Micropterus salmoides L.) is generally considered to comprise two subspecies, Florida bass (M. floridanus) and Northern Largemouth bass , which have biological characteristic differences because of their geographical distribution. In this study, whole-genome re-sequencing was performed among 10 Florida and 10 Northern largemouth bass, respectively. In total, 999,793 SNPs and 227,797 InDels were finally identified, and 507,401 SNPs (50.75%) and 116,213 InDels (51.01%) were successfully mapped to annotated 18,629 genes and 14,060 genes, respectively. KEGG classification indicated that most of these genes were focused on the pathways including signal transduction, transport and catabolism, and endocrine system. Genetic diversity analysis indicated that Florida largemouth bass had higher genetic diversity than Northern largemouth bass, indicating that the germplasm quality of Northern largemouth bass had markedly reduced in China. To examine the accuracies of the identified markers, 23 SNPs and eight InDels (the insertions or deletions of more than 45 bp) were randomly selected and detected among Florida largemouth bass, Northern largemouth bass, and their F1 hybrids. The detection efficiencies of all the markers were higher than 95%; nineteen SNPs and three InDels could accurately distinguish the two subspecies and their F1 hybrids with 100% efficiencies. Moreover, the three InDel markers could clearly distinguish the two subspecies and their F1 hybrids with a PCR-based agarose gel electrophoresis. In conclusion, our study established a simple PCR-based method for the germplasm identification of largemouth bass, which will be useful in the germplasm protection, management, and hybridization breeding of largemouth bass.Largemouth bass ( Micropterus salmoides L.) is native to freshwater lakes and rivers in North America. It is considered to comprise two subspecies  distributed in the Florida peninsula, and the Northern largemouth bass  distributed in most central and eastern parts of America, northeast Mexico, and southeast areas of Canada , expected heterozygosity (He), and polymorphic information content (PIC) were all highest in FB ♀ × NB ♂, which were 0.849, 0.639, and 0.571, respectively  provides an advantage for the discovery of SNP and InDel markers between NB and FB. In this study, 10 “pure” FB and 10 “pure” NB were selected and sequenced with a whole-genome re-sequencing technique. Then, the SNP and InDel markers were screened and annotated according to the reference genome of largemouth bass. In addition, genetic diversity analysis was conducted based on the high-quality SNPs, and a series of SNP and InDel markers were randomly selected and detected among FB, NB, and their F1 hybrids. Our study provided a PCR-based method for the germplasm identification of largemouth bass, which would be useful in the protection, management, and hybridization breeding of largemouth bass.Largemouth bass were collected from the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences . FB was originally imported from America in 2010, and NB was the new variety of largemouth bass “Youlu No.3” bred in 2019. Identifications of these fish were conducted based on a subset of microsatellite primers from For each sample, genomic DNA was extracted from 0.1 g fin tissue using a modified CTAB method . The extAfter removing adapter sequences and low-quality reads, the clean reads were further rechecked for quality based on the following criteria: consecutive bases on the ends with base quality <20, read length <50 bp, and the singletons were also removed. High-quality sequences were aligned and mapped to the reference genome of largemouth bass using a BWA program (v0.7.17)  with defSNPs and InDels were called using the Genome Analysis Toolkit (GATK) Haplotype Caller . To reduhttp://treesoft.sourceforge.net/treebest.shtml/) (http:/itol.embl.de). The principal component analysis was performed using PLINK v1.07 software with default parameters (r2) values between the two SNPs and the physical distance between the two SNPs. Nucleotide diversity (π) and inbreeding coefficient (F) were calculated using VCFtolls (v.0.1.13) (All the SNPs that passed filtering in the population were used to construct a phylogenetic tree using TreeBeST (.shtml/) . The finrameters . Populatrameters . Linkagerameters , and the.0.1.13) .1 hybrids (NF1 -NF30) bred in 2020, which were obtained by “Youlu No.3” ♀ × re-imported FB in 2010 ♂. Total DNA was extracted from fin tissues using the standard phenol-chloroform procedure and DNA integrity was examined using agarose gel electrophoresis and quantity was determined using an Agilent 2,100 Bioanalyzer .In total, 130 largemouth bass came from three populations were used to validate the accuracies of candidate SNP and InDel markers: 1) 20 FB and 20 NB re-imported from America in 2010; 2) 30 FB progenies of the 2010 re-imported FB bred in 2020, and 30 NB progenies of the “Youlu three” bred in 2020; 3) 30 F2O. The PCR procedure was as followed: 94°C for 3 min, followed by 35 cycles of 94°C for 15 s, 58°C for 15 s, 72°C for 30 s, and finally, 72°C for 4 min. Then, the PCR production was purified with ExoI and FastAP. The reaction mixture was 3 μl PCR production, 0.2 μl ExoI, 0.8 μl FastAP, 0.7 μl ExoI buffer, and 3.3 μl ddH2O, respectively. The PCR procedure was as followed: 37°C for 15 min and 80°C for 15 min. Then, the extension reaction was performed with 2 μl purified PCR production, 1 μl Snapshot Ready ReactionMix, 1 μl extension primer (10 pM), and 3 μl ddH2O. The procedure was as follows: 96°C for 1 min, followed by 30 cycles of 96°C for 10 s, 52°C for 5 s, and 60°C for 30 s. Then, 10 μl Hidi formamide  was added into 1 μl extension production and treated in the ice. Then, the detection of DNA polymorphisms was sequenced with an ABI3730XL Sequencer .The 30 NB, 30 FB, and 30 NF were used for the SNP detection. The polymorphisms of 23 randomly selected SNPs were detected with the Snapshot method. Briefly, primers were designed according to the largemouth bass genome sequences of these SNPs, respectively . Then, m2O. The PCR procedure was as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and finally, 72°C for 10 min. Then, electrophoresis on a 2.0% agarose gel was used to detect the sizes of PCR amplification products.To develop the InDel markers for the germplasm identification of largemouth bass, eight InDels were randomly selected from the identified InDels, with a size number >45 bp of the insertions or deletions bases in each of the InDel makers. These InDel markers were detected in the aforementioned NB, FB, and NF individuals. qPCR was performed in a 20 μl reaction mixture including 10 μl of Premix Taq™ , 0.5 μl of each primer (10 μm), 1 μl of DNA, and 8 μl of ddHMicropterus salmoides). The overall mapping rate was 97.33% for NB and 95.57% for FB, respectively, with an average of 96.45%. Moreover, for NB and FB, 95.37 and 94.03% paired-end reads, and 0.09 and 0.17% single-end reads were mapped to the reference chromosomes of the largemouth bass genome, indicating the high quality of the sequencing data. In addition, nucleotide statistics on the assembled scaffolds showed that the GC content is nearly 40% , and varied from 10,398 on Chr10 to 77,288 on Chr1 . The denIn total, 227,797 filtered InDels were finally identified between NB and FB. These InDels varied from 2,669 on Chr12 to 16,845 on Chr1 . The denKEGG classification suggested that these SNPs and InDel markers are widely involved in transport and catabolism, and the cellular community of cellular processes; signal transduction, signaling molecules, and interaction of environmental information processes; folding, sorting and degradation, and transcription of genetic information processes; carbohydrate metabolism and lipid metabolism of metabolism; immune system and endocrine system of organismal systems . SpecialTo explore the relationships between the 10 NB and 10 FB, a neighbor-joining phylogenetic tree was constructed using all the filtered SNPs. The phylogenetic tree classified the 20 LMB into two groups: the 10 FB were clustered into one group and the 10 NB clustered into another group . A populπ) of FB was higher than that of NB, while the inbreeding coefficient (F) of FB was lower than that of NB. Taken together, our results indicated that the NB population experienced a longer breeding process.Principal component analysis (PCA) was used to further confirm the relationship between the 10 NB and 10 FB. As shown in the principal component plot for the first two principal components, which accounted for 60.24% of the total variation observed in 20 largemouth bass. PC1 explained 55.71% of the overall variation and separated 20 largemouth bass into two groups named NB and FB, and PC2 explained 4.53% of the overall variation . The LD To test the efficiency of the identified SNPs, a total of 23 randomly selected SNPs were conducted in 30 NB, 30 FB, and their 30 F1 progenies (NF), with one on each chromosome. The results indicated that all the 23 SNPs performed good polymorphisms and 19 SNPs (82.60%) could distinguish these fish with 100% efficiencies . The accIn order to develop a PCR-based method for the germplasm identification of largemouth bass, a total of eight InDel markers were selected randomly from the aforementioned Indels, which were longer than 45 bp . After PTo better understand the genetic variation in largemouth bass subspecies, 10 NB and 10 FB were selected for whole-genome re-sequencing. With the guidance of the largemouth bass reference genome, 999,793 SNPs were finally identified, which was much more than that in the previous studies, without the largemouth bass reference genome. For example, 3,674 SNPs were discovered through the transcriptomes analysis between NB and FB ; and 58,Similar to SNPs, InDels are widely distributed in the genome and have been used in various biological process analyses in animals and plants . HoweverHo values of the three populations were 0.519, 0.480, and 0.4206, respectively; the He values of the three populations were 0.491, 0.454, 0.3916, respectively; the PIC values of the three populations were 0.407, 0.412, and 0.3257, respectively (π) of NB was lower than those in FB, corresponding to the lower genetic diversity of the NB. In addition, the inbreeding coefficient (F) of NB was higher than FB, indicating the long-time inbreeding resulted in low genetic heterogeneities in NB.In the past two decades, we analyzed the genetic structure of collected NB largemouth bass populations with microsatellite DNA markers, including the imported NB population in 2010, the new variety “Youlu No.1” bred in 2011, and the offspring of “Youlu No.1” bred in 2016. The ectively . In thisBecause of their geographical distribution differences, it was reported that NB is more tolerant to low temperature, ammonia nitrogen, and low oxygen, while FB is more resistant to high temperatures and has a stronger stress response . AbioticThrough gene combination of two or more species, populations, or varieties, which have different genetic bases, hybridization alters the genetic structure of the offspring and thus increases their heterozygosity levels and genetic variation . In thisThrough whole-genome resequencing of 10 NB and 10 FB, 999,793 SNPs and 227,797 InDels were finally identified, which contributed to exploring the biological characteristic differences between NB and FB. The genetic structure analysis indicated that FB had higher genetic diversity than NB, indicating that the germplasm quality of cultured largemouth bass had markedly reduced in China. In addition, three identified InDels provided a simple PCR-based method to distinguish NB, FB, and their F1-based progenies. Our study provides an effective and accurate method for the germplasm identification of largemouth bass, which will be useful in the molecular-assisted breeding of largemouth bass in the future.


