight at 37 °C, the survival rate was calculated by colony counting. The final bacterial concentration was 1 × 107 CFU/mL.Antioxidants were freshly prepared as mentioned below before use . Stock sE. coli solution and crp4 were mixed and incubated at 37 °C for 1 h. For cells treatment, 0.5×, 0.375×, 0.25×, 0.125×, and 0.0625× MBC of crp4oxi (MBC = 5 μg/mL) and crp4red (MBC = 10 μg/mL) were used. The mixture was then diluted and added to a 3% TSB medium containing 1.5% agar and 15 mM GSH. TSB agar plates were prepared by adding stock solutions (250 mM) of glutathione to the medium before pouring the plates.Cultivated Circular dichroism (CD) data were collected using a Jasco J 725 spectropolarimeter . Spectra were collected from 250 to 190 nm and scanned at 20 nm/min at 25 °C. The bandwidth was 1.0 nm, and each data point was scanned four times under nitrogen gas. The spectra were measured in three different environments: 10 mM sodium phosphate buffer (pH 7.4), 10, 20, 30%, and 40% trifluoroethanol (TFE), and 10 mM sodium dodecyl sulfate (SDS), and 113 μg/mL peptide was used.θ, were calculated using the formula below:n is the number of amino acid residues, C is the peptide concentration, and l is the optical passage length of the cell.The mean residue ellipticity values, This study suggests that different antimicrobial activities and mechanisms depend on the redox-dependent structure of crp4 and the growth conditions of the target bacteria. This complexity is likely to contribute to the success and stability of this highly conserved host defense system in the constant fight against microbes.


Medicago sativa L.), white clover (Trifolium repens L.), orchardgrass (Dactylis glomerata L.), and tall fescue (Festuca arundinacea Schreb.) were grown in monocultures and mixtures  under three N inputs  in a split plot arrangement. The results highlight that A1 mixture under N2 input had a greater forage yield of 13.88 t ha−1 year−1 than the other N inputs, whereas A2 mixture under N3 input had a greater forage of 14.39 t ha−1 year−1 than N1 input, but it was not substantially greater than N2 input (13.80 t ha−1 year−1). The crude protein (CP) content of grass monocultures and mixtures significantly (P < 0.05) increased with an increase in the rate of N input, and A1 and A2 mixtures under N3 input had a greater CP content of 18.91% and 18.94% dry matter, respectively, than those of grass monocultures under various N inputs. The A1 mixture under N2 and N3 inputs had a substantially greater (P < 0.05) ammonium N content of 16.01 and 16.75 mg kg−1, respectively, whereas A2 mixture under N3 had a greater nitrate N content of 4.20 mg kg−1 than the other cropping systems under various N inputs. The A1 and A2 mixtures under N2 input had a substantial higher (P < 0.05) urease enzyme activity of 0.39 and 0.39 mg g−1 24 h−1 and hydroxylamine oxidoreductase enzyme activity of 0.45 and 0.46 mg g−1 5 h−1, respectively, than the other cropping systems under various N inputs. Taken together, growing legume–grass mixtures under N2 input is cost-effective, sustainable, and eco-friendly, which provide greater forage yield and improved nutritional quality by the better utilization of resources.Information regarding relationships between forage yield and soil enzymes of legume–grass mixtures under nitrogen (N) fertilization can guide the decision-making during sustainable forage production. The objective was to evaluate the responses of forage yield, nutritional quality, soil nutrients, and soil enzyme activities of different cropping systems under various N inputs. Alfalfa ( With the instant development of animal husbandry, the demand for forages has emerged frequently . A consiForage production has been tightly bound up with the environment, as it is a basic industry that requires resources including land, energy, water, and labor. The cultivation of legume–grass mixtures emerged as a practical approach for increasing the forage biomass production and nutritional quality and sustaining the soil nutrient balance with minimal environmental impact . AdditioThe Sichuan province of China is regarded as one of the leading producers of livestock husbandry, but forage deficit, both as quantitative and qualitative, is the main constraint on the advancement of livestock husbandry in this region . This shConsequently, the current study aimed to investigate the responses of forage yield and quality, soil nutrients, and soil enzyme activities to different planting patterns and N inputs. The results of this study may provide guidance for forage production in Sichuan, China, by mixed planting and optimal N inputs that could mitigate the negative environmental impacts leading towards sustainable agricultural systems.22.1Medicago sativa L. (cv. Xibuzhixing)] and white clover [Trifolium repens L. (cv. Ladino)] and grasses orchardgrass [Dactylis glomerata L. (cv. Amba)] and tall fescue [Festuca arundinacea Schreb. (cv. Meishijia)] were selected as forage materials.A field experiment was conducted on September 15, 2018 at Modern Agriculture Research and Development Base of Sichuan Agricultural University, Chongzhou, China . The legumes alfalfa [2.2−1; alkali hydrolyzed nitrogen, 135.70 mg kg−1; total nitrogen, 1.81 g kg−1; available phosphorous, 10.20 mg kg−1; and available potassium, 101.10 mg kg-1. The climate of the experimental site is subtropical monsoon humid with an annual average temperature of 15.9°C, rainfall of 1,012.4 mm, and sunlight of 1,161.5 h.The soil in the upper 20 cm of the experimental field is purple clay loam with uniform fertility and has the following properties: pH, 6.30; organic matter, 37.6 g kg2.3−1, N2, 300 kg ha−1, and N3: 450 kg ha−1). The main plots included three N levels, while the subplots included two legume–grass mixtures and four monocultures. The first N dose was applied at the emergence stage, while the rest of the doses were applied after each mowing. The basal inputs of P2O5 and K2O fertilizers were applied at 96 and 160 kg ha−1 year−1 to all plots, respectively. The