Chenopodium quinoa Willd.) is a herb within the Quinoa subfamily of Amaranthaceae, with remarkable environmental adaptability. Its edible young leaves and grains are rich in protein, amino acids, microorganisms, and minerals. Although assessing the effects of fertilization on quinoa yield and quality has become an intensive area of research focus, the associated underlying mechanisms remain unclear. As one of the three macro nutrients in plants, potassium has an important impact on plant growth and development. In this study, extensive metabolome and transcriptome analyses were conducted in quinoa seedlings 30 days after fertilizer application to characterize the growth response mechanism to potassium. Quinoa (The differential metabolites and genes present in the seedlings of white and red quinoa cultivars were significantly enriched in the photosynthetic pathway. Moreover, the PsbQ enzyme on photosystem II and delta enzyme on ATP synthase were significantly down regulated in quinoa seedlings under potassium deficiency. Additionally, the differential metabolites and genes of red quinoa seedlings were significantly enriched in the arginine biosynthetic pathway.These findings provide a more thorough understanding of the molecular changes in quinoa seedlings that occur under deficient, relative to normal, potassium levels. Furthermore, this study provides a theoretical basis regarding the importance of potassium fertilizers, as well as their efficient utilization by growing quinoa seedlings.The online version contains supplementary material available at 10.1186/s12870-022-03928-8. Chenopodium quinoa Willd, also known as quinoa, South American quinoa, and Indian wheat, among other names, is a dicotyledonous plant that belongs to the subfamily Quinoa of the family Amaranthaceae. According to Food and Agriculture Organization, quinoa has rich and uniform nutritional value and is capable of meeting many of the nutritional needs of humans. Additionally, it has been described as an excellent “functional food” with the capacity to reduce the risk of various diseases [diseases –3. Quinodiseases –6. Concodiseases .Given that potassium (K) is present in nearly 5% of all higher plant tissues, it is considered an essential macronutrient. Although it is not a structural component of organic compounds, it is closely related to plant growth, development, and metabolism, with a particularly important impact on plant yield, quality, and stress resistance. For instance, K is a necessary component of the stomatal opening mechanism and, therefore, directly impacts stomatal conductance and photosynthesis, phloem transport, and cell ion balance. Moreover, K can promote chlorophyll synthesis in leaves, thereby improving the structure of chloroplasts and promoting plant photosynthesis, while also promoting the transportation and storage of various nutrients, increasing the sugar and starch contents in the crop, and promoting oil formation . ImportaUnder an unlimited K supply, the transport rate in plant vascular tissues is increased, plant metabolism is promoted, and plant resistance to lodging, disease and insect attack, and drought and cold are effectively enhanced , 11–14. Numerous studies have characterized the relationships between fertilizers and metabolism, including the responses of maize and barley to low nitrogen stress , 17; com2O gradient of 0 , 112.5 , or 337.5 kg/hm2 . R2 and W2 represented the control treatments. Collectively, our findings provide theoretical support for the research of high-yield cultivation technology for quinoa.In this study, we aimed to determine the effect of K fertilizer rate on metabolome and transcriptome profiles of quinoa and analyze the response mechanism of quinoa seedlings to K fertilizer application through correlation analysis. To avoid one-sided results of a single cultivar, two quinoa cultivars, namely, red (dianli-1299) and white (dianli-71), were analyzed in this study. Each cultivar was treated under three conditions with a KThe superposition diagram Fig. Sa.b for tSample metabolites were qualitatively and quantitatively detected using extensive targeted metabolome analysis. Eighteen samples were divided into six groups; biological triplicates were included for each sample. A total of 1057 metabolites were detected based on the Ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) detection platform and a self-built database. Among them were 98 amino acids and their derivatives, 166 phenolic acids, 69 nucleotides and their derivatives, 172 flavonoids, 24 lignans and coumarins, 9 tannins, 78 alkaloids, 39 terpenoids, 94 organic acids, 177 lipids, 11 quinones, and 120 other metabolites , the top 10 DEMs in R2 vs R6 primarily included organic acids. The top included four organic acids; additionally, three of the top 10 downregulated metabolites were phenolic acids, three were sugars and alcohols, two were organic acids, and one corresponded to lipids, nucleotides, and their derivatives  in the samples was concentrated in log10 2.5 Fig. a. Furthe2.5 Fig. b, sample2.5 Fig. c, and cl2.5 Fig. d, the biTo assess gene expression patterns under different K supply levels, we first centralized and standardized the FPKM of genes and then performed K-Means cluster analysis. Considering that the same types of genes exhibited similar expression trends under different experimental treatments, we hypothesized that they might share similar functions Fig. S. AccordiThe results of KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation of DEGs revealed that the DEGs of each group were primarily enriched in “cellular process,” “environmental information process,” and “genetic information process.” Moreover, the common DEGs between R2 vs R6 and W2 vs W6 were mainly enriched in “photosynthesis,” “photosynthetic antenna protein,” “arginine biosynthesis,” and “carbon metabolism pathway.” Meanwhile, the common DEGs between R2 vs R7 and W2 vs W7 were largely enriched in “pentose phosphate pathway” .Fig. 7PIn R2 vs R6, R2 vs R7, and R6 vs R7, DEGs were also enriched in the “arginine biosynthesis pathway.” Furthermore, the three groups shared three key metabolites in this pathway, namely, N-acetylcholine, citrulline, and arginine, which were downregulated in R2 vs R7 and R6 vs R7 and upregulated in R2 vs R6.chlorophyll a, the content of R6 was significantly higher than R2 and R7, and the lowest in R7; The content of chlorophyll a in W6 was significantly higher than that in W2 and W7, and the lowest was W7. In chlorophyll b, the content of chlorophyll b in R6 was significantly higher than that in R2 and R7, and there was no significant difference between R2 and R7 groups; the content of chlorophyll b in W6 was the highest and significantly higher than that in W7, but the difference between W6 and W2 was not significant. The total chlorophyll content of R6 was significantly higher than that of R2 and R7, and the lowest was R7. The total chlorophyll content of W6 was significantly higher than that of W2 and W7, and the lowest was W7. Through the detection of chlorophyll content, we found that the expression trends of chlorophyll a, chlorophyll b, and total chlorophyll of different varieties were consistent under the same treatment, and the chlorophyll content would be significantly reduced in the case of potassium deficiency.Through the comparison of the six treatments Fig. , it can Tub1 was selected as the internal reference gene. Table STo determine the authenticity and reliability of transcriptome data and extent of differential candidate gene expression, RT-qPCR verification was performed on several key genes. 