Lactobacillus acidophilus NRRL B-4495 or Lactiplantibacillus plantarum NRRL B-4496 by oil-in-water (O/W) emulsification and to evaluate the stability under storage conditions. The results showed that L. acidophilus and L. plantarum encapsulated in gelatin (LAEG and LPEG) presented diameters of 26.08 ± 1.74 μm and 21.56 ± 4.17 μm and encapsulation efficiencies of 89.6 ± 4.2% and 81.1 ± 9.7%, respectively. However, those encapsulated in alginate (LAEA and LPEA) showed an encapsulation efficiency of <1.0%. Furthermore, LAEG was stable for 120 days of storage at 5 °C and 25 °C. Therefore, encapsulation in gelatin by O/W emulsification is a promising strategy for protecting and stabilizing probiotic bacteria, enabling future application in foods.Probiotics are associated with health benefits to the host. However, their application can be limited due to a decrease in cell viability during processing, storage, and passage through the gastrointestinal tract. Microencapsulation is a simple and efficient alternative to improve the physical protection and stability of probiotics. The present study aimed to produce and characterize alginate or gelatin-based microparticles containing  Probiotics are live microorganisms that confer a health benefit to the host when administered in adequate amounts . Probiot6–107 colony forming units (CFU) per gram (g) or milliliter (mL) to promote health benefits to the host ..−1 for b−1 for the asymmetric and symmetrical elongation of the amide I bond (C=O), bands at 1617 cm−1 corresponding to amide II (NH), and at 3549 cm−1 and 3412 cm−1 concerning stretches of (NH) and (OH) bonds, respectively, which can form hydrogen bonds with the carbonyl group of the peptide bond [−1, which corresponded to the presence of the carbonyl group (C=O).The spectrum obtained for porcine gelatin A,B showside bond ,32. Sodi−1, referring to dimers of secondary amides (NH) in s-cis or s-trans conformations [−1 for phosphate-bearing compounds, and at 1162–916 cm−1 attributed to microbial cell wall carbohydrates [−1 and 1744 cm−1 may be associated with the CH2 elongation of fatty acids from the probiotic’s lipid membranes and the presence of corn oil [Characteristic bands in the formulation LAEG A were obrmations , at 1235hydrates . Furthercorn oil , used as−1 and 3416 cm−1) and vibrational stretching at 2926 cm−1 and 2853 cm−1 for Tween 20.It can be seen that there was a chemical interaction between the gelatin and Tween 20 surfactant after attenuating the absorption bands in the gelatin for the amide group –C groups [−1, 3479 cm−1, and 3412 cm−1 (N–H) and vibrational stretching of the bands at 2926 cm−1 and 2853 cm−1 for Tween 20. These results confirm the chemical interactions in the system, indicating that the probiotics were encapsulated in porcine gelatin.Characteristic bands of the chemical constituents present in the microorganism and the oil for the encapsulated LPEG B were obC groups ,33. Furt−1 and 2856 cm−1) and alginate bands (3545–3239 cm−1 and 1469 cm−1), but without stretching or the formation of new bands. Similarly, L. plantarum in alginate , however, not attenuated. It is assumed that the low chemical interaction between the system’s constituents for LAEA and LPEA did not favor incorporating microorganisms, which were more exposed during the encapsulation process, resulting in a loss of viability.On the other hand, it was impossible to observe characteristic bands of the chemical constituents present in the probiotic in the LAEA formulation spectra C such asalginate D also diThe diffractograms showed structures of a semi-crystalline nature, with noise characterizing amorphous regions and defined peaks in the crystalline regions for porcine gelatin (PG) 2θ = 22.07°, and alginate (AG) 2θ = 19.94°, 20.79°, 23.30°, 25.51°, 28.46°, 31.40°, 35.51°, 36.34° . De OlivThose encapsulated in gelatin (LAEG and LPEG) presented a single translocated peak at 2θ = 19.86° and 20.65° , which mL. acidophilus-LAEG showed a reduction of 1.2 Log CFU/g in viability, corresponding to an EE of 89.6 ± 4.2% (L. plantarum-LPEG (81.1%), with a reduction of 2.4 Log CFU/g. Using an Ultra-Turrax dispenser for homogenization during the encapsulation process represents a risk factor since the homogenization can cause physical damage to the microbial cells. However, the high EE values indicated that O/W emulsification is compatible with probiotic encapsulation. Similarly, Matos-Jr et al. [L. rhamnosus 64 and L. paracasei BGP1 cells when submitted to homogenization using an Ultra-Turrax at 5500 and 7000 rpm.After encapsulation using gelatin, 6 ± 4.2% . A similr et al.  did not The EE results using gelatin corroborate data obtained from SEM, in which it was possible to observe the probiotic cells arranged under gelatin-based particle structures (LAEG and LPEG). Furthermore, the FTIR results showed the chemical interactions between the raw agents (gelatin and Tween 20) and the microorganisms, indicating the protection of probiotics.Bifidobacterium adolescentis 15703T probiotic encapsulated in type A porcine gelatin (13%) coated with 1% alginate by the calcium gelation technique, Annan et al. [B. pseudocatenulatum G4 in Type B gelatin resulted in an EE of only 45.9%.According to Sagiri et al. , studiesn et al.  obtainedn et al.  reportedL. acidophilus and L. plantarum was observed when using alginate as an encapsulating agent, with an encapsulation efficiency < 1.0% . Solubility directly influences food application, favoring (or not) acceptance. The laser diffraction results showed that LAEG and LPEG had smaller particle diameters associated with the physical and chemical characteristics of the encapsulating agent, possibly influencing the good solubility of the particles.The dispersibility test showed that the LAEG and LPEG obtained a water solubilization of 69.9 ± 9.7% and 69.0 ± 8.4%  at 420 °C in melting events A, which L. plantarum, L. casei, and S. boulardii to survive in edible films based on gelatin and low methoxyl pectin (LMP). However, the gelatin used in this study was Type B (bovine gelatin).These results differ from those found by Khodaei et al. , who obsL. acidophilus showed a loss of 55% and L. plantarum of 56% (TG). This can be explained by the degradation of cellular constituents of probiotics such as proteins, lipids, and polysaccharides [Other constituent materials also partially lost mass in the melting event such as Tween 20 surfactant (56%) B and corcharides . Their fThe mass loss was considerably lower (4%) for gelatin encapsulates up to 65 °C F,G. As tAccording to the results , encapsuL. plantarum by double emulsification followed by complex coacervation using gelatin and gum Arabic.The data showed that the encapsulates maintained superior viability even at room temperature (25 °C) compared to other studies ,41 that The X-ray diffraction technique was used to evaluate the effects of storage on the porcine gelatin-based encapsulate structures. The diffractograms showed  that theIt is possible to notice changes in the structure of the microparticles at 45 H,I and 6More pronounced changes in the structure were observed for both storage conditions from day 90 L,N, withAgglomeration and crystallization can lead to a release of components encapsulated in the matrix , which iAn essential aspect of maintaining probiotic viability is carefully selecting the encapsulation technique and materials . The gooFinally, lyophilization is a drying strategy used to manufacture food products, which enables the preservation of probiotic cells . This teL. acidophilus and L. plantarum by O/W emulsification using gelatin has been proven to be a suitable technology, resulting in higher encapsulation efficiencies. In addition, the present work showed that gelatin-based microparticles presented favorable characteristics for protecting and maintaining probiotic viability during 120 days of storage at 5 °C and 25 °C. Therefore, the results obtained in the present study are important for the food industry as support for using O/W emulsification to apply probiotics in food matrices.Microencapsulation of


Prunus salicina L. is an important fruit tree species of great economic value which is mainly distributed in the northern hemisphere. Prunus salicina L. were collected from 8 provinces in China, Japan, USA, and New Zealand. The genetic variations of these samples were characterized by the random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) technique, respectively, and in combination.  25 samples of  Totally, 257 RAPD bands ranging 200∼2300 bp was found, and 81.59% of these bands were polymorphic. ISSR analysis identified 179 bands ranging 300∼2500 bp, and 87.74% of the bands were polymorphic. ISSR results showed that the similarity coefficient index between samples P10  and P13  was lowest, while that between samples P10  and P15 (Baili in Japan) was highest. Combined analysis of RAPD and ISSR demonstrated that the similarity coefficient index between samples P4  and P13  was lowest, while that between samples P19 (Laroda in USA) and P20 (Red heart in USA) was highest. Prunus L. species. RAPD combined with ISSR analysis can be used for genetic characterization of  Prunus salicina L., belonging to the family of Rosaceae, are one of the most important economical fruit trees and are widely cultivated all over the world. They are mainly distributed in the northern hemisphere, especially in the temperate zone [Prunus L. species. Prunus L. species contain more than 430 species and are first segregated into six genera according to the morphology of fruit: Amygdalus L., Armeniaca Scop., Cerasus Mill., Laurocerasus, Padus Mill., Prunus species, and Tourn. ex Duh. However, phylogenetic analysis showed that Cerasus, Laurocerasus, and Padus were not monophyletic [Prunus L. species to cultivate new breed with improved quality characteristics.ate zone , 2. ChinPrunus L. species [Lonicera japonica Thunb. [Atractylodes lancea [Ocimum basilicum L. [Litchi chinensis Sonn. [Allium species [Cymbopogon [Prunus L. species [DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence has been widely used for genetic diversity analysis of species . Several species –8. Recen species , 6, 9–11 species . RAPD is species . RAPD is species . ISSR is species . RAPD ana Thunb. , syntheta Thunb. , Atractys lancea , and Ocilicum L. . Howeveris Sonn. , Allium  species , date pa species , and Cymmbopogon . However species –25.P. salicina from China and other countries. This study may provide valuable insight into the genetic diversity of P. salicina L. and provide information to cultivate new breed with improved traits.In this study, we applied the RAPD and ISSR technique for the genetic characterization