seeds were handed-scattered into the soil and were covered with a thin layer of soil. Weeds that appeared in all plots were removed by hand. The seeding rates used for growing legume–grass mixtures and their corresponding monocultures are presented in The field experiment was carried out in a split plot arrangement with three biological replications. A total of two legume–grass mixtures  and four monocultures  were planted in a net plot size of 5 m × 3 m under three pure N inputs  content, which was later used to calculate the DM yield.The first, second, third, and fourth cuttings for forage yield were performed during the initial flowering stage of alfalfa on March 24, May 6, July 23, and September 25 in 2019, respectively. Before harvesting, the side rows of each plot were removed, 50 cm of both sides was removed, and the area of (5–0.5 × 2) × (3–0.6) = 9.6 m2.4.2The dried samples were ground to pass a 1-mm screen for nutritional quality analysis. The water-soluble carbohydrate (WSC) content analysis was referred to the thracenone–sulphuric acid method, while the crude protein (CP) content was measured by the Kjeldahl method . The neu2.4.3The soil samples at 15 cm depth were randomly collected from the experimental site prior to seeding and after each mowing. The five sub-samples were taken and then bulked to one sample for each replication. The soil samples were stored in cloth bags and air-dried at room temperature to a constant weight. The roots, stones, and other debris in the soil samples were removed, and the soil samples were passed through a 2 mm sieve and then stored in the laboratory until analysis.2.4.4The pH was measured in 1/5 (w/v) aqueous extract using a pH meter. The contents of phosphorus and potassium were determined by inductively coupled plasma spectrometry after nitric-perchloric acid digestion. Soil organic matter was determined by the dilution heat method, while that of alkali hydrolyzed nitrogen was determined by the alkaline hydrolysis diffusion method . The tot2.4.52)2, and 20 ml of citrate buffer were added. The samples were placed in an incubator for 24 h at 38°C. After incubation, CH2O was added into each sample to a constant volume of 100 ml and mixed well. The supernatant (1 ml) was taken and mixed with deionized water (9 ml), phenol (4 ml), and sodium hypochlorite solution (3 ml). The samples were placed at room temperate for 20 min. The absorbance of the samples was measured at 578 nm using a spectrophotometer.The phenol-sodium hypochlorite colorimetry method was followed to measure the urease enzyme activity . Briefly2.4.63 was added and mixed well. Then, 1 ml of 0.5% hydroxylamine hydrochloride, 1 ml of 1% glucose as hydrogen donor, and 5 ml of H2O were added. The samples were incubated for 5 h at 30°C in an incubator. The control sample was set without adding hydroxylamine hydrochloride and soil. After the incubation, the mixture was transferred in different test tubes, and 2 ml of alumina potassium alum saturated reagent was added. The samples were vortexed, and 1 ml of supernatant was taken and mixed with 1 ml buffer (1 mol/L CH3COONa and 1 mol/L CH3COOH), 1 ml ferric ammonium sulfate solution (0.004 mol/L), and 1 ml o-phenanthroline ethanol solution (0.01 mol/L). The color of the solution was developed for 10 min. A spectrophotometer was used to measure the absorbance at 510 nm.Hydroxylamine oxidoreductase (HAO) enzyme activity was determined by the ammonium ferric sulfate o-phenanthroline method. Briefly, 1 g of dried soil was taken in a test tube, and 20 mg of CaCO2.4.73 was added and mixed well into the former. Then, 1 ml 0.8 mmol/L 2,4-DNP solution, 1 ml 1% KNO3 solution, and 1 ml 1% grape poplar solution as hydrogen donor were added, and these were also mixed well. After mixing, 15 ml H2O was added to form the liquid seal, and the solution was incubated for 24 h at 30°C in the incubator. The control sample was set without adding 1% KNO3 and soil. After incubation, the solution was transferred to a different test tube, and 1 ml alumina potassium alum saturated reagent was added. Then, 1 ml of supernatant was taken and mixed with deionized water, 4 ml 0.1% α-naphthylamine solution, and 0.5% p-aminobenzenesulfonic acid. The color of the solution was developed for 15 min, and then a spectrophotometer was used to measure the absorbance at 520 nm.The α-naphthylamine-p-aminobenzene sulfonic acid colorimetry method was followed to determine the nitrate reductase (Nar) enzyme activity . Briefly2.4.83 was added, and these were mixed well. Then, 2 ml 0.25% NaNO2 solution, 1 ml 1% glucose as hydrogen donor, and 15 ml H2O were added. The samples were incubated for 24 h at 30°C in the incubator. The control sample was set without adding 0.25% NaNO2 solution and soil. After incubation, the mixture was transferred in a different test tube, and 1 ml alumina potassium alum saturated reagent was added. Then, 1 ml of supernatant was taken and mixed with deionized water, 4 ml of 0.1% α-naphthylamine solution, and 0.5% p-aminobenzenesulfonic acid. Color was developed for 15 min, and then absorbance was measured at 520 nm with a spectrophotometer.Nitrite reductase (Nir) enzyme activity was also determined using the α-naphthylamine-p-aminobenzene sulfonic acid colorimetry method . Briefly2.5All the results reported are the mean of three replicates, and the relevant data were analyzed using the SPSS software . The forage yield, nutritional quality, soil nutrients, and soil enzyme activities were analyzed using a two-way analysis of variance with Duncan’s multiple-range test. The relationships between the variables—forage yield, nutritional quality, soil nutrients, and soil enzyme activities—were determined by calculating the Pearson’s correlation coefficients and were plotted by using Origin 2022. The tables and graphics were created using Excel 2019 and GraphPad Prism 8, respectively.33.1P < 0.001) and N inputs (P < 0.001), and the interaction between the cropping systems and N inputs was significant (P < 0.001) (P < 