2 assimilation during photosynthesis [In the current study, the DEMs and DEGs identified in the potassium-deficiency treatment and control treatment groups were significantly enriched in the photosynthetic pathway. Moreover, we show that the photosynthetic system of quinoa is impacted by potassium deficiency; inhibition of this system leads to a significant decrease in the accumulation of organic matter, which is not conducive to normal plant growth, and may partially account the dwarfism of potassium-deficient crops. Meanwhile, Naciri et al.  showed tynthesis , which i+) gradient is established on either side of the thylakoid membrane through water oxidation and PQB2− reduction. ATP synthase, also known as FoF1 ATPase, then catalyzes the synthesis of energy-rich ATP. During respiration or photosynthesis, the energy released through the electron transport chain is first converted into the transmembrane proton (H +) ladder, along which the proton flows. ADP + PI can synthesize ATP through ATP synthase [In this study, red and white quinoa were grown under three different K fertilization conditions, among which, R2 vs R7 and W2 vs W7 showed no common significant pathway differences; hence, our analysis focused on R2 vs R6 and W2 vs W6, the metabolome and transcriptome results for which revealed enrichment of the photosynthetic pathway as the most significant. In particular, R2 vs R6 and W2 vs W6 shared three common enzymes in the photosynthetic pathway, namely, PsbQ, delta, and atpB, which we speculated to be key enzymes affecting photosynthetic carbon fixation under potassium deficiency. PsbQ is a photosystem II enzyme, while both delta and atpB are components of ATP synthase. PsbQ and the delta subunit were significantly downregulated in the R2 vs R6 analysis compared with W2 vs W6. The function of photosystem II is to use the absorbed light energy to split a water molecule and transfer the released electron to plastid quinone through the electron transport chain. Concomitantly, a proton  nitrogen to carbon (N/C) ratio and performs the most functions in plants. In fact, it is not only a component of proteins and a medium for N storage and transport but also an osmotic regulator. Additionally, arginine participates in proline and NO synthesis through the urea cycle and citrulline NO cycle .Arginine synthesis and its regulation have been well studied in prokaryotes, fungi, and animals –39; in cN-Acetylcholine is an intermediate in the enzymatic synthesis of L-arginine . CitrullIn this study, we discussed a response mechanism of quinoa seedlings under potassium deficiency, the primary components of which included photosynthesis and arginine biosynthetic pathway. When quinoa seedlings are deficient in potassium, the PsbQ enzyme in photosystem II and delta subunit of ATP synthase become significantly downregulated; the subsequent series of chain reactions reduce the photosynthetic efficiency. In addition, the identified differential genes and metabolites are significantly enriched in the arginine biosynthetic pathway; arginine and its derivatives have important roles in the resistance of quinoa to low potassium stress. Therefore, we speculate that altering the photosynthesis pathway is a passive response of quinoa seedlings when exposed to low potassium stress, whereas enriching arginine and its derivatives is an active response to these conditions.Collectively, the findings of this study provide novel insights into the molecular mechanisms initiated by quinoa seedlings in potassium-deficient and normal conditions, thereby, describing a new pathway by which quinoa establishes resistance to low potassium stress. 2O treatment rates applied to two lines were 0 , 112.5 , and 337.5 kg ha−1 . The P2O5 and N fertilization rates for all K treatments were both 112.5 kg ha−2 with three repetitions. Conventional water management was used during the experimental period . Fertilization was initiated when seedlings reached the second-leaf stage. The aboveground portions of quinoa seedlings were randomly sampled 30 days after fertilizer application .Two high-generation cultivars of red (dianli-1299) and white Chenopodium (dianli-71) independently bred by Yunnan Agricultural University were planted in the modern Agricultural Education and Scientific Research Base of Yunnan Agricultural University in Xundian County, Kunming . Uniform grains were selected and evenly planted in pots (117 cm × 39 cm × 65 cm). The KThe biological samples were placed in a freeze dryer (Scientz-100F) for vacuum freeze-drying, and the samples were ground  to a powder with a grinder . For analysis, 100 mg of sample powder was weighed and dissolved in 1.2 ml of 70% methanol for extraction. They were then vortexed once every 30 min for 30 s, six times in total, and placed at 4 °C overnight. After centrifugation for 10 min at 12,000 rpm, the supernatant was absorbed and filtered through microporous filter membranes (0.22-μM pore size) prior to storing in injection bottles for UPLC-MS/MS analysis.www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions were as follows: UPLC: column, Agilent SB-C18 . The mobile phase comprised solvent A, pure water with 0.1% formic acid, and solvent B, acetonitrile with 0.1% formic acid. Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and maintained for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.10 min and maintained for 2.9 min. The flow velocity was set as 0.35 ml/min. The column oven was set to 40 °C, and the injection volume was 4 μl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.The sample extracts were analyzed using an UPLC-ESI–MS/MS system , mzVault, and MassList databases to obtain accurate qualitative and relative quantitative results. Python (Python version 2.7.6), R statistical software (r version r-3.4.3), and CentOS (CentOS release 6.6) were used for statistical analysis. When the data were not normally distributed, an area-normalization method was applied.The raw data file generated using UPLC-MS/MS was processed with Compound Discoverer 3.1 ; the peaks represent the collection and quantification of each metabolite. Retention time tolerance, actual quality tolerance, signal strength tolerance, signal-to-noise ratio, and minimum strength were 0.2 min, 5 ppm, 30%, 3, and 100,000, respectively. Peak intensities were normalized to the total spectral intensity. Based on the molecular formula prediction of additive ions, the ion and fragment ion peaks of normalized data were used, and matched with mzCloud (http://www.genome.jp/kegg/), HMDB database (http://www.hmdb.ca/), and LipidMaps database (http://www.lipidmaps.org/). Metax is a flexible and comprehensive metabolomics data processing software that can support PCA and partial least-squares discriminant analysis (PLS-DA). Statistical significance  was calculated by univariate analysis (t test). Metabolites with VIP > 1, p < 0.05, fold change (FC) ≥ 2 or ≤ 0.5 were selected as DEMs. Pearson’s correlation coefficients were calculated among all metabolites to identify relationships among all metabolites.Metabolites were annotated using the KEGG database . RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system .The library was prepared for transcriptome sequencing by first employing fastp (v0.19.3) to filter the original data and remove reads with adapters. When the N content in any sequencing read exceeded 10% of the base number of reads or when the low-quality (Q ≤ 20) base number contained in any sequencing read exceeded 50% of the total base read number, the paired reads were removed to obtain clean reads. Next, the reference genome and its annotation files were downloaded from the designated website, the index was constructed using HISAT (v2.1.0), and clean reads were aligned to the reference genome. StringTie (v1.3.4d) was used to predict new genes. StringTie applied a network flow algorithm and optional de novo assembly to splice transcripts. Additionally, featureCounts (v1.6.2) was employed to calculate the gene alignment and the FPKM of each gene according to the gene length. Differential expression analysis between two groups was performed using DESeq2 (v1.22.1), and p-values were corrected using the Benjamini & Hochberg method. The corrected p-value and | log2 foldchange | were established as the thresholds for significant differential expression.For KEGG enrichment analysis, hypergeometric distribution testing was performed for the pathway unit. For GO, the analysis is based on GO terms.The batch data from the transcriptome and metabolome analyses were normalized and statistically analyzed to establish the relationship between molecules at different levels. Using combined biological function analysis, metabolic pathway enrichment, and correlation analysis, we systematically analyzed the biological molecular functions and corresponding regulatory mechanisms to develop a comprehensive understanding of the general trend and direction of biological variation. Finally, we proposed a model of the mechanism accounting for molecular biological variation and selected key metabolic pathways or genes and metabolites for subsequent in-depth experimental analysis and application.Tub1 gene was used as the internal control gene, and the relative transcription level was calculated using the 2−∆∆CT method.RNA was extracted from the aboveground portion of treated quinoa seedlings for RT-qPCR. The specific primers for the 15 genes were designed by Beacon Designer 7.9. RT-qPCR was performed on a 96-well StepOnePlus instrument  using PerfectStart™ SYBR qPCR SuperMix , according to manufacturer’s instructions. The reaction system comprised a 20 µL volume including 10 µl of 2 × PerfectStart™ SYBR qPCR SuperMix, 0.4 µL of calibration solution, 5.8 µL of nuclease-free water, 0.4 µL of each primer (10 mm), and 3 μL (200 μg/μL) of cDNA. The thermal cycle was as follows: 94 °C (30 s), 94 °C (5 s) 40 cycles, 60 °C 30 s. The Additional file 1:Figure S1. a,b QC sample mass spectrometry TIC overlap diagram. Figure S2 kmeans cluster analysis diagram. Figure S3. KEGG channel histogram a) R2 vs R6; b) W2 vs W6; c) R2 vs R7; d) W2 vs W7. Table S1. Analysis of metabolites. Table S2. Pecific primer pairs of selected genes.