of 25 P. salicina L. which were collected from 14 different regions from China (13 samples), Japan (4 samples), USA (7 samples), and New Zealand (1 sample)  . Among tP. salicina L. was extracted from fresh leaves using a modified cetyl trimethylammonium bromide (CTAB) method as described previously [µl for PCR and stored at −20°C until use.The genomic DNA of 25 eviously , 26. DNAμL volume contains 1 μL of 2.5 μmol/L primers, 1 μL of DNA template, 5 μL of 2 × PCR Taq Mastermix , and 3 μL of deionized water. The PCR was executed on Applied Biosystems Veriti 96-Well Thermal Cycler  in the following procedure: initial denaturation at 95°C for 90 s, followed by 40 cycles of 40 s at 94°C, 60 s at 36°C, 90 s at 72°C, and final extension of 5 min at 72°C.The random RAPD primers were selected randomly for PCR amplification . The PCRμL reactions including 1 μL of 2.5 umol/L primers, 1 μL of DNA template, 5 μL of 29 PCR Taq Mastermix , and 3 μL of deionized water. PCR was executed on Applied Biosystems Veriti 96-Well Thermal Cycler using the following procedure: initial denaturation at 95°C for 90 s, followed by 35 cycles of 30 s at 94°C, 30 s at 50°C, 90 s at 72°C, and final extension of 5 min at 72°C [Fifteen ISSR primers were synthesized by Thermo Fisher (USA) . ISSR am at 72°C .The amplified PCR products were separated by electrophoresis on 1.8% agarose gel in 1 × TAE buffer. Gels were visualized by 0.5 g/ml ethidium bromide staining, and the images were documented using the ChemiDoc XR . Bands that were unambiguous and reproducible in successive amplifications were selected for scoring.All PCRs were repeated five times for each of five samples. Bands in the gel profiles were scored as 1 for present and 0 for absent. The similarity matrix (SM) and the similarity index (SI) were calculated using SM coefficient in Numerical Taxonomy Multivariate Analysis System (NTSYS pc 2.1) software. The dendrogram based on the unweighted pair group method with arithmetic mean algorithm (UPGMA) was generated using the SAHN module in the NTSYS pc 2.1 software.A total of nineteen RAPD primers and fifteen ISSR primers were used in this study for the evaluation of DNA polymorphism . All RAPFor the ISSR primers, a total of 204 bands with an average of 13.60 bands per primer were produced; of them, 179 (87.74%) were polymorphic. The approximate range of band size was 300 bp to 2500 bp . The minP. salicina samples formed four clusters at a cutoff of 0.692. The similarity coefficient between sample P4  and P13  was lowest (0.584), while that between sample P19 (Laroda in USA) and P20 (Red heart in USA) was highest (0.860)  .P. salicina samples were divided into five clusters at a cutoff of 0.692. The similarity coefficient between sample P10  and P13  was lowest (0.558), while that between sample P10  and P15 (Baili in Japan) was highest (0.892)  .The dendrogram results of RAPD combined with ISSR showed that the similarity coefficients ranged from 0.597 to 0.865. Total 519 DNA fragments were yielded, of which 435 (84.7%) were polymorphic. The average number of PF per primer was 12.7. The mean PIC, RP, EMR, and MI values observed for all primers were 0.42, 17.77, 10.80, and 4.54, respectively . The simPrunus L. samples  and P13  was lowest (0.584), while that between sample P19 (Laroda in USA) and P20 (Red heart in USA) was highest 0.860). However, the ISSR results showed that the index of similarity coefficient between sample P10  and P13  was lowest (0.558), while that between sample P10  and P15 (Baili in Japan) was highest (0.892). In addition, the analysis of RAPD combined with ISSR showed that the similarity coefficient between sample P10  and P13  was lowest (0.597), while that between sample P10  and P15 (Baili in Japan) was highest (0.865), which was consistent with the RAPD analysis. These findings demonstrated that the RAPD technique not only increased the resolution and yield but also was a reliable molecular tool for the genetic characterization of various organisms, which was reported in previous studies [0. HoweveP. salicina L. Our results might assist in parental gametophytes selection for hybrid breeding of P. salicina L.In summary, our study indicates that the RAPD combined with ISSR techniques would be used for the genetic diversity, molecular-assisted breeding, and genetic characterization of


Juniperus thurifera is a native species to the mountains of the western Mediterranean region. It is used in traditional medicine as a natural treatment against infections. The present study aimed to carry out the chemical analysis and evaluate the antioxidant, antimicrobial, as well as in silico inhibition studies of the essential oils from Juniperus thurifera bark (EOEJT). Chemical characterization of EOEJT was done by gas chromatography (GC-MS). We have performed three antioxidant assays (Reducing power (FRAP), 2, 2-diphenylpicrylhydrazyl (DPPH), and total antioxidant capacity (TAC)) of the EOEJT. We next evaluated the antimicrobial activity against in silico study, which was carried out to help evaluate the inhibitory effect of EOEJT against NADPH oxidase. Results of the GC/MS analysis revealed seven major compounds in EOEJT wherein muurolol (36%) and elemol (26%) were the major components. Moreover, EOEJT possessed interesting antioxidant potential with an IC50 respectively of 21.25 ± 1.02 μg/mL, 481.02 ± 5.25 μg/mL, and 271 μg EAA/mg in DPPH, FRAP, and total antioxidant capacity systems. Molecular docking of EOEJT in NADPH oxidase active site showed inhibitory activity of α-cadinol and muurolol with a glide score of −6.041 and −5.956 Kcal/mol, respectively. As regards the antibacterial and antifungal capacities, EOEJT was active against all tested bacteria and all fungi, notably, against Escherichia coli K12 with an inhibition diameter of 21 mm and a MIC value of 0.67 mg/mL, as well as against Proteus mirabilis ATCC 29906 with an inhibition diameter of 18.33 ± 1.15 mm and a MIC value of 1.34 mg/mL. A more pronounced effect was recorded for the fungal pathogens Fusarium oxysporum MTCC 9913 with inhibition of 37.44 ± 0.28% and MIC value of 6.45 mg/mL, as well as against Candida albicans ATCC 10231 with an inhibition diameter of 20.33 ± 1.15 mm and a MIC value of 0.67 ± 0.00 mg/mL. Altogether, these results highlight the importance of EOEJT as a source of natural antibacterial and antioxidant drugs to fight clinically important pathogenic strains. Juniperus thurifera [Juniperus thurifera is a variably sized tree that belongs to the family Cupressaceae. This species is native to mountainous areas of the Mediterranean basin's western region [Juniperus thurifera grows more particularly in the High and Middle-Atlas Mountains [Quercus ilex and Cedrus atlantica. Such hardy species may tolerate a wide range of weather conditions and may reach up to 19 meters tall and live for up to 800 years. The thurifer covers an estimated area of almost 20 000 hectares. It is the most regressed forest species in Morocco, with a 90 percent regression from its potential range [Morocco is the most biodiverse country in the western Mediterranean region . Within hurifera . The Bouhurifera . Junipern region . In Moroountains , 6. The al range .Different phytoconstituents have been isolated from this species, and include mainly polyphenols, essential oils, and sterols , 9. SuchMultidrug-resistant organisms have become a major concern and the situation is far from being mastered. Therefore, the research for antibacterial from natural origins has become a vivid area of exploration. Essential oils are combinations of volatile secondary metabolites that occur naturally in plants, which mostly represent the various smells and scents of a variety of plants . A myriaJuniperus thurifera by GC/MS. Next, we conducted the antioxidant potential of essential oils in vitro and explore in silico as NADPH oxidase inhibitors. Finally, the antibacterial and antifungal effects against drug-resistant microbial strains were also described.This study aimed at characterizing the bark essential oils of Juniperus thurifera (hurifera  was coll3), sodium phosphate, ammonium molybdate, butylated hydroxytoluene (BHT), 2, 2-diphenylpicrylhydrazyl (DPPH), and 2, 3, 5-triphenyltetrazolium chloride (TTC). Malt extract (ME), sodium chloride (NaCl), trichloroacetic acid (TCA), potassium ferricyanide (K3Fe(CN)6), agar, erythromycin, fluconazole, and dimethylsulfoxide (DMSO) were bought from Sigma-Aldrich .Ferric chloride  were recovered and placed at 4°C until further use , 14.μm Film thickness). One microliter of EOEJT was used for chromatographic analysis after being diluted in hexane (10 : 100 dilution). The oven temperature program was initially set to 40°C for 2 minutes and followed by 260°C for 10 minutes, then increased by 5°C/min up to 280°C. Finally, the temperature was held at 280°C for 10 minutes. The carrier gas was nitrogen and the flow rate was 1 mL/min. The temperatures of the injector and detector were adjusted to 250 and 280°C. The ionization energy was set to 70 eV, the ion source temperature was set at 200°C, the interface line temperature was 280°C, and the scan mass range was m/z 40–650.00. Volatile compounds of essential oil were identified by the use of the Kovats index and NIST-MS Search Version 2.0 [Analysis was done using a gas chromatograph (GCMS-TQ8040 NX Shimadzu brand) with an apolar capillary column  were diluted in methanol before being added to 750 μL DPPH, while a solution consisting of 750 μL DPPH and 100 μL methanol was used as a negative control. The reaction media were incubated for 25 minutes, and the optical density (DO) was measured at λ = 517 nm. The antioxidant efficiency was determined according to the following formula:Free radical powder was prepared in methanol ,17. BrieEA: antioxidant efficiency in percent (%); Ds: optical density of sample; Ct: optical density of negative control (White).3Fe(CN)6 (1%) [μL EOEJT before being incubated at 50°C for 30 minutes. To prepare for analysis, the sample was remixed with 200 μL of trichloroacetic acid (10 mg/mL), 500 μL of distilled water (H2O), and 100 μL of FeCl3 (1 mg/mL). At 700 nm, absorbance was measured against a negative control (50 μL of methanol without EOEJT). The EC-50 effective concentration was used to present the results.This test was assayed by preparing a reaction medium using phosphate buffer with 200 mM-pH = 6.6 and KN)6 (1%) ,18. Brieλ = 695 nm against a negative control containing 25 μL of methanol without EOEJT, while ascorbic acid was used as a positive reference. Results of the TAC assay were expressed in micrograms equivalent to ascorbic acid per milligram of the sample (μg AAE/mg) [Briefly, EOEJT was combined with 1 mL of a reagent solution before being heated at 95°C for one hour and then cooled at room temperature for 20 minutes. The optical density was measured at  AAE/mg) .