0.05), while the forage yield of white clover and A1 mixture first substantially increased and then decreased (P < 0.05). The alfalfa forage yield of N3 was greater than N2 and N1 by 1.3% and 7.9%, respectively; the white clover forage yield of N2 was greater than N1 and N3 by 15.2% and 15.7%, respectively; the orchardgrass forage yield of N3 was greater than N1 and N2 by 27.5% and 4.4%, respectively; the tall fescue forage yield of N3 was greater than N1 and N2 by 23.5% and 2.9%, respectively; the A1 forage yield of N2 was greater than N1 and N3 by 6.9% and 7.4%, respectively; and the A2 forage yield of N3 was greater than N1 and N2 by 25.9% and 4.1%, respectively. Taken together, the A1 mixture had a greater forage yield of 13.88 t ha−1 year−1 than the other N inputs, while that of A2 mixture under N3 had a greater forage yield of 14.39 t ha−1 year−1 than N1, but it was not substantially greater than the N2 input (13.80 t ha−1 year−1). These findings highlight that growing legume–grass mixtures under N2 input is sustainable and cost-effective, which provides greater forage yield by efficient resource utilization.The forage yield differed substantially among the cropping systems (< 0.001) Table 2.3.2P < 0.001), and the interaction among the cropping system and N level was also significant (P < 0.001) (P < 0.05) with an increase in the rate of N input. Additionally, the A1 and A2 mixtures had a greater CP content of 18.91% and 18.94% of DM, respectively, under N3 input than the grass monocultures under various N inputs. The cropping system substantially affected the WSC content (P < 0.001), and while the N level did not influence the WSC content, but their interaction was significant on the WSC content (P < 0.05) (P < 0.001), while the N level had a non-significant effect, but their interaction was significant on the fiber contents (P < 0.001)  was numerically greater than those of other cropping systems under various N inputs. The ammonium N content was substantially affected by the cropping system (P < 0.001) and N level (P < 0.001), and their interaction was also significant for ammonium N content (P < 0.001) (P < 0.05), while the ammonium N content of white clover and grass monocultures first increased and then decreased (P < 0.05). The A1 mixture had a greater ammonium N content of 16.01 and 16.75 mg kg−1 under N2 and N3 inputs than the other cropping systems (except white clover under N2 input: 17.02 mg kg−1) under various N inputs. The nitrate N content was significantly influenced by the cropping system (P < 0.001) and N level (P < 0.001), and their interaction was also significant for nitrate N content (P < 0.001) (P < 0.05), whereas the nitrate content of legume monocultures first increased and then decreased (P < 0.05). A1 and A2 had a greater nitrate N content of 3.6 and 4.2 mg kg−1 under N3 input compared with the other cropping systems under various N inputs. Taken together, the legume–grass mixed cultivation substantially increases the contents of available N in the soil when the N input rate was ≥300 kg ha−1.The effect of N input on the soil nutrients of the different cropping systems is shown in 3.4P < 0.001), cropping system (P < 0.001), and their interaction (P < 0.001) (P < 0.05) with an increase in the rate of N input, whereas no substantial difference was observed among the N2 and N3 inputs of the cropping systems. The A1 and A2 mixture had a greater urease activity of 0.39 and 0.39 mg g−1 24 h−1 under N2 input than the other cropping systems under various N inputs. The HAO enzyme activity was significantly influenced by the N level (P < 0.001) and cropping system (P < 0.001), and their interaction was also significant for the HAO enzyme activity (P < 0.001)  and N level (P < 0.001), and the interaction of the cropping system and N level was significant for the Nar enzyme activity (P < 0.001) (P < 0.05), whereas the Nar enzyme activity of the A1 mixture substantially decreased (P < 0.05) with an increase in the rate of N input. The A1 mixture had a lower Nar enzyme activity of 0.48 mg g−1 24 h−1 under N3 input than the other cropping systems under various N inputs . The Nir enzyme activity was significantly influenced by the cropping systems (P < 0.001) and N level (P < 0.001) and their interaction (P < 0.001)  with soil enzyme activities, nitrate N, ADF, and ADF, while it was significantly negatively correlated (P < 0.05) with WSC. The CP had substantially positive relationships (P < 0.05) with nitrate N and WSC, while it had significantly negative correlations (P < 0.05) with NDF, ADF, and Nir activity. The Nir enzyme activity was significantly positively correlated (P < 0.05) with fiber contents, total N, ammonium N, urease, and HAO activity. The urease activity was substantially positively associated (P < 0.05) with soil nutrients, while the HAO activity was significantly positively correlated (P < 0.05) with total N, urease activity, and ammonium N.The relationships among forage yield, nutritional quality, soil nutrients, and soil enzyme activities are presented in 44.1+4 that suppressed the urease activity . Therefore, the growing of legume–grass mixtures under the N input of 300 kg ha−1 is recommended, which provides guidance for eco-friendly, sustainable, and cost-effective forage production in Sichuan, China.N addition and legume–grass mixed seeding significantly influenced the forage yield, nutritional quality, soil nutrients, and soil enzyme activities. The A1 mixture under N2 had a greater forage yield of 13.88 t haThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.MT: writing—original draft preparation, methodology, and software. XW: methodology, resources, and formal analysis. HL: investigation and data curation. JL: writing—review and editing and visualization. JZ: investigation and data curation. BK: validation and formal analysis. DJ: resources and validation. YY: project administration, funding acquisition, supervision, and investigation. All authors contributed to the article and approved the submitted version.