Compared with control, ETH treatment not only accelerated the breakdown of chlorophyll with higher activities of Chlase and MDCase but also induced the synthesis of carotenoid and anthocyanin with higher activities of PAL, CHI, DFR, and UFGT. Moreover, the changes in DFR and UFGT activities coincided with the increase in ETH concentration. Further, correlation analysis showed that the production of endogenous ethylene induced by ETH was significantly negatively correlated with firmness and chlorophyll content, whereas positively correlated with MDA content and anthocyanin content. This study suggests that the positive effect of ETH on “Guifei” mango color transformation is concentration-dependent within a certain concentration range. Anthocyanin is the main pigment for the red formation of “Guifei” mango, and DFR and UFGT may play critical roles in anthocyanin synthesis. ETH promoted the red coloration by promoting the release of endogenous ethylene and enhancing the activities of anthocyanin synthesis enzymes.Due to geographical location and climatic factors, postharvest storage and preservation of tropical fruits and vegetables are still facing huge challenges. Ethephon (ETH) is widely used as an ethylene donor to achieve the commercial color and flavor of climacteric fruits. However, the effect of ETH on fruit coloration was affected by many factors, such as fruit species, plant hormones, and storage conditions. In this study, the main mango variety “Guifei” in Hainan, China, was used to study the effects of different concentrations of ETH on fruit ripening and coloration during storage at 25°C. Results showed that postharvest treatment with ETH (300, 500, and 900 mg·L Mangifera indica) is very popular because of its pleasure color, characteristic aroma, delicate flavor, and rich nutrition phosphonic acid, ETH] serves as one of the artificial analogs of ethylene and the role of ETH in fruit ripening has been reported in extensive literature . PreviouFruit color is an important trait contributing to fruit quality and market value. Mango fruits are generally classified into green, yellow, and red types based on peel color. Carotenoids and anthocyanins are the important pigments responsible for the yellow or red color of fruits . ChloropAnthocyanin is another photosynthetic pigment responsible for the color of fruits . The bioFaUFGT and FaANS genes  fruits used in this study were picked up at the mature green stage from a commercial orchard located in Baise City, Guangxi province of China. To avoid mechanical injury, harvested mango fruits were packed in plastic crates, wherein the fruit of each layer was separated with soft fabric, and air-transported to the postharvest laboratory with air conditioning at 25°C and 75–80% relative humidity (RH). Only well-formed fruits with uniform maturity, size, and color were soaked with 0.1% sodium hypochlorite, dried at room temperature, and used for further experiments.All mango . Then, each group was divided into two categories: one category (10 fruits) was used for monitoring the color change of mango fruit on 0, 3, 6, 9, 12, and 15 days after treatment. The other category (about 120 fruits) was used for the measurement of related physiological parameters.All mango fruits were divided into four groups, and each group consisted of 130 mangoes. The four groups were immersed in 0 (water as control), 300, 500, and 900 mg·LMango flesh and peel were separated at each sampling time point. Subsequently, each fresh sample was divided into two parts: one part was used for measurement of the accumulation of MDA and pigments; the other part was frozen in liquid nitrogen, ground into powder, and kept at −80°C to further measure the activities of ACS, ACO, and pigment metabolizing enzymes. Each treatment comprised three replicates and each replicate comprised nine mangoes.Fruit firmness was measured using a handheld penetrometer  equipped with a 5-mm diameter probe. Two opposite points on the equator axis of each peeled fruit were monitored and firmness was expressed as force in newtons (N).g for 30 min at 4°C. The supernatant (2 mL) was added to 3 mL of 10% TCA with 0.5% thiobarbituric acid, the mixture was rapidly put into a boiling water bath for 10 min, and then centrifuged at 12,000 × g for 30 min. The absorbances of the supernatant at 532 and 600 nm were used for MDA content.MDA content was determined based on the protocol reported by Lin et al.  with minTwo mango fruits were put in a 10-L sealed jar for 2 h at 20°C to collect the released ethylene. A 1-mL gas mixture was extracted to test the production of ethylene with a gas chromatograph (GC) system  equipped with TG-5MS column (30.0 m × 0.32 mm × 0.25 μm) and a hydrogen ion flame detector. Each treatment comprised three replicates, and each replicate was sampled and measured three times.2HPO4/KH2PO4 buffer (pH = 8.5), containing 10 μM pyridoxal-5-phosphate (P-5-P), 5 mM dithiothreitol (DTT), 1 mM Na2-EDTA, and 5% (m·v−1) polyvinylpyrrolidone (PVP) at 4°C. The mixture was centrifuged at 9,000 × g at 4°C for 15 min. After that, 1 mL supernatant was mixed with 1.5 mL of 50 mM HEPES-KOH buffer (pH = 8.5), containing 10 μM P-5-P, 0.25 mM S-adenosyl methionine (SAM), and 1% Triton X-100. The mixture was incubated in a 10-mL sealed test tube at 30°C for 30 min. Finally, 1 mL gas was withdrawn from the headspace to analyze the level of ethylene by GC. One unit (U) of ACS activity was defined as the amount of enzyme that produced 1 nM C2H4 g−1 fresh weight min−1.The activity of ACS was measured according to the methods in Liu et al.  with som−1) glycerin, 5% (m·v−1) PVP, 5 mM DTT, 0.1 mM FeSO4, and 30 mM sodium ascorbic acid. The mixture was centrifuged at 9,000 × g for 15 min at 4°C. Afterwards, 1 mL supernatant was mixed with 3 mL of 0.1 M Tris-HCl (pH = 7.5), containing 30 mM NaHCO3, 10% (m·v−1) glycerin, 0.1 mM FeSO4, 1 mM ACC, and 30 mM sodium ascorbic acid. The reaction solution was incubated at 30°C for 1 h, and then 1 mL gas was collected from the headspace for measuring the production of ethylene by GC. One unit (U) of ACO activity was defined as the amount of enzyme that produced 1 nM C2H4 g−1 fresh weight min−1. Activities of ACS and ACO were expressed as U·g−1·min−1.ACO activity was measured according to the methods in Zhang et al.  with somL*, a*, and b* at each site were recorded with a chromameter . L* represents the degree of lightness and its values generally stay within a range from 0 to 100; a* represents redness (+) and greenness (-) on the positive and negative axes; b* represents yellowness (+) and blueness (-) on the positive and negative axes. Each time point comprised six mangoes.Fruit color was measured at three evenly distributed equatorial sites of every fruit. g for 30 min. After centrifugation, the pigment solution was mixed with 80% acetone and brought to a final volume of 10 mL. Contents of chlorophyll and carotenoid were calculated according to the absorbances at 470, 645, and 663 nm.The contents of chlorophyll and carotenoid were measured with an ultraviolet spectrophotometer  according to the methods in Xie et al.  with minAnthocyanin content was determined based on the methods reported by Zhang et al.  with som−1) TritonX-100, 30 g·L−1 PVP, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 5 mM L-cysteine. The mixture was homogenized on ice and centrifuged at 12,000 × g for 20 min at 4°C. Activities of Chlase and MDCase were assayed based on the absorbance at 667 and 692 nm, respectively.The crude enzyme