μL of EOEJT before being placed on Petri dishes surfaces that were previously inoculated with bacteria (106 to 108 CFU/mL) and fungi. Erythromycin and fluconazole served as positive controls, respectively, for bacteria and fungi. Subsequently, Petri dishes inoculated with bacteria and fungi were meticulously incubated at 37°C and 30°C for bacterial and fungi strains, respectively [C. albicans. However, after 7 days postinoculation, these calculations were performed for A. flavus, F. oxysporum, and A. niger [The disk diffusion assay was used to determine whether or not EOEJT has antimicrobial capabilities . In a nuectively . After 2A. niger .μL of EOEJT with a 1 : 10 (V/V) dilution of DMSO (10 %) was pipetted into the first column of the plate to perform a series of dilution using sterile MH for bacteria and sterile ME for fungi, and lastly, 30 μL of microbial suspension of each strain (108 CFU/mL) was added to wells. After an incubation period ranging from 48 hours to 7 days for fungi and 24 hours for bacteria at 37°C [The MIC of EOEJT against microbial strains was determined using the microdilution method . Briefly at 37°C , 22, the at 37°C .For ligand preparations, we used the PubChem database to download all the molecules identified by GC/MS in EOEJT in SDF format. Next, the OPLS3 force field was applied, and the LigPrep tool in Schrödinger's Maestro 11.5 software was used to prepare the structures. Based on the ionization states at pH 7.0 2.0, a total of 32 stereoisomers were generated for each ligand.For receptor preparation, the three-dimensional crystal structure of NADPH oxidase was downloaded from the PDB data bank in PDB format. Protein Preparation Wizard of Schrödinger-Maestro v11.5 was used to add hydrogen atoms to heavy atoms, transform selenomethionine into methionine, and remove water. Next, the OPLS3 force was used for minimization, and fixing the maximum heavy atoms RMSD (root mean square deviation) at 0.30 Å .X = 17.584, Y = 9.05, and Z = 51.541. When the volumetric spacing performed was 20 × 20 × 20, the glide of Schrödinger-Maestro v11.5 was used for SP flexible ligand docking [The receptor grid was set at the following coordinates:  docking .X = 17.584, Y = 9.05, and Z = 51.541. When the volumetric spacing performed was 20 × 20 × 20, the glide of Schrödinger-Maestro v11.5 was used for SP flexible ligand docking. The ligand was coupled to the grid box produced from protein using “Standard precision” (SP), while SP GScore was used to evaluate the results [The receptor grid was set at the following coordinates:  results .In ligand docking, the penalties were applied to noncis/trans amide bonds. The partial charge cutoff was set to 0.15 and the Van der Waals scaling factor and partial charge cutoff were set to 0.80 for each ligand atom, The energy-minimized poses presented by the glide score were used to calculate the final score. The best-docked pose with the lowest glide score value was recorded .p values lower than 0.05 were regarded to indicate a statistically significant difference.Data were all provided as the mean value and standard deviation of tests performed in triplicate. GraphPad Prism was used to perform statistical analysis. Levine's test was used to verify homogeneity, while Shapiro–Wilks test was used to verify normality. Analysis of variance (ANOVA) flowed by Tukey's HSD test was used to arrange multiple comparisons. of Juniperus thurifera [Juniperus thurifera is shown in The yield of EOEJTs was about 0.89%, and it is somewhat comparable to what was previously found in the leaves (0.96%) hurifera . Seven cμg/mL scored an inhibition of about 75%, 85%, and 70%, respectively, for EOEJT, BHT, and quercetin. At a higher concentration, 250 μg/mL, the inhibition percentage was found to be 89.21% (EOEJT), 94% (BHT), and 92% (quercetin), 50 were 21.25 ± 1.02 μg/mL, 17.25 ± 1.20 μg/mL, and 20.15 ± 1.30 μg/mL, respectively, for EOEJT, BHT, and quercetin  method. Results showed that EOEJT had a good antioxidant capacity since it scored 271 g EAA/mg . These rg AAE/mg .E. coli wherein we noted an inhibition zone diameter of 21 ± 0.00 mm and MIC of 0.67 ± 0.00 mg/mL, and against P. mirabilis with an inhibition diameter of 18.33 ± 1.15 mm and a MIC of 1.34 ± 0.00 mg/mL  and one fungal (fluconazole) specific antimicrobials. The results showed that the EOEJT test was more effective against Gram-negative or Gram-positive bacterial strains. Moreover, the principal component analysis  indicateα-cadinol, muurolol, gamma-eudesmol, elemol, and α-pinene present the highest glide score value of −6.041, −5.956, −5.542, −4.538, and −4.358 kcal/mol, respectively , elemol (12%), and muurolol (12%) [Juniperus thurifera EOs [Juniperus thurifera of terpinene, which is considered among the compounds that have been shown to increase the antioxidant power [In the present work, EOs extracted from ol (12%) . Previoufera EOs . Such difera EOs . The assfera EOs –32. It hfera EOs –38, whicfera EOs . Recent nt power .P. aeruginosa and other multidrug-resistant bacteria [S. aureus and E. coli strains [Juniperus thurifera essential oils exhibited substantial antibacterial capacity against bacteria strains, particularly against S. aureus (31.12 ± 3.11 mm of inhibition), E. coli (13.23 ± 2.59 mm of inhibition), and P. aeruginosa (18.27 ± 2.29 mm of inhibition). Our results are also in agreement with several other studies such as the study by Zeraib et al. [S. aureus was highly sensitive to the EO of Algerian Juniperus thurifera, and also the study by Bahri et al. [Juniperus thurifera had potent antibacterial activity against S. aureus ATCC 33862 , E. coli ATCC 25922 , and P. mirabilis ATCC 7002 (inhibition zone diameter: 18.8 mm).Concerning the antibacterial power, taking a closer look at the published literature supported that muurolol exerted significant antibacterial activity against pathogenic bacteria . Elemol bacteria ,43. More strains . The cur strains , who shob et al. , which ri et al.  demonstrA. niger, C. albicans, and F. oxysporum may be due to their active ingredients, especially the high content of bioactive molecules in EOEJT such as muurolol, elemol, and pinene. Many studies have reported that these molecules have a strong antifungal activity, most notably, the study by Chang et al. [α-pinene against Candida spp, and Shi et al. [C. gloeosporioides, F. proliferatum, A. kikuchiana, and Phomopsis sp.The interesting antifungal capacity of EOEJT against g et al. , which sg et al. . For ping et al. , showed i et al. , reporteA. flavus, F. oxysporum, A. niger, and C. albicans using different types of substances, either natural or chemically synthesized. Our results are in agreement with previous reports e.g., the study of Jemli et al. [A. alternata, F. solani, F. oxysporum, V. dahlia, and R. solani with a percentage of inhibition ranging from 24% to 92.1%. Lafraxo et al. [Juniperus thurifera leaves exhibited potent antifungal activity vs. F. oxysporum and C. albicans at a concentration of 0.095 g/mL.Many strategies had been devoted to the control of i et al. , which so et al.  showed tJuniperus thurifera bark essential is rich in terpene compounds, which were extracted and characterized by GC-MS. Additionally, the Juniperus thurifera essential oil also had proven antimicrobial, antifungal, and antioxidant capacity. This opens up the possibility of encapsulating this essential oil through complex biotechnology applications with antibiotics to enhance their effects against pathogen resistance. However, before any potential application, further studies dealing with toxicity are warranted.This study showed that


Compound Turkish gall ointment (CTGO) has a long history of being widely used as a folk medicine in Xinjiang for the treatment of eczema. CTGO is currently in the pre-investigational new drug application stage, but its pharmacological mechanisms of action have not yet been clarified.First, a sensitive and reliable ultra-high performance liquid chromatography-Q exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) technique was established. Second, an integrative strategy of network analysis and molecular docking based on identified and retrieved ingredients was implemented to investigate the potential targets and pathways involved in the treatment of eczema with CTGO. Finally, Sprague–Dawley (SD) rats with eczema were prepared to verify the predicted results. The skin conditions of the rats were observed, evaluated, and scored. Skin tissues were observed by hematoxylin–eosin (HE) staining, and the levels of serum interferon-γ (IFN-γ) and interleukin-4 (IL-4) were determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were detected by real-time quantitative polymerase chain reaction (RT-qPCR).A total of 29 compounds were identified. We found 38 active components and 58 targets for the treatment of eczema, which included 118 signaling pathways related to inflammation, immunity, and apoptosis. CTGO significantly improved the skin surface and histopathological characteristics of eczema-affected rats, downregulated the expression of IL-4, TLR4, NF-κB (p65), IL-1β, and TNF-α, and upregulated the expression level of IFN-γ.We predicted and validated our prediction that CTGO may be used to treat eczema by affecting the TLR4/NF-κB signaling pathway, which provides guidance for future experimental studies. Eczema is a chronic inflammatory skin disease with exudative tendency and clinical manifestations including erythema, blisters, erosion, and pruritus . Modern We systematically expounded the possible targets and related pathways of CTGO in treating eczema by integrating UHPLC-Q-Orbitrap HRMS, network analysis, molecular docking analysis, and experimental evaluation using molecular biology. Figure Low , medium , and high  dose CTGO were purchased from NEW CICON Pharmaceutical Co., Ltd.; Gallic acid , Ellagic acid , and β,β-Dimethylacrylalkannin  were purchased from China Institute for Food and Drug Control; Gallic acid methyl ester  and Ethyl gallate  were purchased from Chengdu Pufei De Biotech Co., Ltd.; L-shikonin (Batch No. 110769–200506) was purchased from China Institute for the Control of Pharmaceutical and Biological Products; Methanol and acetonitrile of HPLC grade were purchased from Sigma-Aldrich Company. All other reagents were of analytical grade and supplied by Tianjin Beilian Fine Chemicals Development Co., Ltd. Compound dexamethasone acetate cream (20201024X) was purchased from China Resources Sanjiu Medical and Pharmaceutical Co., Ltd.; 2, 4-dinitrochlorobenzene  was