Introduction: Fatty acids are a major nutrient in dietary fat, some of which are ligands of long-chain fatty acid receptors, including G-protein-coupled receptor (GPR) 40 and GPR120. Pretreatment with GPR40 agonists enhanced the secretion of insulin in response to elevating blood glucose levels after glucose load in a diabetes model, but pretreatment with GPR120 agonist did not ameliorate postprandial hyperglycemia. This study examined whether oral administration of linoleic acid (LA), a GPR40 and GPR120 agonist, immediately before glucose load would affect the elevation of postprandial blood glucose levels in rats.Methods: Male rats and rats with type 1 diabetes administered streptozocin were orally administered LA, trilinolein, α-linolenic acid (α-LA), oleic acid, TAK-875, or TUG-891 immediately before glucose load. Blood glucose levels were measured before, then 15, 30, 60 and 120 min after glucose load. CACO-2 cells were used to measure the uptake of [14C] α-MDG for 30 min with or without LA. Gastric content from rats administered LA was collected 15 and 30 min after glucose load, and blood samples were collected for measurement of glucagon-like peptide 1 (GLP-1) and cholecystokinin concentrations.Results: The elevation of postprandial blood glucose levels was slowed by LA but not by trilinolein in rats without promotion of insulin secretion, and this effect was also observed in rats with type 1 diabetes. The uptake of α-MDG, an SGLT-specific substrate, was, however, not inhibited by LA. Gastric emptying was slowed by LA 15 min after glucose load, and GLP-1, but not cholecystokinin, level was elevated by LA 15 min after glucose load. TUG-891, a GPR120 agonist, ameliorated postprandial hyperglycemia but TAK-875, a GPR40 agonist, did not. Pretreatment with AH7614, a GPR120 antagonist, partially canceled the improvement of postprandial hyperglycemia induced by LA. α-LA, which has high affinity with GPR120 as well as LA, slowed the elevation of postprandial blood glucose levels, but oleic acid, which has lower affinity with GPR120 than LA, did not.Conclusion: Oral administration of LA immediately after glucose load ameliorated postprandial hyperglycemia due to slowing of gastric emptying via promotion of GLP-1 secretion. The mechanisms may be associated with GPR120 pathway. Diet is an important factor in maintaining energy homeostasis, and it is taken orally by animals. Fat, a major nutrient within a diet, is digested into fatty acids and glycerol by pancreatic lipase in the small intestine . Fatty aGPR120 is expressed in endocrine cells from the small intestine to the large intestine . In miceGastric emptying is controlled by various mechanisms including the endocrine system and/or nervous system, and it regulates the sending of gastric content in the gastric cavity to the small intestine . PostpraThis study investigates whether the oral administration of LA taken immediately before glucose load slows the elevation of postprandial blood glucose level. We also examine whether the beneficial effect is induced by GLP-1 or CCK, and whether their secretions are associated with the GPR40 or GPR120 pathway.Five-week-old male Sprague–Dawley (SD) rats were purchased from CLEA Japan, Inc. . Two or three rats were housed in a plastic rat cage (24.7 cm × 40.9 cm × 19.7 cm) with free access to tap water and laboratory animal feed  under a 12 h light/dark cycle (lights on/off at 8:00 a.m./p.m.) at 25°C ± 1°C and 50%–60% humidity. All animals were used for experiments after an acclimation period of 1 week. The experimental protocol was approved by the Wakayama Medical University Animal Experiment Committee (Nos 883 and 1050).14C]-Labelled methyl-α-D-glucopyranoside  , phlorizin , TAK-875 , TUG-891 , and AH7614 .The following chemicals were purchased: LA , trilinolein , oleic acid , α-linolenic acid , streptozocin , olive oil ,  α-MDG with compounds  were used as incubated medium. CACO-2 cells were incubated at 37°C for 30 min when the uptake of α-MDG in HBSS with Na+ was remarkably different from one in HBSS without Na+ . The difference of α-MDG uptake between HBSS with Na+ and HBSS without Na+ was the α-MDG uptake via SGLT1. The experiments were performed three times.CACO-2 cells were seeded at a cell density of 2.0 × 10hout Na+ . Each ceFive rats were allocated to each group, and we examined oral glucose tolerance test (OGTT) in the normal rats at 6 weeks of age. OGTT was performed by oral administration of glucose solution (2 g/kg body weight). Just before the glucose load, rats fasted overnight were orally administered one of the oil solutions (each 5 mL/kg body weight). Oil solutions were either olive oil, LA dissolved with olive oil (2.5 g/mL), trilinolein dissolved with olive oil (2.5 g/mL), oleic acid dissolved with olive oil (2.5 g/mL), or α-linolenic acid dissolved with olive oil (2.5 g/mL). Blood samples were collected from the tail vein before administration of the oils and then 15, 30, 60, and 120 min after glucose load. Blood glucose levels were directly measured in blood samples with Freestyle Libre . After measuring blood glucose levels, plasma samples were collected from the tail vein and added to aprotinin , and plasma insulin, glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK) levels were measured using rat insulin measurement kit , and