solution of Chlase and MDCase was extracted according to the methods described by Fukasawa et al.  with min2HPO4/KH2PO4 (pH = 7.0), containing 0.05 M ascorbic acid and 0.018 M β-mercaptoethanol. For DFR and UFGT, 0.5 g of peel powder were homogenized with 0.1 M Tris-HCl (pH = 7.5), containing 0.01 M ascorbic acid, 0.005 M dithiothreitol, 0.1% β-mercaptoethanol, and 0.01 M PMSF. The mixture was centrifuged at 12,000 × g for 20 min at 4°C. After centrifugation, the enzyme extract was concentrated with ammonium sulfate. Activities of the four enzymes were assayed following the methods described in Chen et al.  method according to our previous reports . The inthttps://primerexplorer.jp/e/), and the primers used in this study are listed in 3 G Real-Time PCR System  was used for RT-qPCR. The thermal cycling protocol was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The relative expression was calculated using the 2−ΔΔCt method  according to its manufacturer's instructions. The primers were designed using the Primer explorer v5 online website (t method . Each saP < 0.05). Correlation analysis was carried out via OriginPro 2021  with differences being considered significant .All data were presented as mean ± standard error (SE). Duncan's multiple range test was used for statistical evaluation using SPSS statistics 20.0  with differences being considered significant . Like the results of peel color, the ETH-mediated softening process of mango fruit was positively correlated with its concentration. For example, the firmness of 300 mg·L−1 ETH-treated fruit at 3 days was significantly lower than those of 500 and 900 mg·L−1 ETH-treated fruit (P < 0.05).In this study, firmness and MDA content were used as two important indicators of fruit ripening. After treatment, the firmness of the control fruit were 15.74 N and 13.23 N at 3 and 6 days, respectively . However−1 ETH-treated groups exhibited significantly higher contents of MDA compared with control during the whole storage time (P < 0.05).As shown in −1·min−1. Compared with control, ETH-treated fruit exhibited earlier and stronger production of endogenous ethylene, which peaked at 6 days with 140.16 μL·g−1·min−1, 143.87 μL·g−1·min−1, and 149.41 μL·g−1·min−1, respectively (P < 0.05). In addition, ETH-induced production of endogenous ethylene was closely related to its application concentration. As shown in −1 ETH-treated fruit exhibited significantly higher production of endogenous ethylene than the other two ETH-treated groups.After treatment, the production of endogenous ethylene in the control fruit peaked at 9 days with 131.36 μL·gP < 0.05). Similar to ACS, ACO activity of control and ETH-treated groups all peaked at 6 days, but ETH-treated fruit showed significantly higher activities of ACO at 3, 6, 9, and 12 days than control fruit (P < 0.05). The enhanced activities of ACS and ACO contributed to an earlier and stronger accumulation of endogenous ethylene in mango fruit . ETH-treated fruit exhibited significantly lower levels of chlorophyll from 3 to 15 days. However, the chlorophyll content of the control fruit showed a relative high level until 6 days. During the mid-storage time, the chlorophyll content of fruit treated with 900 mg·L−1 ETH was significantly lower than those of the other two ETH-treated groups. At the end of the storage period, there was no significant difference in chlorophyll content among the three ETH-treated groups (P < 0.05).Compared with control, the fruits treated with ETH all showed significantly decreased content of chlorophyll during the whole storage time (P < 0.05). The highest content of carotenoid was found in fruit treated with 900 mg·L−1 ETH at 15 days. Similar to carotenoid, ETH treatment increased the accumulation of anthocyanin in mango fruit (P < 0.05). In addition, 900 mg·L−1 ETH-treated fruit exhibited the highest anthocyanin content at 15 days.In general, the content of carotenoids in the four groups slowly increased after treatment. Carotenoid contents of three ETH-treated groups were significantly higher than that of control from 6 to 15 days . Furthermore, the MDCase activity of each group showed an increasing trend during the first 6 days, followed by a decrease from 6 to 12 days (P < 0.05).In this study, control and ETH-treated fruit showed a similar change trend of Chlase activity, which peaked at 6 or 9 days, and then slowly decreased . The fru 12 days . At 6 da−1 ETH treatment was most effective in activating the activity of PAL. However, there was no difference in PAL activity between 300 and 500 mg·L−1 ETH treatment groups (P < 0.05). As shown in P < 0.05), but the control fruit exhibited significantly higher activity of CHI than ETH-treated fruit at 15 days (P < 0.05).PAL, CHI, DFR, and UFGT were four critical enzymes associated with the synthesis of anthocyanin. Compared with control, ETH treatment significantly increased the activity of PAL during the first 9 days, and 900 mg·L−1 ETH-treated fruit was significantly higher than those in 500 and 300 mg·L−1 ETH-treated fruit at 6 days (P < 0.05). From 9 days, the control fruit exhibited significantly higher activity of DFR compared with ETH-treated fruit (P < 0.05). Generally, UFGT activity of the four groups all peaked at 9 days, but UFGT activity in ETH-treated fruit was significantly higher than that in control fruit during the whole storage time (P < 0.05).Similar to Chlase, the activity of DFR in ETH-treated fruit peaked at 6 days, while the peak of control fruit occurred at 9 days. Moreover, DFR activity in 900 mg·LMiACS2, MiACO2, and MiACO3. Moreover, ETH treatment significantly upregulated the relative expression of MiChl2 and MiMDC2, which were involved in the degradation of chlorophyll. ETH-treated mango fruit exhibited higher expression of MiPAL4, MiCHI3, MiDFR1, and MiUFGT4, which contributed to the synthesis of anthocyanin.As shown in R2 value between ethylene production and activity of ACS or ACO were 0.89 and 0.85, respectively  between them was 0.71 (P < 0.01). Chlorophyll content was negatively correlated with a* and b*, and the R values were −0.87 and −0.61, respectively (P < 0.05). Moreover, the R-value between the production of endogenous ethylene and chlorophyll content was −0.95 (P < 0.001). These results supported that the production of endogenous ethylene was positively correlated with a* and b*, and the R values between them were 0.87 and 0.63 (P < 0.05), respectively. The R-value between the content of carotenoid and a* was 0.67, but the R-value between the content of anthocyanin and a* was 0.83.As shown in P < 0.001). The R values between anthocyanin content and activities of anthocyanin metabolizing enzymes, including PAL, CHI, DFR, and UFGT were 0.84, 0.92, 0.91, and 0.91 (P < 0.001), respectively. Moreover, the production of endogenous ethylene was also significantly positively correlated with the activities of pigment metabolizing enzymes (P < 0.001). In addition, the activity of Chlase and MDCase was negatively correlated with chlorophyll content, and the R-value was −0.84 and −0.86, respectively (P < 0.001).Furthermore, the production of endogenous ethylene was significantly positively correlated with the content of anthocyanin, and the R-value was 0.91 , the Construction Program of Preservation Technology Association for Tropical Fruits and Vegetables in Hainan Province (Grant No. HDLM201901), and the Start-Up Grant Program in Hainan University (Grant No. KYQD(ZR)-22127).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. 