purchased from Shanghai Jiudin Chemical Technology Co., Ltd.; IL-4 and INF-γ test box  were purchased from Shanghai Enzyme Linked Biotechnology Co., Ltd.; the PCR kit (AQ601-01) was purchased from Beijing Quanshijin; the microscope (EQ2016-037) was purchased from Leica, Germany; the Multiskan FC was purchased from Thermo Fisher; and the electronic balance (CPA124S) was purchased from Sartorius, Germany.Sixty Sprague–Dawley (SD) rats  were purchased from the Laboratory Animal Center of Xinjiang Medical University (License number: SCXK (new) 2018–0002). All of the rats were fed in an environment free of specific pathogens, at a temperature of 20–26 °C, humidity 40%– 70%, artificial light, 12-h light/dark cycle, and had free access to food and water.http://tcmspw.com/index.php/), CNKI (https://www.cnki.net/), CTD (http://ctdbase.org/), ETCM (http://www.tcmip.cn/ETCM/index.php/Home/Index/The following databases were used: TCMSP (http://47.100.169.139:8000/tcmid/), BATMA.), TCMID (http://bionet.ncpsb.org.cn/batman-tcm/), GEO (https://www.ncbi.nlm.nih.gN-TCM (https://go.drugbank.com/), STRING (https://string-db.org/), Pu.ov/geo/), DrugBank (https://pubchem.ncbi.nlm.nih.gov/), and PDB (http://www1.rcsb.org/). The software Cytoscape 3.7.2, R3.6.1 language, Chem3D, PyMOL, AutoDock Vina-1.5.6, and BIOVIA Discovery Studio 2020 were used.bChem  using a Kromasil Classic 100–5-C18 column  at 30 °C, with a flow rate of 1.0 mL/min and a wavelength of 300 nm. The mobile phase consisted of water containing 0.1% formic acid (solvent system A) and acetonitrile (solvent system B). Gradient elution was conducted as follows: 0–5 min, 5% B; 5–45 min, 5–12% B; 45–50 min, 12% B; 50–80 min, 12–20% B; 80–95 min, 20–25% B; 95–105 min, 25–67% B; 105–115 min, 67–70% B; 115–125 min, 70–75% B; 125–135 min, 75% B. Ten microliters of the sample solution was injected for analysis.2 resolution was 17,600 FWHM (200 m/z). The Xcalibur 4.0 software package was used for data acquisition and analysis .Mass detection was performed using an UHPLC-Q-Orbitrap HRMS equipped with a Dual ESI source operating in both positive and negative modes with the following operating parameters: ESI spray voltage, –2.8 kV ; sheath gas, 40 arb; auxiliary gas, 10 arb; air curtain gas (CUR), 35; ion source temperature (heat temp), 350 °C; capillary temperature, 300 °C; –70 V; focusing voltage (FP), –350 V; and DP2, –10 V. The data dependent acquisition (DDA) mode was adopted for sample analysis; the collection range of the q-orbitrap was 100–1500 m/z, and the fragment ion scanning range was 50–1500 m/z. The collision gas (NCE): 20, 40, 60. The mass spectrometry (MS) resolution was 70,000 full width at half maximum (FWHM) (200 m/z), and the MSTo prepare the sample, 0.05 g of Turkish gall was weighed and placed into a conical flask. Next, 25 mL of 80% methanol was added before a 1-h reflux in a water bath at 75 °C. Then, a pipette was used to transfer 1 mL of the supernatant into a 5 mL volumetric flask. Simultaneously, 0.1 g of Comfrey was weighed precisely and placed into another conical flask, before adding 50 mL of chloroform and subjecting it to a 1 h reflux in a water bath at 75 °C. The sample was then filtered, and the solvent was evaporated in the water bath. The residue was transferred into the abovementioned 5-mL volumetric flask with approximately 4 mL of acetonitrile to bring the total volume to 5 mL.“Turkish gall” and “Comfrey” were searched for in the TCMSP, CTD, TCMID, ETCM, and BATMAN-TCM databases and the literature to obtain the components of Turkish gall and Comfrey. These agents were combined with the compounds identified by UHPLC-Q-Orbitrap-HRMS to build the active ingredient database of CTGO. Targets corresponding to the components were obtained through the TCMSP and BATMAN-TCM databases, and a target database of CTGO was constructed. “Eczema” was searched in the GEO and DrugBank databases to obtain eczema targets. R 3.6.1 language software was used to obtain the intersection targets of drugs and diseases.To clarify the relationship between intersection targets, an intersection target PPI network of eczema and CTGO components was constructed and analyzed using the STRING database. Species were defined as Homo sapiens. PPIs with a confidence score > 0.40 were selected and topology analyses were conducted to ensure the accuracy of the results. The PPI network was imported into the Cytoscape 3.7.2 software for use with PPI topology analysis.q < 0.05), the top 10 GO biological processes, cellular composition, and molecular functions were selected to draw bubble maps; the intersection targets of CTGO and eczema were analyzed by KEGG enrichment. According to the number and significance of gene enrichment (q < 0.05), the top 20 pathways were selected and bar graphs were drawn. The size of the bubbles and strips in the figure represents the number of enriched genes on this pathway, and the color difference of the bubbles and strips represents the level of enrichment of target genes on this pathway.GO enrichment analysis was performed on the intersection targets of CTGO with eczema using R language software. According to the number and significance of gene enrichment  in the PPI network. First, the composition and target files were downloaded from PubChem and PDB. Next, energy minimization of the component file was completed by Chem3D, and dewatering and impurity removal of the target file were completed by PyMOL. The file was then converted to PDBQT format using AutoDock Vina. The active pocket of the target was determined, molecular docking was performed, and binding energy values were obtained. The best docking model diagram was drawn by BIOVIA Discovery Studio. Finally, the binding ability was evaluated by the affinity of the component and the target.Sixty rats were randomly divided into a normal group, a model group, a positive group (compound dexamethasone acetate cream), a CTGO high-dose group, a CTGO medium-dose group, and a CTGO low-dose group, with 10 rats per group. The rats in all groups, except for the normal group, were treated with 7% DNCB solution by rubbing the solution on the back to establish the eczema model. The hair on the neck (A: 2 cm × 2 cm) and back (B: 4 cm × 4 cm) of the animals was removed using an electric shaver. The rats were sensitized by applying 100 μL of 7% DNCB acetone solution with a pipetting gun on the skin of site A. Severe pruritus, frequent scratching, and rolling behaviors were observed in rats, lasting for approximately 2 h. After 1 week, 200 μL of 7% DNCB acetone solution was applied to site B for excitation. The rats were stimulated once every 5 days, and in each stimulation, severe pruritus could be seen in rats, with frequent scratching and rolling behavior. Erythema, papules, edema, scratches, and desquamation gradually appeared on the skin at site B. After each stimulation, the skin lesions were recorded and scored. After six sessions of stimulation, erythema, papules, scab, and exudation appeared in skin lesions on the back of each rat, at which point the stimulation was stopped, indicating that the model had been successfully established .Each group was administered a low (0.035 g/g), medium (0.07 g/g), or high (0.14 g/g) dose of CTGO, and the positive group was administered compound dexamethasone acetate cream . The normal and model groups were administered with saline. After smearing the experimental drug, 2–3 layers of medical gauze were wrapped and fixed with a non-irritating adhesive tape. After 4 h, the covering was removed and washed with warm pure water for continuous administration for 14 days.After the final administration, the rats were fasted with water for 12 h. The rats were mildly anesthetized with 1% sodium pentobarbital . Blood samples were collected from the abdominal aorta and placed in a centrifuge tube for 30 min at 3000 rpm at 4 °C, before storing the supernatant at –20 °C. One part of the obtained skin tissue was immersed in 10% neutral formalin for fixation, and the other part was stored at − 80 °C.On days 1, 5, 10, 15, 20, 25, 30, and 35 (administration D0) of modeling, and on days 0, 9, and 14 of administration, the grades of erythema, edema, exudation, desquamation, liche-like change, desquamation were scored from 0 to 2. A score of 0 represents no symptoms; 1 represents mild symptoms; and 2 represents significant symptoms After 14 days of administration, the skin conditions were photographed observed with the naked eye.Skin tissue was removed from 10% neutral formalin solution, dehydrated, permeated, paraffin impregnated, embedded, sectioned, stained, and sealed. Histomorphological observation was performed with a microscope, and radiographs were taken.The levels of IFN-γ and IL-4 in the serum of rats were determined according to the instructions of the ELISA kit.−△△CT method was used to analyze the difference in target gene expression between the control group and test groups.The mRNA expression levels of TLR4, NF-κB (p65), IL-1β, and TNF-α in skin tissue samples were detected by RT-qPCR. RNA was extracted, its purity was determined, and cDNA was obtained by reverse transcription. PCR upstream and downstream primers were added , IL1β, and TLR4. In PPI topology analysis, the six topological eigenvalues of “closeness centrality (CC),” “betweenness centrality (BC),” “eigenvector centrality (EC),” “local average connectivity (LAC),” “degree centrality (DC),” and “network centrality (NC)” were used as filter conditions. Targets with topological eigenvalues greater than their corresponding medians were used as core targets, and two rounds of analysis were performed. Fifteen core targets were obtained in the first round and four were obtained in the second round, as shown in Fig. PPI topology analysis results showed that after importing the intersection targets into the STRING data platform, the PPI relationship between the intersection targets was obtained. The PPI analysis network was obtained using Cytoscape 3.7.2 for topology analysis of the relationship. The network consisted of 58 nodes and 390 edges. The average node degree value was 13.4, and the PPI enrichment q < 0.05) GO functional enrichment items were obtained, including 1316 biological processes (BPs), 75 molecular functions (MFs), and 29 cellular components (CCs), as shown in Fig. q < 0.05) signaling pathways were obtained, as shown in Fig. GO and KEGG enrichment analyses and visualization processing were performed on 58 intersections of CTGO and eczema using the Bioconductor bioinformatics software package. GO functional annotation showed that 1420 . Compared to the model group, the apparent scores in each administration group were significantly decreased (p < 