rat GLP-1 ELISA kit  rat CCK Enzyme Immunoassay Kit . T1DM rats were subjected to OGTT at 8 weeks of age. Blood glucose levels were directly measured in blood samples from the tail with Freestyle Libre before the administration of oils, and then at 15, 30, 60, and 120 min after glucose load.To measure the amount of glucose in the gastric tract, we collected stomachs ligated at the cardia and the pylorus from anesthetized rats with isoflurane 15 or 30 min after glucose load following blood glucose level in portal vein with Freestyle Libre, and then collected gastric content in cold phosphate-buffered saline. The volume of the supernatant was measured after centrifuging gastric content with phosphate-buffered saline at 400 g for 10 min, and the glucose concentration in the supernatant was measured with Lab Assay Glucose . The amount of glucose in the gastric tract was calculated from the volume and concentration. Residual ratio of glucose in the stomach was calculated from the ratio of the amount of glucose in the gastric tract to the amount of orally-administered glucose.To analyze the effect of GPR40 or GPR120 agonists, rats fasted overnight were orally administered either TAK-875 suspension (5 and 10 mg/kg body weight) or TUG-891 suspension (20 mg/kg body weight) in olive oil immediately before glucose load or 60 min before glucose load. Dose of TAK-875 was 1, 3 and 10 mg/kg, and dose of TUG-891 was 30 mg/kg, as in previous studies . Blood gTo examine the inhibitory effect of GPR120 antagonist in rats administered LA immediately before glucose load, rats fasted overnight were orally administered AH7614 solved with 0.5% CMC solution , GPR120 antagonist, 10 min before LA and glucose load. Blood glucose levels were measured in the same way as other OGTT experiments. Dose of AH7614 in this study was 28.5 mmol/L suspension, because in a previous study, 1 µM of AH7614 fully inhibited GPR120 agonistic activity of LA .t-test was used for two-group comparison and Tukey-HSD test for three-group comparison, and significance level was 0.05. Results are shown as means ± SDs in figures.Statistical analyses were performed using JMP 14 . Repeated measures analysis of variance (ANOVA) was used in analysis of blood glucose level changes after glucose load, and the significance level was 0.05. The significance level was 0.0166, calculated with Bonferroni correction in multiple comparison of three groups. Student’s  = 2.9, p = 0.13]. The effect of time was significant  and their interaction was also significant   = 23.2, p < 0.01]. We therefore examined the hypothesis that LA slowed the elevation of the postprandial glucose levels by 30 min after glucose load. The dose-dependent manner of LA administration was confirmed in our preliminary data  = 11.2, p < 0.01], but dose of LA (313 mg/dL) did not decrease it .Focusing on the oral administration of linoleic acid immediately before glucose load, we measured the postprandial glucose levels 15, 30, 60 and 120 min after glucose load in rats orally administered LA or olive oil . Repeate < 0.01] . The pos < 0.01] . Thus, oary data . Repeate = 0.1, p = 0.75]  = 31.0, p < 0.01]. We made T1DM model rats using streptozocin and measured blood glucose concentration in T1DM model rats administered LA or olive oil  = 8.4, p = 0.03] , but LA did not affect the uptake of α-MDG  and 30 min (p < 0.01). LA had therefore already slowed gastric motility 15 min after glucose load and LA indirectly inhibited glucose intake.To examine whether LA inhibited the glucose intake in sodium-dependent glucose transporter (SGLT) 1 located on the brush border membrane of intestinal epithelium cells, we measured the glucose uptake under LA or phlorizin, an SGLT inhibitor, in CACO-2 cells. Phlorizin inhibited the uptake of α-MDG, an SGLT1-specific substrate  = 11.3, p < 0.01]  = 0.9, p = 0.37]  = 0.0, p = 0.92]  = 14.3, p < 0.01], but TAK-875 (10 mg/kg), a GPR40 agonist, did not decrease it   = 4.2, p = 0.07]  = 24.3, p < 0.01].The effect of GPR40 or GPR120 agonists on the postprandial blood glucose in rodents administered GPR40 or GPR120 agonist before glucose load has been previously reported . We exam = 0.52] . A half  = 0.52] . The ele = 0.07] . Further = 41.6, p < 0.01], but it was not decreased by LA + AH7614 compared with in the olive oil group   = 2.1, p = 0.19]  = 8.4, p = 0.03]  = 1.2, p = 0.30]  . LA mighLA is a dual ligand of both GPR40 and GPR120 , but preThis study used olive oil as control and solvent of fatty acids or TriLA to minimize the difference of nutrition intakes. However, olive oil contains polyphenols, vitamin E, phytosterols, and carotenoids derived from olives as well as triglyceride, and these substances might affect the postprandial glucose levels. In this study, the solvent in the LA (625 mg/kg) group containing 87.5% olive oil and 12.5% LA also slowed the postprandial glucose levels , but theThis study indicated that the oral administration of GPR120 agonist immediately before glucose load decreased the postprandial blood glucose levels up to 120 min after glucose load . In caseOral administration of free LA, a GPR120 agonist, immediately before glucose load improved postprandial glucose levels independent from insulin. LA contained in dietary fat may therefore improve postprandial hyperglycemia in patients with T1DM.