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Solanum lycopersicum L.) were investigated via field trial. In addition, the application prospect in China was also evaluated. In the experiment, five treatments with three replications and a randomized complete block design were followed. The treatments were: furrow application of fluopimomide  at the dosage of 375, 750, and 1,500 g ha−1, which was recommended, double recommended, and quadruple recommended dosages, respectively. Besides, common control fungicide fluopicolide (5% SC) furrow was applied at recommended application dosages of 750 mL ha−1, and a non-treated control was also undertaken. Results indicated that fluopimomide exhibited no effects on the amount of soil bacteria and actinomycetes, and its inhibition effect on fungi amount could be recovered at 60 days after treatment (DAT). With the recommended application dosage, fluopimomide could efficiently reduce the number of plant pathogens in soil by 79.56–85.80%, significantly reduce the disease incidences in tomato plants by 80.00–88.24%, and improve plant height by 13.25–24.05% and marketable yield by 16.88%. Furthermore, soil enzymes exhibited a complex response to fluopimomide, and AOB and nifH gene copy numbers were increased by the double and quadruple recommended dosage of fluopimomide. Based on the above results, fluopimomide could be recommended as an efficient fungicide for the tomato field.Fluopimomide is a novel acid amide fungicide registered for the control of many plant pathogens. In the present study, the effects of soil-treated fluopimomide on soil micro biomass, disease incidence, plant growth, soil enzyme activity, and marketable yield of tomato ( Ltd, China in 2010, it has a similar structure to fluopicolide  Figure . Its chenzamide) . In Chin control . Taking in vitro bioassays, and exhibited EC50 values of 0.97, 2.36, and 3.59 μg mL−1 for B. cinerea, Phytophthora, and F. oxysporum, respectively [F. oxysporum, Phytophthora, and B. cinerea, (b) to investigate its micro-ecology effect on tomato planted soil, and (c) to identify the influence of fluopimomide on tomato plant growth and marketable yield.In the previous study, bactericidal spectrum of the innovative fungicide fluopimomide was measured by ectively . However22.1Fluopimomide (purity = 98%) and fluopicolide (purity = 96%) were both provided by Shandong United Pesticide Industry Co. Ltd, China.2.2Field experiments were arranged in the autumn cropping seasons of 2018–2019 and 2019–2020 in a commercial greenhouse near Fang county, Tai’an, China . Six-week-old “Jinpeng” tomato seedlings were transplanted on August 23, 2018 and August 18, 2019.−1 soil, and pH 7.1. This site suffered heavily from soil-borne diseases, such as F. oxysporum, Phytophthora, and B. cinerea. In conventional farm operations, compound fertilizer of 15N–15P2O5–15K2O was broadcast applied at the dosage of 750 kg ha−1 as base.The soil physicochemical properties of the experimental sites were: sand 29.17%, silt 70.49%, clay 0.34%, organic matter 18.95 g kg−1, it is the field recommended application dose; (b) fluopimomide  furrow-application dose of 750 g ha−1; (c) fluopimomide  furrow-application dose of 1,500 g ha−1; (d) laboratory-made fluopicolide  furrow-application dose of 750 mL ha−1, it is the field recommended dose; and (e) non-treated control. Evaluation of 2 and 4 folds of recommended application dosage is necessary for novel pesticide crop safety verification.Five treatments with 3 replicates in each were arranged in a random block design, where 25 seedlings were planted per plot (7.20 m × 0.75 m). The treatment programs were: (a) laboratory-made fluopimomide  furrow-application was done at a dosage of 375 g ha2.320, 40, and 60 DAT, a soil column cylinder with a diameter of 5 cm was used to sample soil nearby tomato plants at a depth of 0–10 cm, and random 20 points were included for each plot. Each collected soil sample (0.5–1.0 kg) was sieved (1 mm mesh) and separated into two parts. Part one was stored at 4°C for the microbiological and enzyme activities test, and the test was completed within less than a week. Part two was treated with liquid nitrogen and stored at −80°C for DNA extraction.2.4−1 dilution. Fungi, including yeast, were counted on Martin’s medium, with pH 6 and containing 150 mg L−1 streptomycin. Bacteria were counted on a selective medium prepared with glucose 1 g L−1, proteose peptone 3 g L−1, yeast extract 1 g L−1, K2PO4 1 g L−1, agar 15 g L−1, and cycloheximide 100 mg L−1. Actinomycetes were counted on improved GAO No.1 medium with pH 7.4–7.6 [F. oxysporum in soil was counted on the PCNB selective medium containing KH2PO4 1.0 g L−1, MgSO4·7H2O 0.5 g L−1, peptone 5.0 g L−1, agar 20.0 g L−1, with streptomycin 0.30 g L−1 and 75% quintozene wettable powder 1.0 g L−1 added before usage [Phytophthora was measured in a PDA medium with 3-hydroxy-5-methylisoxazole, benomyl, nyastatin, pentachloronitrobenzen, rifampicin, and ampicillin added at the concentration of 100, 10, 25, 25, 10, and 50 mg L−1, respectively [B. cinerea selective medium included basic components  as well as fungicide components  added after sterilization [−1.The amounts of fungi, bacteria, and actinomycetes were evaluated using the serial ten-fold dilution (10-2–10-7) method , where 4 7.4–7.6 . The numre usage . The amoectively . The B. 2.5After the transplant, incidences of wilt, blight, and gray mold of ten random plants in each plot were recorded at intervals of 20, 40, and 60 days. Levels of disease severity were assessed by visually estimating the percentage of diseased surface, and graded as: 0 = 0%, 1 = 1–9%, 2 = 10–24%, 3 = 25–49%, 4 = >50% of surface affected. Wilt was calculated by spot on stem base, while blight and gray mold were weighted by round and angle spots on leaves, respectively. The disease index was calculated using the following formula:2.6Analysis of soil dehydrogenase and urease activity was done according to the methods of Lebrun et al. . Soil ph2.7nifH, and nirS was performed with ABI 7500 Real-Time PCR System and 7500 System Software-SDS 2.2 by absolute quantification method, and the primer information is shown in −1, and cDNA template 2.5 μL. The procedure of qPCR was performed at 95°C for 15 min and followed by 40 cycles of 95°C for 10 s, annealing  for 20 s, and 72°C for 1 min.The qPCR analysis of AOA, AOB, 2.820, 40, and 60 DAT, the plant height of randomly chosen ten plants in each plot was measured. Fruits were harvested twice (85 and 120 DAT) when they were mature, and the marketable yield was calculated.2.9p < 0.05.As there were no significant differences between experiments over the 2 years, data from the two experiments were combined for analysis and interpretation. The data were analyzed statistically by the Duncan test with a significance level of 33.1p < 0.05), while it showed no influence on bacteria and actinomycetes. This result falls following Ji et al., who had reported the excellent compatibility of fluopimomide with Bacillus. 