0.01), as shown in Table After 14 days of administration, the back skin of rats in the normal group was pale red, delicate, and tender, with a soft texture. The back skin lesions of the rats in the model group were obvious, with skin erythema, infiltration, scales, scabs, rough and thickening texture, pigmentation, and scratches, which still met the diagnostic criteria for chronic eczema. Regarding the observation record of apparent indicators, as shown in Fig. The thickness of the cuticle, epidermis, and dermis in the normal group was normal. Model group rats showed chronic eczema, including hyperkeratosis, parakeratosis, hypertrophy of the granular layer, hypertrophy of the spinous layer, and infiltration of numerous inflammatory cells in the epidermis/dermis. After 14 days of administration, hyperkeratosis, parakeratosis, hypertrophy of the granular layer, and hypertrophy of the spinous layer were inhibited in each administration group, and the infiltration of inflammatory cells in the epidermis/dermis was reduced. The results are shown in Fig. . Among t#p < 0.05) and the levels of IL-4 in the model group were significantly increased (#p < 0.05). Compared to the model group, the level of IFN-γ in the CTGO group was significantly increased (**p < 0.01) and that of IL-4 was significantly decreased  , and the level of IL-4 in the low dose group decreased significantly (*p < 0.05).Compared to the normal group, the levels of IFN-γ in the model group were significantly decreased (01) Fig. . Compare##p < 0.01, ###p < 0.001, or ####p < 0.0001). Compared to the model group, the contents of IL-1β and TNFα in the skin tissue of rats in the positive group and CTGO low-, medium-, and high-dose groups were significantly decreased , and the contents of TLR4 and NF-κB in the skin tissue of rats in the CTGO low-, medium-, and high-dose groups were significantly decreased . Compared to the positive group, the contents of TLR4 and NF-κB in the skin tissue of rats in the medium- and high-dose CTGO groups were significantly decreased   Fig. .Fig. 12mTCM compounds have multi-component and multi-target synergistic effects, and have been widely used in clinical practice because of their clear curative effect and minor side effects. CTGO has shown good clinical efficacy in the treatment of eczema, but its mechanism of action remains unclear. Based on network pharmacological analysis, we investigated the pharmacodynamics and action mechanism of CTGO in a rat model of eczema.In this study, a combined strategy of phytochemistry, network analysis, molecular docking, and basic experiments was used to assess the active constituents and potential molecular mechanisms of CTGO on eczema. First, the chemical composition of CTGO was characterized by UHPLC-Q-Orbitrap HRMS for the first time. A total of 29 constituents, mainly belonging to polyphenols, naphthalene quinones, and anthraquinones, were identified. Due to the small number of chemical components identified by mass spectrometry, no more components were identified. To make the results of network pharmacological prediction and analysis more comprehensive, we performed a database and literature search of the chemical constituents of gallic gall and Comfrey, and retrieved 57 chemical constituents. The components identified by mass spectrometry were combined with the retrieved components, and after removing duplicates, 77 components were finally obtained and used as a basis for network pharmacological analysis.Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and E. coli [The component-target-pathway network contained 38 active ingredients. The common active components of gallic gall and Comfrey are palmitic acid and β-sitosterol. Their common targets are the core targets of TNF and IL-1β, which rank highly in degree value. The top two components in degree value were gallic acid and ellagic acid, which are the main components of gallic gall, and have 11 common targets, namely, TNF, TGFβ1, RELA, PTGS1, PTGS2, NOS2, MMP9, IL-1β, CYP1B1, CYP1A1, and Alb. All of the targets are closely related to inflammatory, immune, and metabolic reactions. Shikonin is the main chemical component of Comfrey; it can act on nine eczema-related targets, among which TNF and PTGS2 are the common targets of shikonin, gallic acid, and ellagic acid. Gallic acid has a good antibacterial effect, and can effectively inhibit  E. coli . Gallic  E. coli , 15. Ell E. coli . Studies E. coli . Shikoni E. coli . Studies E. coli .The PPI network contains 58 core targets for the treatment of eczema, reflecting the multi-target characteristics of CTGO in the treatment of eczema. TNF-α, IL1β, and IL-4 are important cytokines in the process of skin immunity and inflammation. Under the action of allergens in the skin system, numerous inflammation-mediated factors, such as TNF-α, IL1β, and IL-4, are secreted during epidermal keratinization, all of which interact with each other in the pathogenesis of eczema, thus influencing the development and prognosis of the disease , 21. TLROur molecular docking results showed that the overall scores of TNF, TLR4, IL1β, and Alb when docking with four key components were all lower than –5.0 kcal/mol, which suggested that they may play an essential role in CTGO against eczema.The 38 active ingredients act on 58 targets related to eczema; all of these 58 targets are connected with 118 pathways closely related to inflammation and immunity. All of the above components, targets, and pathways form a network, which may be the mechanism by which CTGO functions to treat eczema. The toll-like and NF-κB pathways are among the first 30 pathways significantly associated with CTGO in the treatment of eczema and are closely related to inflammatory responses. The toll-like pathway affects the NF-κB pathway through regulating NF-κB, and the NF-κB pathway then regulates the downstream of the toll-like pathway. As NF-κB connects the two pathways, they are closely related and mutually regulated. Eight of the 58 intersection targets were enriched on the Toll-like pathway, namely TNF, IL1B, TLR4, RELA, CD14, MAP2K1, RAC1, and IRF3. Seven of the 58 intersection targets were enriched in the NF-κB pathway, namely TNF, IL1B, TLR4, RELA, CD14, PTGS2, and PLAU. PPI topology analysis yielded four core targets, namely TNF, IL1β, TLR4, and Alb, of which TNF, IL1β, and TLR4 were key upstream and downstream targets involved in both of the above pathways. Therefore, we speculate that the toll-like and NF-κB signaling pathways may be the main way by which CTGO functions in the treatment of eczema. The TLR4/NF-κB signaling pathway was selected for validation, and TLR4, NF-κB, TNF-α, and IL1β were finally selected as indicators to be validated in this pathway.Toll receptors are a class of highly conserved pattern recognition receptors. TLR4 recognizes various ligands of microorganisms and undergoes dimerization upon ligation. MyD88 and non-MyD88 pathways activate mitogen-bound protein kinase, activate NF-κB, upregulate inflammatory cytokines and chemokines, and induce inflammation. These pathways play an important role in the pathogenesis of eczema , 25. NF-In this study, the rat eczema model was induced by DNCB. When the body is re-exposed to the same antigen after being stimulated by DNCB through the skin, a type IV allergic reaction characterized by skin damage occurs. Repeated stimulation can produce clinical manifestations similar to eczema, and the coincidence degree with the main symptoms of eczema in Chinese medicine is > 70%. Following establishment of the model, many indicators can be used to judge whether the model has been established successfully and whether the drug has a therapeutic effect after administration. Among them, the apparent indexes belong to the core indexes, such as the skin lesion area, itching condition, fur gloss, and skin erythema and edema. Pathological indexes are directly related indexes, and local histopathological changes of skin are reliable indexes to evaluate the degree of skin lesions. Biochemical indexes are indirectly related indexes, and IL-4 and other biochemical indexes will increase after the animal eczema model has been successfully established . After CImmune imbalance, especially Th1/Th2 imbalance, plays an important role in the pathogenesis of eczema. IFN-γ is mainly secreted by Th1 cells and can inhibit IL-4 secretion by Th2 cells, as well as inhibit IgE production by B cells. IL-4 is mainly secreted by Th2 cells and mainly inhibits Th1 cell function, and promotes IgE production by B cells, which inhibit and antagonize each other and maintain the Th1/Th2 balance. The expression levels of IFN-γ and IL-4 in normal subjects maintained a relatively balanced state, which is lost in patients with eczema. The experimental results showed that CTGO could significantly reduce IL-4 levels and increase IFN-γ levels, suggesting that CTGO may play a role in the treatment of eczema by promoting Th1/Th2 homeostasis by down-regulating IL-4 levels and up-regulating IFN-γ levels , 31.Patients with eczema have a severe inflammatory response, and many inflammatory factors play a key role in the occurrence and development of eczema. The TLR4/NF-κB signaling pathway is involved in the inflammatory response, where TLR4 recognizes endogenous ligands and is activated via identifying related ligands, and the signal conduction through the MyD88 pathway leads to the activation of NF-κB. This signaling ultimately results in the production of a large amount of IL-1β, TNF-α, and other inflammatory factors, which promote cell proliferation and repair. However, with the excessive activation of NF-κB comes excessive release of these inflammatory factors; then, the responsive activation of TLR4 forms a vicious cycle, which leads to uncontrollable inflammatory response. Consequently, the epidermis produces excessive inflammatory reactions, and the tissue organs are damaged, which result in the occurrence and development of eczema. The results of the animal experiments indicated that CTGO could significantly reduce the expression of IL-1β, TNFα, TLR4, and NF-κB. These changes in key genes suggest that the mechanism of CTGO in treating eczema may be related to the effect of the TLR4/NF-κB signaling pathway , 33.In conclusion, our experiments showed that CTGO could reduce the expression of IL-1β, TNFα, TLR4, and NF-κB, and significantly promote the expression of IFN-γ, which is consistent with the results of the prediction of network pharmacology and molecular docking. The mechanism of CTGO in treating eczema may be related to regulating related inflammatory factors, promoting the Th1 and Th2 immune balance, and affecting the TLR4/NF-κB signaling pathway.