Bis(indolyl)methanes (BIMs) are a class of compounds that have been recognized as an important core in the design of drugs with important pharmacological properties, such as promising anticancer and antiparasitic activities. Here, we explored the biological activity of the BIM core functionalized with different (hetero)aromatic moieties. We synthesized substituted BIM derivatives with triphenylamine, N,N-dimethyl-1-naphthylamine and 8-hydroxylquinolyl groups, studied their photophysical properties and evaluated their in vitro antiproliferative and antiparasitic activities. The triphenylamine BIM derivative 2a displayed an IC50 of 3.21, 3.30 and 3.93 μM against Trypanosoma brucei, Leishmania major and HT-29 cancer cell line, respectively. The selectivity index demonstrated that compound 2a was up to eight-fold more active against the parasites and HT-29 than against the healthy cell line MRC-5. Fluorescence microscopy studies with MRC-5 cells and T. brucei parasites incubated with derivative 2a indicate that the compound seems to accumulate in the cell’s mitochondria and in the parasite’s nucleus. In conclusion, the BIM scaffold functionalized with the triphenylamine moiety proved to be the most promising antiparasitic and anticancer agent of this series. Trypanosoma brucei) and leishmaniasis (caused by more than 20 species of Leishmania spp.). Currently, these parasitic diseases do not have a FDA-approved vaccine .,7,8.Bis(tionship .bis(indolyl)methane and its derivatives have been widely investigated in several cancer cell lines + calcd for C29H26N3 416.2127, found 416.2126.Brown solid (80%); mp: 204.8–205.4 °C. 1H NMR (DMSO-d6) δ = 6.19 , 6.85 , 7.01 , 7.04 , 7.06 , 7.29–7.36 , 7.56 , 10.89 . The signal of OH is not visible in the 1H NMR. IR (Nujol) ν = 3184, 1634, 1599, 1537, 1378, 1091, 1040, 779, 744 cm−1. HRMS (ESI) m/z: [M + H]+ calcd for C26H20N3O 390.1606, found 390.1593.Green solid (37%); mp: 207.8–208.4 °C. T. brucei ) [2 in HMI-9 medium supplemented with 10% heat-inactivated fetal bovine serum , as previously described [7 (S16))  were culescribed .L. major (MHOM/IL/80/Friedlin) promastigotes were cultured at 28 °C, 5% CO2 in modified RPMI-1640 medium  supplemented with 10% hiFBS [T. brucei and 10 million parasites per mL for L. major).0% hiFBS . Parasit2 in DMEM medium (1 g/L glucose) supplemented with 10% hiFBS, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine [MRC-5 cell line (human lung fibroblast) was grown in monolayer at 37 °C, 5% COlutamine .2 in high glucose DMEM (4.5 g/L glucose) supplemented with 10% hiFBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine and non-essential amino acids (1X). Cells were plated and passaged according to ATCC recommendations and were used for the experiments while in the exponential growth phase.HeLa  and HT-29  cell lines were maintained at 37 °C, 5% CO® assay  [4 parasites per mL were incubated at 37 °C, 5% CO2 in 96-well plates (50 μL/well) alone or in the presence of an increasing concentration of compounds for 72 h. A total of 20 μL of resazurin solution (110 ng/mL) was then added to each well, and the parasites were incubated for 4 h at 37 °C. Finally, cells were lysed with 50 μL per well of SDS 3%. The plate was incubated at 37 °C for an extra hour; then, the fluorescence intensity was measured with an Infinite F200 plate reader , exciting at 550 nm and recording the emission at 590 nm. The results are expressed as the concentration of the compound that reduces cell growth by 50% versus untreated control cells (IC50). Data are presented as the average of at least three independent measurements all conducted in triplicate.The trypanocidal activity of the compounds was assessed using the alamarBlueMA, USA) ,47. The 6 parasites per mL were incubated at 28 °C in 96-well plates (50 μL/well) alone or in the presence of an increasing concentration of compounds DMSO for 72 h. A total of 10 μL of MTT (5 mg/mL) was added to each well, and parasites were incubated for 4 h at 28 °C. Finally, cells were lysed with 50 μL/well of 20% SDS. The plate was incubated at 37 °C for an extra hour; then, the absorbance was measured with the Infinite F200 plate reader  at a wavelength of 540 nm. The IC50 was calculated as described above. Data are presented as the average of at least three independent measurements all conducted in triplicate.The leishmanicidal activity of the compounds was determined using MTT-based assay  . The sto® assay (ThermoFisher Scientific) [3 cells per mL  were seeded in 96-well plates (100 μL/well) with 24 h of incubation time before compound addition at 37 °C, 5% CO2. Then, the compounds were added at increasing concentrations (from 0 to 100 μM), and the plates were incubated for 72 h. A total of 20 μL of resazurin solution (110 ng/mL) was added to each well, and cells were incubated for 4 h. Then, cells were lysed with 50 μL/well of 3% SDS. The plate was incubated at 37 °C for an extra hour; then, the fluorescence intensity was measured with the Infinite F200 plate reader , exciting at 550 nm and recording the emission at 590 nm. The results are expressed as the concentration of compound that reduces cell growth by 50% versus untreated control cells (IC50). Data are presented as the average of at least three independent measurements all conducted in triplicate.Cytotoxicity was measured in MRC-5, HeLa and HT-29 cell lines using alamarBlueentific) ,47. The https://admetmesh.scbdd.com/ (accessed on 10 September 2023) [The physicochemical and pharmacokinetic properties of the compounds were predicted using ADMET  profiles available in the ADMET lab 2.0 online server er 2023) .4/mL) were incubated with 5 μM of compound 2a in 0.5 mL of their respective medium for 30 min and 1 h at 37 °C and 100% humidity. Mitochondrial and nuclear staining was performed with MitoTracker deep red (200 nM) and green nuclear dye (2 drops/mL), respectively, for 30 min. Cells were washed five times with room temperature PBS and fixed with 2% paraformaldehyde for 20 min and washed two extra times with PBS. Then, the cover slides were immerged in water, and ethanol immersion of the cover slide was also necessary prior to sample processing for microscopy.MRC-5 cells (2 × 10T. Brucei parasites (2 × 107/mL) were incubated with 5 μM of compound 2a in 0.5 mL of their respective medium for 30 min, 1 h and 2 h at 37 °C and 100% humidity. Mitochondrial and nuclear staining was performed with MitoTracker