20, 40, and 60 DAT, the amounts of fungi treated by fluopimomide increased gradually with the time-lapse and recovered to the control level at 60 DAT under the recommended dosages. However, the treatments with 2- and 4-fold still inhibited the amount of fungal population. The control fungicide fluopicolide exhibited similar effects with fluopimomide on the amounts of fungi, bacteria, and actinomycetes. Overall, fluopimomide had no significant negative effects on soil bacteria and actinomycetes, and the effects on fungal populations could be recovered within 60 days, indicating a less negative effect on soil microbe.Soil microbes are basic components of soil ecology and are highly sensitive to environmental changes . The div3.2F. oxysporum), blight (P. infestans), and grey mold (B. cinerea), have caused severe yield loss throughout the world [Fusarium gene level in tomato planted soil suffered from wilt [B. cinerea and F. oxysporum (the inhibition ratios >80%). As for Phytophthora, fluopimomide exhibited similar efficiency (79.56–89.21%) to fluopicolide (84.64–87.59%). As time passed, the inhibition efficiency of fluopimomide remained constant until 60 DAT. Combined with the results of As a greenhouse vegetable, disease management is one of the most essential components of tomato production. The primary soil-borne diseases, wilt , while quadruple recommended dosage exhibited a significant inhibition effect. At 40 days after treatment, the dehydrogenase activities of soil treated with a quadruple recommended dosage of fluopimomide increased drastically. This may be because of the soil ecosystem alteration with the increasing fungicide concentration, and microorganisms can increase their metabolic activity in response to xenobiotics in the soil [Dehydrogenase, representative of soil organism metabolism , can trasticides ,29. As sthe soil . This isthe soil , who hadthe soil  had founthe soil , the var3.4.2As shown in Our result is in accordance with that of Jastrzębska et al. , which r3.4.3As reported, urease is externalized due to parent cell death and lysis. This enzyme plays an important role in the nitrogen cycle in soils. Its substrate, urea, is incorporated into the soil from fertilizer, animal excreta, or nucleic acids . In this3.4.4Soil invertase is of particular importance in carbon cycles . A previOverall, the effects of fluopimomide on the four soil enzymes are temporary and reversible, indicating relatively high safety to the soil environment .3.5nifH encodes for N2 fixation, AOA and AOB for ammonia oxidation, and nirS for denitrification [amoA gene abundance in soil and had different effects on nirS [nirS fluctuated greatly, but the differences among treatments were not obvious, indicating that the effects of chemical treatment on AOA and denitrifying bacteria in soil were not regular. At 40 and 60 DAT, the AOB and nifH gene copy numbers were higher in the double and quadruple dosages of fluopimomide treatments than in the control, indicating that fluopimomide could promote the proliferation of soil AOB, nitrogen-fixing bacteria, and other bacteria (Soil microbes are essential in soil nutrient mineralization and accumulation . Soil N fication . Jiang e on nirS . In thisbacteria , which m3.6−1, respectively. Furthermore, fluopicolide exhibited lower stimulation efficiency by 9.87%. Thus, the conclusion could be drawn out that fungicide fluopimomide could improve tomato yield, possibly via the inhibition of soil-borne pathogens.Soil management for disease suppressive could help to improve plant growth . Jakl et4nifH gene copy numbers were increased by the double and quadruple dosages of fluopimomide treatment. Compared with the control fungicide fluopicolide, fluopimomide exhibited more efficiency in tomato plant height and marketable yield. Therefore, as a broad-spectrum fungicide, fluopimomide could be popularized to manage tomato diseases. Still, the broader application scope remains to be investigated.Via soil treatment, the new fluorinated benzamide fungicide fluopimomide could significantly reduce the amounts of soil-borne pathogens in soil, reduce the disease incidences in tomato plants, and eventually increase the marketable yield of tomatoes. During the inoculation period, the soil enzymes had been influenced differently, and AOB and


Lattice structures for implants can be printed using metal three-dimensional (3D)-printing and used as a porous microstructures to enhance bone ingrowth as orthopedic implants. However, designs and 3D-printed products can vary. Thus, we aimed to investigate whether targeted pores can be consistently obtained despite printing errors. The cube-shaped specimen was printed with one side 15 mm long and a full lattice with a dode-thin structure of 1.15, 1.5, and 2.0 mm made using selective laser melting. Beam compensation was applied, increasing it until the vector was lost. For each specimen, the actual unit size and strut thickness were measured 50 times. Pore size was calculated from unit size and strut thickness, and porosity was determined from the specimen’s weight. The actual average pore sizes for 1.15, 1.5, and 2.0 mm outputs were 257.9, 406.2, and 633.6 μm, and volume porosity was 62, 70, and 80%, respectively. No strut breakage or gross deformation was observed in any 3D-printed specimens, and the pores were uniformly fabricated with < 10% standard deviation. The actual micrometer-scaled printed structures were significantly different to the design, but this error was not random. Although the accuracy was low, precision was high for pore cells, so reproducibility was confirmed. Current10. However, lattice structures used for securing biocompatibility and bone ingrowth have poor mechanical properties and microstructural weakness11, so load bearing can be difficult to achieve with them5. The bone-inducing ability of the lattice structure with Ti6Al4V has been previously reported in animal experiments and human studies15. For example, Fig. Implants must have both body strength and bone growth potential at the junction to the host bone. Considering practical use, a solid and periodic cellular structure, which is called the lattice structure, should have a clear division of roles in orthopedic implants and be properly mixed in each customized implant Fig. . The mec19. Previous studies have reported that titanium scaffolds with open pores with appropriate pore size and volume porosity demonstrate an osteoconduction effect24. Both EBM and SLM types of 3D-printing have been applied for cellular lattice structures, and SLM was found to fabricate finer lattice than EBM16. However, even using the SLM method, differences were noted between the design and actual products, especially in micrometer-scaled structure fabrication28. In our experience, reliable pore size is decreased by 675 μm using the EBM method, and lattice structures with open pores of 675 μm are commonly used for customized 3D-printed Ti6Al4V implants. However, the lattice structure was designed as a dode-thin structure with unit size of 2.0 mm by Magics software  and a pore size of 790 μm. From a technical point of view, developing a structure in which the design and 3D-product match as much as possible is important; however, if the error is predictable, it may be possible to select and use a lattice structure with an appropriate unit size despite errors, in the clinical field.Targeted microstructures with open pores to enhance bone ingrowth used in orthopedics have an average pore size of 300–600 μm, with a volume porosity of 70%This study aimed to quantify fabrication errors between designs and actual 3D products with various pore sizes, and to determine if printing errors are random or reproducible, that is, whether targeted pores can be consistently obtained despite printing errors.26  with unit sizes of 1.15 mm (A), 1.5 mm (B), and 2.0 mm (C). Specimens were printed using an SLM method printer Dpert M200  and Ti6Al4V ELI powder  with a particle size between 15 and 53 μm. The basic process conditions of the SLM method were as