Adverse events were consistent with known effects of chemotherapy. This international, multicenter study provides real‐world data on the safety and effectiveness profile of CT‐P10 for DLBCL treatment and supports the adoption of CT‐P10 for the treatment of DLBCL.The rituximab biosimilar CT‐P10 is approved for the treatment of non‐Hodgkin lymphoma. Previous studies have demonstrated clinical similarity between CT‐P10 and reference rituximab. However, real‐world data relating to treatment in patients with DLBCL with rituximab biosimilars are limited. This study collected real‐world data relating to the effectiveness and safety of CT‐P10 treatment from the medical records of 389 patients with DLBCL . For the primary outcome , overall survival (OS), progression‐free survival (PFS), and best response (BR) were assessed. The percentage  of patients alive at 12‐, 18‐, and 30 months postindex (initiation of CT‐P10) was 86% (82.4%–89.4%), 81% (76.9%–84.9%), and 76% (71.2%–80.1%), respectively. The PFS rate  at 12‐, 18‐, and 30 months postindex was 78% (74.2%–82.5%), 72% (67.9%–76.9%), and 67% (61.9%–71.7%), respectively. Median OS/PFS was not reached. For 82% ( The curTwo real‐world studies of CT‐P10 in DLBCL patients have been published thus far. A single‐center real‐world study conducted in Korea investigated the safety and effectiveness of CT‐P10 in combination with CHOP (T[Truxima]‐CHOP) in patients with DLBCL and observed no significant difference in terms of response, overall survival (OS) or progression‐free survival (PFS) in patients treated with R‐CHOP compared to reference rituximab . In addiTo date, no multinational, multicenter studies have investigated the effectiveness or safety of CT‐P10 treatment in patients with DLBCL in a real‐world clinical setting. Additional real‐world data will help inform clinical management decisions and support the adoption of CT‐P10 for treatment of DLBCL in routine practice. The present study addressed this evidence gap by collecting real‐world data relating to the effectiveness and safety of CT‐P10 treatment in patients with DLBCL in several European countries.22.1This study was a multicenter, noninterventional postauthorization safety study conducted in 24 European specialist treatment centers or hospitals , which routinely used CT‐P10 for DLBCL treatment. In the preindex observation period, patient‐level data for patients with DLBCL were collected from hospital medical records from the date of diagnosis until the index date (date of CT‐P10 initiation), to acquire demographic and clinical characteristics data. Safety and clinical outcome data were collected in the 30‐month postindex observation period or until patient death, if sooner.2.2Patients were eligible for inclusion if they had a confirmed diagnosis of DLBCL, received CT‐P10 for the treatment of DLBCL, were aged ≥18 years at date of DLBCL diagnosis, provided written informed consent for data collection , and had preindex medical history available, with at least one clinical response assessment in the 30‐month postindex period (unless the patient was deceased). Patients receiving reference rituximab for previous treatment cycles  within the same line of treatment were excluded. In addition, patients were excluded if their medical records were unavailable.2.3Approval was sought from institutional review boards and/or independent ethics committees and local hospitals, as appropriate for each country in the study. Further information can be found in the 2.4The primary objective was to describe the clinical effectiveness of CT‐P10 for the treatment of DLBCL. Endpoints addressing the primary objective were OS and PFS rate at 12‐, 18‐, and 30‐month postindex date, best response to CT‐P10 and time to complete or partial response. Secondary objectives included a description of baseline demographic, clinical and disease characteristics and assessment of the CT‐P10 safety profile and treatment patterns in the 30‐month postindex.2.5The initial target sample size for this study was 500 patients, to provide adequate precision for key descriptive outcomes in the overall sample and relevant subgroups. Demographic, clinical and disease characteristics data were described using summary statistics: distributions and descriptive statistics of both central tendency (medians and/or means) and dispersion  were presented for quantitative variables, as appropriate to the data distribution. For the primary outcome analysis , time‐to‐event analyses were conducted using the Kaplan–Meier method, where OS and PFS were defined as the time from index until death (any cause), or the time from index until death from any cause or first documented evidence of disease progression, respectively. Patients were censored at 30‐month postindex date or the date of the last recorded hospital visit. Subgroup analysis for OS and PFS of patients who received CT‐P10 as the first line of therapy was also conducted. Disease progression and response data were assessed as documented in the patients’ medical records. Percentages were reported to the nearest whole number; hence, percentages may not total 100% due to rounding. Detailed information can be found in the 33.1n = 227/389] male patients). The median duration of disease at index (days from diagnosis to index date) was 23 (IQR 12.0–46.0) days. The most common recorded comorbidities at index were diabetes  and peripheral vascular disease , and the most common Charlson Comorbidity Index score at index was “3” . Most patients had an Eastern Cooperative Oncology Group score at index of “0”  or “1” . Disease stage at index was recorded using the Ann Arbor or Lugano staging system, with 11% (n = 25/218) of patients recorded as stage I, 18% (n = 39/218) as stage II or stage III (n = 40/218), 52% (n = 113/218) as stage IV and <1% (n = 1/218) as other (unknown). Eighty‐four percent (n = 328/389) of patients received CT‐P10 as first‐line treatment. Most patients had an International Prognostic Index score at index of 2  or 3  years at index  and 75% (n = 224/299) of patients achieved a complete response (CR) to CT‐P10 on or after 3‐ and 6 months, respectively. For patients where CT‐P10 was the first line of therapy, a total of 60% (n = 219/301) and 62% (n = 186/246) of patients achieved a CR to CT‐P10 on or after 3‐ and 6 months, respectively  of patients, PR in 12% (n = 46/382), no response or stable disease in 4% (n = 16/382) and progressive disease in 2% (n = 8/382) Figure The median (IQR) follow‐up was 30 22.3–30.0) months. For the overall sample, the percentage (95% CI) of patients alive at 12‐, 18‐, and 30 months postindex was 86% (82.4%–89.4%), 81% (76.9%–84.9%), and 76% (71.2%–80.1%), respectively. The percentage (95% CI) of patients alive and free from disease progression at 12‐, 18‐, and 30 months was 78% (74.2%–82.5%), 72% (67.9%–76.9%), and 67% (61.9%–71.7%), respectively. For patients where CT‐P10 was the first line of therapy at index, the percentage (95% CI) of patients alive at 12‐, 18‐, and 30 months postindex was 86% (81.9%–89.5%), 81% (76.3%–85.0%), and 74% (69.2%–79.1%), respectively. Additionally, the percentage (95% CI) of patients alive and free from progression at 12, 18, and 30 months was 78% (73.8%–82.8%), 73% (67.9%–77.7%), and 67% (61.3%–72.1%), respectively; median OS/PFS was not reached . R‐CHOP was the most frequently administered chemotherapy regimen at index . At the end of the observation period 85% (n = 325/384) of patients had discontinued the first line of CT‐P10 treatment commenced at index due to planned completion of the treatment course, 5% (n = 18/384) due to adverse events (AEs), 5% (n = 21/384) due to disease progression and for 4% (n = 16/384) due to other reasons. For three patients (n = 3), the first line of treatment was ongoing at data collection. A complete response was the most common (n = 3/16) “other” reason for discontinuation of first‐line CT‐P10 treatment. Treatment pattern data are summarized in Table Patients received a median of 6.0 (range 1.0–18.0) CT‐P10 infusions during the observation period (including index and post‐index infusions), with almost two thirds of patients receiving 5<10 infusions (61% n = 26/389). R3.4n = 351/389) of patients experienced one or more AEs  at index or postindex, 83% (n = 324/389) experienced nonserious AEs and 43% (n = 168/387) experienced serious AEs (SAEs). Of all AEs with grade recorded , most were classified as either grade 1  or grade 2 (31% [n = 715]). The percentage of AEs classified as grade 3, grade 4, and grade 5 was 16% (n = 369), 3% (n = 78), and 1% (n = 22), respectively. For all SAEs where the relationship to CT‐P10 was recorded (n = 193), SAEs were most commonly considered to be not related to CT‐P10 (64% [n = 123/193]). A total of 7% (n = 13/193), 3% (n = 5/193), 20% (n = 38/193), and 7% (n = 14/193) of SAEs were found to be definitely, probably, possibly or unlikely related to CT‐P10, respectively . IRR and AE data for the overall cohort and for first‐line CT‐P10 patients are summarized in supplementary Tables Overall, 90%  with DLBCL to compare CHOP chemotherapy in combination with rituximab versus CHOP chemotherapy alone. In relation to clinical effectiveness data, the 2‐year OS and PFS rates were 70% (95% CI 63–77 and 57% (95% CI [50–64]), respectively, in the group of patients who received CHOP chemotherapy in combination with rituximab. The percentage of patients achieving a complete and partial response was 52% and 7%, respectively .A retrospective study by Sehn et al. 2005) investigated clinical outcomes in patients with newly diagnosed advanced‐stage DLBCL treated with CHOP plus rituximab . The rep05 investIn some cases, as for OS/PFS and CR rate, our study appears to demonstrate a more favorable clinical effectiveness profile than patients who received reference rituximab in a previous study by Coiffier and authors , even wiIn relation to the safety profile, in the present study, overall AE, SAE, and IRR rates of 90%, 43%, and 16%, respectively, were demonstrated. Select real‐world safety events relating to CT‐P10 treatment in patients with DLBCL were investigated by Lee et al. (2020). In this study, an overall IRR rate of 26.7% was presented for the CT‐P10 group, compared with 16% in the present study. Results from the present study from an SOC perspective indicate that AEs were primarily related to the gastrointestinal organ class, for example, nausea, vomiting, which are AEs consistent with the known effects of chemotherapy Table  19]. In. In19]. Limitations are associated with this study. Due to the real‐world nature of this study, interpretation of data collected retrospectively was dependent on the completeness of the medical records and the reliability of the abstraction of data. For some participating countries, consent was a requirement and may have introduced selection bias (percentage of patients from each country). In addition, a total of 389 patients were included in this study as opposed to 500 patients that was initially planned, due to recruitment feasibility. However, as shown in Table n = 328) was reported.The originally planned number of 500 patients also enabled sufficient precision for subgroups as well as the overall sample, and only a single subgroup , data were stratified by patients who received CT‐P10 as a first‐line treatment. Collectively, the results support the prescription of CT‐P10 for the treatment of DLBCL. Overall, it is evident that previously reported clinical effectiveness and safety data for reference rituximab are broadly similar to the data found in the present study in patients with DLBCL treated with CT‐P10.5In conclusion this is the first multinational real‐world study evaluating the clinical effectiveness and safety of CT‐P10 in patients with DLBCL. CT‐P10 was well tolerated in patients with DLBCL. The majority of AEs were mild or moderate and in line with the expected toxicity of chemotherapy. The CT‐P10 observed response, OS, and PFS rate in this study were similar to the rates found in other studies on reference rituximab and a single centre real‐world study on CT‐P10 treatment in DLBCL patients. Overall, the results from this study provide healthcare professionals with more detailed information on the safety profile CT‐P10 for the treatment of DLBCL, allowing for informed, evidence‐based decisions on the most appropriate treatment strategy for patients with DLBCL.SK, YNL, and PLZ designed the study. All authors performed the research, analyzed the data, and wrote the paper.MB has received research funding from Takeda, Gilead, Roche, Abbvie; Travel/acc/expenses = Roche, Takeda, Gilead, Celltrion, Honararia Tevapharma, Roche, Celltrion; Advisory board = Beigene; Trial Management Group = Roche. GS has received in the last 12 months financial compensations for participating in advisory boards or consulting from: Abbvie, Bayer, Beigene, BMS/Celgene, Epizyme, Genentech/Roche, Genmab, Incyte, Janssen, Kite/Gilead, Loxo, Milteniy, Morphosys, Novartis, Rapt, Regeneron and Takeda. Shareholder: Owkin. PLZ has received personal fees for consulting from Verastem, EUSA Pharma, MSD and Novartis; personal fees for participating in a speaker's bureau from Verastem, Celltrion, Gilead, Janssen‐Cilag, BMS, Servier, MSG, TG Therapeutics, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, Incyte and Beigene; and personal fees for participating in advisory boards from Verastem, Celltrion, Gilead, Janssen‐Cilag, BMS, Servier, Sandoz, MSG, TG Therapeutics, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, ADC Therapeutics, Incyte and Beigene. SM has received educational grants to attend educational meetings, educational lecturing and attendance of pharmaceutical advisory boards for Novartis, Abbvie and AstraZeneca in the last year. KL has received research grants from Celltrion. Outside this work, KL has received research grants from AbbVie, Novartis, Takeda, Roche, Amgen, and Sandoz as well as personal fees from AbbVie, Novartis, Sandoz, Celgene, Jansen, and Amgen. SKK and YNL are employees of Celltrion Healthcare. CG, WK, MBo, DT, BS, JH, AB, JJB, and MW have no conflict of interest to disclose.Supp Table informationClick here for additional data file.Supplementary methodsClick here for additional data file.