deep red (200 nM) and green nuclear dye (2 drops/mL), respectively, for 30 min. Parasites were washed five times with cold PBS and fixed with paraformaldehyde 4% for 20 min, washed two extra times with cold PBS and processed by microscope observation.2a and MitoTracker, respectively. Emission was detected at 512–548 nm for green nuclear dye, 420–450 nm for compound 2a and 666–724 nm for MitoTracker. The images were processed and analyzed with Fiji software 64 bits version for windows .Images were acquired using a widefield Olympus ix81 microscope. Excitation was carried out with the 490 nm, 385 nm and 633 nm filters for green nuclear dye, compound bis(indolyl)methane derivatives functionalized with triphenylamine, N,N-dimethyl-1-naphthylamine and 8-hydroxylquinolyl groups. The influence of the hetero(aromatic) moieties on their biological activity in the pathogenic parasites T. brucei and L. major, and as well as in the cancer cell lines HT-29 and HeLa, was investigated.In this work, we reported the synthesis of 2a) displayed the best toxicity and selectivity of the series against Trypanosoma brucei and Leishmania major . Compound 2a also showed a higher inhibitory effect against HT-29 cells than against HeLa cells, with an IC50 of 3.93 μM and selectivity index of 8.57. Its antiproliferative activity was found to be remarkably more active than its unsubstituted analogue, 3,3-diindolylmethane (IC50 > 100 μM against HT-29 cell line). Moreover, the internalization studies using fluorescence microscopy suggested that compound 2a localizes in the mitochondria in MRC-5 cells and in the nucleus in T. brucei parasites. In conclusion, the bis(indolyl)methane derivative 2a was demonstrated to be a potential candidate as a trypanocidal and leishmanicidal agent and as a chemotherapy drug against colon cancer.The antiparasitic activity studies revealed that the BIM substituted with triphenylamine (


BackgroundGlomerular filtration rate (GFR) estimation is pivotal in the evaluation of kidney donors. There are various methods available for assessing GFR, but there has been a lack of consensus on the measurement of GFR and the frequency of renal evaluation after kidney donation. Our study aims to analyze the measured GFR (m-GFR) before and three months after kidney donation and note the compensatory abilities of the remnant kidney in live related kidney donors.MethodsThis prospective observational study was conducted at the Department of Nephrology, Dr. D. Y. Patil Medical College, Hospital & Research Centre, Pune, from April 2021 to December 2022. The study included 30 donors from both genders aged between 23 and 73 years. The measured GFR was calculated using a technetium-99m diethylene triamine pentaacetic acid (Tc-99m DTPA) scan. We analyzed donor characteristics and various parameters that included demography, anthropometry, blood pressure, and serum creatinine and measured GFR (m-GFR) using a Tc-99m DTPA scan, which was compared before and three months after donor nephrectomy.Results2, whereas the mean body surface area (BSA) was 1.59 ± 0.12 m2. In terms of the measured GFR by DTPA scan, pre-donation and post-donation, the average GFR for our population was 103.83 ± 10.07 mL/minute/1.73 m2 and 60.47±6.57 mL/minute/1.73 m2, respectively. The mean measured GFR of remnant kidney increased by 9.21 ± 4.39 mL/minute/1.73 m2 in 28 donors, while two donors had a fall in the mean measured GFR by 6.8 ± 1.69 mL/minute/1.73 m2.Of the 30 donors, 25 (83.3%) were females and five (16.7%) were males. The mean age of donors was 49.23 ± 12.29 years. The mean body mass index (BMI) was noted to be 24.73 ± 5.58 kg/mConclusionsTo safeguard donor health, accurate measurement of GFR at various timelines after kidney donation should be considered as there are various limitations associated with the use of serum creatinine-based GFR estimating equations for solitary kidneys. However, long-term studies are required to analyze the changes in GFR after nephrectomy and determine the adequacy of compensatory changes in the remnant kidney post-kidney donation. Living kidney donor transplantation has been considered to have superior outcomes as compared to deceased donor transplantation for patients with end-stage kidney disease (ESKD) . From 20Although many studies have emphasized that there is a lower likelihood of developing chronic kidney disease after living kidney donation , minimizAlthough there are various methods available for measuring GFR, there has been a lack of consensus on the assessment of GFR after kidney donation, its method of measurement, and the frequency of renal evaluation. In this study, we aimed to measure the GFR using a technetium-99m diethylene triamine pentaacetic acid (Tc-99m DTPA) scan done up to 14 weeks pre-nephrectomy and 11-13 weeks post-nephrectomy, and changes in the measured GFR (m-GFR) were analyzed before and three months after donor nephrectomy.A part of this study was previously presented as a poster at the American Society of Nephrology Kidney Week 2022 on November 5, 2022.2. Donors with ages more than 75 years, uncontrolled hypertension, diabetes mellitus, microalbuminuria, and measured GFR by Tc-99m DTPA scan of less than 80 mL/minute/1.73 m2 were excluded from the study. A comprehensive medical history along with demographic parameters and detailed clinical examination that encompassed height, weight, BMI (kg/m2), and body surface area (BSA) (m2) calculated using the Du Bois method [This was a prospective observational study of a cohort of 30 donors who, after appropriate medical and surgical evaluation, underwent donor nephrectomy for living related kidney transplants between April 2021 and December 2022 at Dr. D. Y. Patil Medical College, Hospital & Research Centre, Pune, India. For the study, an institutional ethical committee approval was obtained with reference number IESC/S.SP/2020/03. All participants had to sign a written informed consent in accordance with the ethical standards and guidelines. All the healthy live related kidney donors were near relatives of the recipients, which included parents, siblings, and spouses, aged between 23 and 73 years and with a body mass index (BMI) of less than 35 kg/ms method  were nots method .2.Further evaluation of all donors for this study included serum creatinine and GFR measurements by Tc-99m DTPA scan done up to 14 weeks prior to donor nephrectomy, followed by 11-13 weeks after donor