follows: laser power (110 W), laser scan velocity , laser diameter (0.05 mm), and layer thickness (30 μm). The beam compensation value was applied and sequentially increased by 5-μm intervals from 0 to printing failure due to vector loss. The beam compensation was a correction value of reducing the design by a few micrometers to prevent over-sizing due to the melting pool of the laser beam in the printing margin. In microstructure printing, the strut of the lattice structure is a few micrometers, so printing vectors were lost when the beam compensation value exceeded a certain value, resulting in printing errorsThe strut and unit sizes were measured using a scanning electron microscope (SEM) EM-30AX . For each specimen, 50 units of lattice were scanned and measured. All measurements were performed independently by two engineers (HMP and CHY) and averaged.Pore size was calculated as follows Fig. d:\documeThe density of the solid part was 4.37 g Continuous variables were compared using the independent-samples t-test. To examine the beam compensation effects on pore size, one-way analysis of variance (ANOVA) was performed. Statistical analysis was performed using the SPSS v. 21.0 software . All reported P values were two-tailed, and significance was set at < 0.05.In total, 14 specimens were printed. By increasing beam compensation, vector loss occurred at 20 μm for specimen A  and 25 μm for specimens B and C  Fig. . No strup < 0.01). In particular, actual pore sizes were all significantly smaller than the original designs . The means of differences between design and measurements for pore size were − 202.1, − 183.8, and − 156.4 for specimens A, B, and C, respectively , B  and C , respectively. Negligible global enlargement or reduction in unit size was observed. Meanwhile, the means of strut thickness were 228.8 ± 16.4, 245.8 ± 11.9, and 261.6 ± 16.7 μm for specimens A (designed strut 80 μm), B (designed strut 110 μm), and C (designed strut 150 μm), respectively. Therefore, most of the pore size reduction may have been due to the increased strut thickness . Maximal differences in pore size by beam compensation were 20.4, 10.7, and 22.5 for specimens A, B, and C, respectively. However, the pore size changes by beam compensation were random, without exhibiting any clear tendency.The effects of beam compensation for pore size in each specimen had significant differences  and maximal pore size (0.178–0.85 mm) varied among 10 different laboratories, even when using the same micro-CT analysis protocols34. Sources of deviation included scanning and image analysis errors. In the present study, 14 specimens of a cube composed of lattice structures were scanned using micro-CT with a pixel size of 10 μm and upper and lower grey thresholds were 60 and 255, respectively. All specimens were non-destructively analyzed by micro-CT and confirmed to have no strut breakage. However, micrometer-scaled measurements were not reliable. For example, for specimen B , the average strut thickness measurements were 290.4, 289.2, 290.3, 291.5, and 291.5 μm, with beam compensation of 0, 5, 10, 15, and 20 μm, respectively. The strut thickness measurements obtained by micro-CT scans were greater than those from SEM scans by approximately 45 μm. Pore size and porosity (%) of the cubes obtained by micro-CT scans were subsequently lower than those from SEM scans. The calculated mass (g), which was obtained by multiplying the volume of all struts in each cubic specimens , by the density (4.43 g/cm3) of Ti6Al4V ELI, was larger than the actual mass of the specimens; therefore, it was concluded that the struts in micro-CT were over-measured. In other words, although micro-CT was useful for non-destructive evaluation of internal defects such as strut breakage, it was unreliable as a micrometer-scale measuring tool.CT offers many advantages in the inspection of 3D-printed products and processes; however, there is a limitation in micrometer-scaled measurements. The inspection method for geometry or internal defects of 3D-printed products in real-time during printing or after fabrication has not been standardized yetThis study had several limitations. First, the specimens were examined in as-built status. This method was appropriate for observing errors due to printing itself, but the pore sizes might have changed due to deformation after heat treatment. Second, the targeted open pore, which has the best osteoconductivity, was well implemented by SLM; however, an additional in-vivo study is required to confirm this. Last, measurement errors may have occurred. To overcome this problem, each type of lattice was randomly measured 50 times and averaged by two independent engineers.In metal 3D printing of micrometer-scaled structures, a reproducible error was observed between the design and actual product. Nevertheless, in the SLM method, producing an optimal porous structure with a pore size of 300–600 μm and a porosity of 70% was possible, considering repeated errors.Supplementary Information.


Grewia tiliaefolia Vahl., plant known for its ameliorative effect on brain related diseases such as anxiety, depression, cognitive disorders and Parkinson’s disease. Plant extract was subjected to isolation of compound(s) using column chromatography and isolated compound was characterized by NMR FTIR and LCMS. The isolated compound was novel with the IUPAC name of the compound is propyl 3-hydroxy-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylate, designated as A-1 and has not been reported before. A-1 was further evaluated for its antioxidant potential using in vitro antioxidant assays . Also, Acetylcholinesterase (AChE) inhibitory potential of A-1 and extract was analysed. Results showed that A-1 exhibited significantly higher antioxidant activity in both DPPH and RPA assay as compared to plant extract. In case of AChE inhibitory activity again, A-1 has shown significantly higher activity as compared to plant extract. In silico study was conducted to predict its action on proteins playing crucial role in neurological and neurodegenerative disorders such as gamma amino butyric acid (GABA) receptor and glutamate α amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (Glu AMPA) receptor in epilepsy and AChE enzyme in Alzheimer’s diseases. The compound has shown interaction in following order: AChE > GABA receptor > Glu AMPA receptor. Further, molecular dynamic simulations and ADME studies of A-1 and AChE enzyme revealed that A-1 yielded good results in all parameters and hence can relieve Alzheimer’s like symptoms.Medicinal plants possess range of phytochemicals accountable for their diverse biological activities. Presently, such compounds have been isolated from medicinal plants, characterized and evaluated for their pharmacological potential. In the present study, the efforts have been made to isolate the compound(s) from  Medicinal plants contain a range of simple to complex secondary metabolites that may have therapeutic applications2. These metabolites are known to successfully prevent a number of chronic diseases such as cancer, alzheimer’s disease, epilepsy, diabetes and many more through a variety of mechanisms, including the reduction of oxidative stress, inhibition or modification of enzymes and receptors, interference with cellular signalling, and others5.Humans have used plant resources for millennia, not only for food and shelter but also for health and well-being. Between 75 and 90 percent of the world's rural population still relies heavily on herbal extracts