The cropping system affects the physicochemical property and microbial community of paddy soil. Previous research mostly focused on the study of soil 0–20 cm underground. However, there may be difference in the laws of nutrient and microorganism distribution at different depths of arable soil. In surface (0–10 cm) and subsurface (10–20 cm) soil, a comparative analysis including soil nutrients, enzymes, and bacterial diversity was carried out between the organic and conventional cultivation patterns, low and high nitrogen levels. Analysis results suggested that under the organic farming pattern, the contents of total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), and soil organic matter (SOM) as well as alkaline phosphatase and sucrose activity increased in surface soil, but the SOM concentration and urease activity decreased in subsurface soil. A moderate reduction of nitrogen applied to soil could enhance soil enzyme activity. It was demonstrated by α diversity indices that high nitrogen levels remarkably undermined soil bacterial richness and diversity. Venn diagrams and NMDS analysis manifested great difference in bacterial communities and an apparent clustering tendency under different treatment conditions. Species composition analysis indicated that the total relative abundance of Proteobacteria, Acidobacteria, and Chloroflexi retained stable in paddy soil. LEfSe results revealed that a low nitrogen organic treatment could elevate the relative abundance of Acidobacteria in surface soil and Nitrosomonadaceae in subsurface soil, thereby tremendously optimizing the community structure. Moreover, Spearman’s correlation analysis was also performed, which proved the significant correlation of diversity with enzyme activity and AN concentration. Additionally, redundancy analysis disclosed that the Acidobacteria abundance in surface soil and Proteobacteria abundance in subsurface soil exerted conspicuous influence on environmental factors and the microbial community structure. According to the findings of this study, it was believed that reasonable nitrogen application together with an organic agriculture cultivation system could effectively improve soil fertility in Gaoyou City, Jiangsu Province, China. The whole world has witnessed the swift growth of its consumption . As one Being critical to material circulating, nutrient transformation, and the mineralization-fixation process of organic carbon, soil microorganisms act as sensitivity indicators of soil quality . It was −1, which is far lower than that of Japan (73 kg·kg−1), the Philippines (49 kg·kg−1), and Thailand  in surface and subsurface paddy soil were analyzed 2.2.1.−1 organic matter, 112.24 mg·kg−1 alkali-hydrolyzable nitrogen, 7.54 mg·kg−1 available phosphorus, 61.16 mg·kg−1 available potassium, and 1.32 g·kg−1 total nitrogen. The pH of the sample was 8.15. The test rice was of Nanjing 46 brand, with a whole growth period of 165 days. The experiment was conducted in the experimental plot of Mapengwan Ecological Agriculture Technology Co., Ltd., Gaoyou City, Jiangsu Province, China  from 2020 to 2021 . The plo2.2.CF and OF) and nitrogen level (N12 and N18) managements combined into four treatments, including the conventional farming with low nitrogen treatment (CFN12), conventional farming with high nitrogen treatment (CFN18), organic farming with low nitrogen treatment (OFN12), and organic farming with high nitrogen treatment (OFN18), respectively. These treatments were randomly arranged within each zone, and each treatment was repeated 3 times. There were 12 zones, each having an area of 49 m2 (7 m × 7 m). Previous crops were wheat, and straw returning was conducted in all the zones. The test rice was sown on 17th May and transplanted by hand on 9th June with row spacing of 30.0 cm × 12.5 cm. Three seedlings were planted per hole.The experiment involved farming pattern  was subjected to the high yield cultivation management according to local production rules. 45% compound fertilizer (containing 15%N) was applied as basal fertilizer 1 day before rice transplanting. Taking urea as topdressing nitrogen fertilizer, urea was applied 3 times as the first tillering fertilizer, the second tillering fertilizer and panicle fertilizer at a ratio of 1:2:3.via once turning-over of soil. Rapeseed cake (containing 4.60% N) and San’an bio-organic fertilizer  was performed as per the National Standards for Organic Product Production (GB/T19630.1). The milk vetch-rice planting model was adopted. Milk vetch (containing 0.33% N) was applied as basal fertilizer 2 weeks before rice transplanting 2.3.The 0–20 cm deep topsoil in each zone was collected by a soil extractor according to the five-point sampling method on October 21, 2021. Four types of soil were obtained, including CFN12, CFN18, OFN12, and OFN18. Each treatment was carried out 3 times. The soil samples were divided into surface soil (0–10 cm) and subsurface soil (10–20 cm). The samples were mixed evenly, from which sundries like rice root residues and stones were removed. A part of the samples (dry soil) were then air-dried and ground prior to filtering through 20-mesh and 100-mesh sieves. Lately, nutrients and enzyme activity of the samples were measured. The other part of the samples (fresh soil) were put into a sealed bag and stored in a refrigerator at −70°C for subsequent analysis of soil microorganisms.2.4.2SO4-mixed accelerator distillation method. Soil organic matter was measured by the K2Cr2O7-H2SO4 external heating approach. The available phosphorus in soil was detected using NaHCO₃ extraction spectrophotometry. The available potassium in soil was detected by CH3COONH4 extraction flame spectrophotometry. The soil pH was measured manually by the COMBI 5000 gauge in accordance with operation manual.The content of each soil nutrient was measured by the conventional analysis method . The alkThe enzyme activity of soil was detected using the method proposed by 2.5.0.5 g soil was taken from each sample and the microorganism DNA was extracted from the soil using the Omega soil DNA kit. The quantity and quality of the DNA extracted were assessed by ultraviolet spectrophotometer , while the DNA integrity was determined by 1.2% agarose gel electrophoresis. The target DNA sequence of bacteria for amplification was the 16S_V3V4 region. PCR amplification of 16S rDNA was carried out with 338F (5′-ACTCCTACGGGAGGCAGCA-3′) as the forward primer and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) as the reverse primer. The amplification system (25 μL) consisted of 5 × reaction buffer 5 μL, 5 × GC buffer 5 μL, dNTP (2.5 mM) 2 μL, forward primer (10 uM) 1 μL, reverse primer (10 uM) 1 μL, DNA template 2 μL, ddH2O 8.75 μL, and Q5 DNA polymerase 0.25 μL. The amplification conditions were pre-denaturation at 98°C for 2 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 5 min, and heat preservation at 10°C. The process was repeated for 25–30 cycles. After amplification, gel electrophoresis was conducted and 2% agarose was prepared to detect the PCR amplification products. Following QIIME2 dada2 analysis process, Illumina MiSeq sequencing was performed for quality control, denoise, mergence, and chimera removal. Shanghai Personalbio Technology Co., Ltd. was entrusted to conduct high-throughput sequencing of soil.2.6.Data were sorted and analyzed by Microsoft Excel 2019 and SPSS 23.0 software and mapped by Origin 8.5. Soil nutrients and enzyme activity data of various treatment modes were analyzed by the univariate method. Differences in data among groups were compared by LSD. Microbial community characteristics were mapped on the Genescloud platform. According to the distribution of amplicon sequence variants or operational taxonomic units (ASV/OTU) in different treatment modes, Alpha diversity of each treatment was calculated, and a rarefaction curve was drawn to assess whether the sequencing depth was reasonable. The Venn diagram was used to count the number of exclusive and shared ASV/OTU of different treatment methods based on ASV/OTU richness. Species composition analysis was made through assessing the feature table with singletons removed, and the phylum-level diversity was shown by a histogram. In non-metric multidimensional scaling (NMDS) analysis, the Bray–Curtis distance matrix was used for dimension reduction and decomposition, and differences in bacterial community composition were described with a two-dimension distribution figure. In LEfSe analysis, with the linear discriminant analysis (LDA) threshold set at 3.5, Kruskal-Wallis test was conducted to analyze simultaneously the difference among varied groups and identify notable biomarkers in each treatment. The correlation of α diversity indices with nutrients and enzyme activity was investigated by Spearman’s method. The relevance of dominant phyla with nutrients and enzyme activity was examined by redundancy method.3.3.1.3.1.1.As is shown in 3.1.2.As shown in 3.2.3.2.1.Bacterial richness was represented by Chao1 and Observed species indices, while bacterial diversity was denoted by Shannon and Simpson indices in this paper. 3.2.2.To investigate the exclusive and shared species of different treatment groups, the Venn diagram  was plot3.2.3.NMDS analysis can simplify the data structure, describe the distribution of samples at a given distance metric, and represent clearly the difference in the bacterial community structure among varied treatment groups. As shown in 3.2.4.The relative abundance of phyla in each treatment group was analyzed after high-throughput sequencing of the bacterial community. The top 5 dominant phyla were compared among different groups. In subsurface soil , the top3.2.5.CF groups. The most influenced species in CFN12, OFN12, and OFN18 type soil were g_SC_I_84, p_Acidobacteria, and f_GAL15, respectively.Biomarkers that were tremendously different among four treatment groups were identified by LEfSe analysis (species with abundance lower than 0.0015 were removed). Results were concluded in a cladogram and a histogram of the LDA scores. The cladogram represents the phylum to genus from the innermost ring to the outermost ring. The diameter of small circles is proportional to the relative abundance of the species, and the biomarkers with significant difference are colored the same as the group. The length of a row in the histogram represents the LDA score, namely, the influence of the biomarker. It was found that in surface soil , f_SC_I_In subsurface soil , the enrIt is noteworthy that among all biomarkers, p_Acidobacteria with the highest relative richness in surface soil and f_Nitrosomonadaceae with the highest relative abundance in subsurface soil were both present in the OFN12 group.3.3.3.3.1.The relationship between soil environmental factors and bacterial α diversity was analyzed by the Spearman’s algorithm . The resIn subsurface soil , a signi3.3.2.p = 0.39) had small influence on community composition. Acidobacteria was positively associated with AN, TN, AP, SOM, Alkaline phosphatase, and Sucrase , while Proteobacteria was negatively related to these factors. There was slight correlation between Chloroflexi and most environmental factors.The relationship of top 10 phyla with environmental factors was investigated by redundancy analysis. The results are summarized in p = 0.07) might impact community composition, but all the other environmental factors had little effect on community composition, with r2 ranging between 0.01 ~ 0.37 and p between 0.13 ~ 0.98. Proteobacteria was positively correlated to AN, TN and SOM, but negatively correlated to other environmental factors. Chloroflexi was not related to a majority of the environmental factors.In subsurface soil , RDA1 an4.4.1.+  can be found at: CX: validation, formal analysis, investigation, data curation, and writing – original draft. YC: validation, investigation, and data curation. QZ: investigation. YL: methodology and investigation. JZ: investigation. XL: investigation. MJ: supervision. HZ: resources and project administration. LH: term, conceptualization, methodology, resources, and writing – review and editing. All authors contributed to the article and approved the submitted version.This study was financially supported by the National Key R&D Program of China Under Grant, projects number 2017YFD0300102. Modern Agricultural Development Projects of Jiangsu Province, projects number 2019-SJ-039-08-11.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.