nephrectomy. Serum creatinine was measured by alkaline picrate kinetic method using Siemens Dimension EXL 200 Integrated Chemistry System . Prior to the DTPA scan, all subjects were ensured to have adequate hydration. The subjects were then injected intravenously with 228 MBq (4 mCi) Tc-99m DTPA, and dynamic images were acquired at two seconds per frame for one minute and subsequently at a rate of 60 seconds/frame for 20 minutes using the Gates protocol. Subsequently, GFR values were calculated after standardization of GFR as per standard body surface area of 1.73 mStatistical analysisData was collected using a prestructured questionnaire entered in Microsoft Excel 2019 . Variables such as age, weight, BMI, GFR, blood pressure readings, and serum creatinine were expressed as mean and standard deviation (SD), while qualitative variables such as comorbidities and gender were represented by frequencies and percentages. The Student’s paired t-test was used for comparison of continuous variables. Statistical tests for data analysis were applied using Statistical Package for the Social Sciences (SPSS) for Windows version 22.0 . Statistical significance was considered at p < 0.05 at a 95% confidence interval.2, and 1.39 to 1.85 m2, respectively. The mean weight, BMI, and BSA recorded were 60.72 kg, 24.73 kg/m2, and 1.59 m2, respectively  and five males 16.66%). Multiple parameters including demography, anthropometry, pre- and post-nephrectomy blood pressure, serum creatinine, and measured GFR were studied. The age of all donors ranged from 23 to 73 years, with a mean age of 49.23 ± 12.29 years. The weight, BMI, and BSA ranged from 43 to 93 kg, 17.3 to 33.3 kg/m6.66%. MuPre-nephrectomy studies2. In our study, we found that the measured GFR was between 80 and 90 mL/minute/1.73 m2 in six donors and >90 mL/minute/1.73 m2 in 24 donors. Pre-donation, all donors had a mean SBP of 122.47 ± 9.07 mmHg and DBP of 80.47 ± 4.05 mmHg  . Also, the pre-nephrectomy total mean measured GFR of 103.83 mL/minute/1.73 mChanges in the GFR of the remnant kidney post-donation2. Thirteen (43.33%) donors had a mean rise in m-GFR of more than 10 mL/minute/1.73 m2, 11 (36.66%) had a rise between 5 and 10 mL/minute/1.73 m2, and four (13.33%) had a rise between 0 and 5 mL/minute/1.73 m2. In two donors, there was a drop in the measured GFR of the remnant kidney by a mean of 6.8 ± 1.69 mL/minute/1.73 m2.Out of 30 donors, 28 (93.33%) had a compensatory increase in the measured GFR of the remnant kidney, while two (6.66%) had a fall in the measured GFR of the remnant kidney. In 28 donors who had an increased compensatory response post-donation, the remnant kidney had a mean increase in the measured GFR of 9.21 ± 4.39 ranging from 0.7 to 18.7 mL/minute/1.73 mRise in GFR in relation to age2 ranging from 4 to 15.1 mL/minute/1.73 m2, while in those with age less than 50 years and no comorbidities, it was 9.72 ± 5.34 ranging from 0.7 to 18.7 mL/minute/1.73 m2. However, the rise in the measured GFR in both groups was statistically insignificant (p = 0.524) , which increased to 61.67 ± 4.52 mL/minute/1.73 m2 (range: 52.3-69.5 mL/minute/1.73 m2) three months post-kidney donation, representing a 16.7% rise in the measured GFR of the remnant kidney. The mean measured GFR of the remnant kidney increased by 9.21 ± 4.39 mL/minute/1.73 m2. The increase in m-GFR in our study was lower as compared to studies by Chen et al. [2 with a serum creatinine of 1.8 mg/dL, and the second donor was a 56-year-old female, who had a drop of 5.6 mL/minute/1.73 m2 with a serum creatinine of 2.1 mg/dL and had developed acute pyelonephritis. Both donors had no known previous comorbidities and were diagnosed with hypertension after nephrectomy and started on a single antihypertensive. Older age group, the presence of pyelonephritis, and new-onset hypertension might have contributed to the drop in the GFR of the remnant kidney post-donation. In the study done by Lam et al. [Twenty-eight (93.33%) donors had a compensatory increase in remnant kidney GFR three months after donation. Among these donors, 13 (46.42%) had an age of less than 50 years, while 15 (53.57%) belonged to a group with an age of more than 50 years and comorbidities. The baseline mean measured GFR of the remnant kidney of these donors was 51.36 ± 5.54 mL/minute/1.73 mn et al.  and Bahin et al. , who foum et al. , older a2 versus 8.63 ± 3.09 mL/minute/1.73 m2, p = 0.524). This highlights the fact that despite the inclusion of donors with greater age and comorbidities, the compensatory rise in the measured GFR three months after donor nephrectomy was equivalent to young donors and donors without any comorbidities.In donors with a good compensatory response post-donation, we analyzed the mean rise in the measured GFR three months after nephrectomy in donors aged more than 50 years compared to donors aged less than 50 years and found that the increase in the measured GFR in this age group had no significant difference (9.72 ± 5.34 mL/minute/1.73 mOur study limitations comprised a limited sample size, a short follow-up period, and a single-center study. We did not measure GFR using a gold standard method such as inulin clearance. Blood pressure measurements were not taken using 24-hour ambulatory BP monitoring. Further studies are required to determine which variables will predict if the donor will have a rise or fall in GFR after donation and compensatory changes in the remnant kidney post-donation.Previous studies on kidney donors have checked long-term outcomes following donor nephrectomy, but there have been very few studies assessing the changes in measured GFR post-donation and analyzing their sufficiency in the short term. Also, this study showed that the compensatory changes in remnant kidneys after nephrectomy at three months in donors with comorbidities and an older age group were comparable with donors aged less than 50 years.Inaccurate and less frequent monitoring of GFR after kidney donation can have a negative impact on clinical decision-making and the health of individuals after kidney donation. To safeguard the health of kidney donors and reduce the risk of end-stage kidney disease in donors, there should be accurate measurement of GFR at various timelines after kidney donation. Long-term studies are necessary to study the changes in GFR at various timelines after kidney donation and determine the adequacy of compensatory changes in the remnant kidney.