and preparations for the majority of their primary healthcareGrewia genus, consists of 150 species, a member of family Tiliaceae and is found in tropical and sub-tropical areas6. There are about 40 species of this genus in India, many of which have medicinal uses. Grewia tiliaefolia is one of these species7. In India, tribal people and traditional healers have utilised this plant as an aphrodisiac, expectorant, antipruritic, and astringent8. In vitro and in vivo tests conducted on this plant have revealed its anti-inflammatory, anti-cancer, and hepatoprotective properties. G. tiliaefolia exhibited anti-amyloidogenic and neuroprotective effect and also regulated glutamate transporters for mitigating the toxicity exerted by glutamate in Neuro-2a cells10. Given that G. tiliaefolia has neuroprotective potential, the objective of the present study was to isolate compound from the plant and evaluate it for antioxidant and AChE inhibitory activity. Further, its role was predicted using the suitable target proteins playing crucial role in the pathogenesis of neurological diseases. Disbalance of GABA receptors and Glu AMPA receptors play crucial role in the pathogenesis of epilepsy. Decreased GABA and increased Glu AMPA expression leads to GABA/Glutamate disbalance, causing seizures11. AChE can hydrolyse acetylcholine (ACh) and diminish the synaptic concentration more than expected. The level of AChE in the hippocampus and cortex of Alzheimer’s patients is higher than that of healthy individuals. Therefore, increasing ACh levels by blocking AChE is a key strategy for treating Alzheimer patients' cognitive impairment14. AChE inhibitors can attenuate neuronal damage and death from cytotoxic insults and therefore might affect Alzheimer Disease pathogenesis15. Hence, GABA receptor, Glu AMPA receptors and AChE were selected for in silico study.All methods were performed in accordance with the relevant guidelines/regulations/legislations required to conduct the study.Methanol, ammonium molybdate, and trichloroacetic acid were purchased from Sigma Aldrich . 2,2-diphenyl-1-picrylhydrazyl, ferric chloride, potassium ferricyanide, hydrogen peroxide, sodium monophosphate was procured from Himedia Laboratories Pvt. Ltd. . The rest of the reagents and chemicals used in the study were of analytical grade.Grewia tiliaefolia were procured from Sri Venkateshwara University, Tirupati, Andhra Pradesh. Dr. K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateshwara University, Tirupati, Andhra Pradesh, has done the authentication of plant material with Voucher no. 0420. The plant material was coarsely powdered and extracted successively using Soxhlet apparatus with hexane, chloroform, and methanol each for 72 h to separate fatty compounds. Each extract was filtered using Whatman filter paper, concentrated using a rotary evaporator , completely dried and stored at 4 °C for further use16. Among all extracts, Grewia tiliaefolia methanol extract has shown higher extractive yield .The dried aerial parts of G. tiliaefolia methanol extract was subjected to isolation using silica column. The column was then run with hexane (mobile phase) and fractions were collected. Compound was isolated from collected fractions and subjected to characterization using different techniques such as NMR , LCMS and FTIR for structure elucidation17.The 18. The different concentrations (25–400 µg/ml) of the samples were mixed with 0.1 mM DPPH solution and incubated at room temperature for 30 min in the dark room. Gallic acid was used as a reference compound. The absorbance was noted at 517 nm using a Biotek microplate reader . Inhibition potential was calculated using the following formula:The free radical scavenging potential was analysed using a DPPH assay19. Increasing concentrations (25–400 µg/ml) of samples (2 ml), phosphate buffer , and potassium ferricyanide  were mixed and then incubated for 20 min at 50 °C. Trichloroacetic acid  was added to the reaction mixture. A volume of 2 ml from each of the reaction mixture was mixed with 2 ml of distilled water and 0.4 ml of 0.1% (w/v) ferric chloride in a test tube. After 10 min incubation, the absorbance was observed at 765 nm using a Biotek microplate reader . Ascorbic acid was used as standard. Inhibition potential was calculated using the following formula:The reducing power assay was conducted according to Benzie and Strain's technique (1996)20.AChE inhibitory activity of the extract and compound A-1 was investigated using Falé method. 7.5 μL of AChE solution containing 0.26 U/mL and 90 μL of 50 mmol/L Tris–HCl buffer, pH = 8.30 were mixed in a microwell plate and were incubated for 15 min. After that 142 μL of 3 mmol/L 5,5′-dithiobis(2-nitrobenzoic acid) and 22.5 μL of a solution of AChI (0.023 mg/mL) were added. The absorbance was taken at 405 nm when the reaction reached equilibrium using Biotek microplate reader . A control reaction was carried out using water in place of extract or compound A-1 and it was considered 100% activityPrepare Receptor” module of Biosolveit Lead it software version 2.3.2 (25.08.17). A co-crystallized ligand in the protein was used to provide the coordinates of the active binding site to the software, to generate a grid for docking the compound. The ligand was loaded into “Define FlexX docking” module of Biosolveit Lead it and was docked on respective proteins simultaneously. The binding energy  and docking score of the protein–ligand complex was calculated from each pose generated using “score a ligand with Hyde” of Biosolveit Lead it. Biovia discovery studio visualizer 2020 was used to export and render the binding poses22.The isolated compound was docked with GABA receptor, AMPA receptor, and AChE which are involved in the pathogenesis of neurological disorders. Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) website was used to download the PDB files of proteins and were loaded into the “23.For generation of system of protein (PDB code: 1EVE) and ligand complex, a SPC model of solvent was selected with orthorhombic box shape with 5 × 5 × 5 dimensions by using “system builder” module of DE Shaw Desmond software. Later, Counter ions (Na + or Cl −) for neutralization were added, system’s concentration was set to 0.15 M and OPLS3e force field was applied to the system. This generated system was simulated for 50 ns by using “Molecular Dynamics” module of DE Shaw Desmond. A 5-stage minimization of model system was performed prior to final MD simulation. These stages are: (a) stage 1—simulates Brownian Dynamics NVT, T = 10 K, small time steps, and restraints on solute heavy atoms, 100 ps; (b) stage 2—simulates NVT, T = 10 K, small time steps, and restraints on solute heavy atoms, 12 ps; (c) stage 3—simulate NPT, T = 10 K, and restraints on solute heavy atoms, 12 ps; (d) stage 4—simulate, NPT and restraints on solute heavy atoms, 12 ps; (d) stage 5—simulate, NPT and no restraints, 24 ps; (e) stage 6—final simulation for 50 ns that covers about 200 K stepshttp://targetnet.scbdd.com/home/index/) was used and for ADME studies, PreADME website (https://preadmet.webservice.bmdrc.org/) used25.For calculation of Lipinski rule of five and ADME descriptors, molecular structure data was uploaded to two online data-servers. For Lipinski parameters, TargetNet website  of A-1 and r1H NMR  δ 7.72 , 7.53 , 4.88 , 4.08 , 2.10–1.97 , 1.53–1.47 , 1.35–1.26 , 1.25 , 1.23 , 0.98 , 0.87 , 0.83 ; 13C NMR  δ 167.71, 132.39, 130.93, 128.86, 77.28, 77.23, 77.03, 76.93, 76.77, 76.61, 71.81, 33.96, 31.93, 29.70, 29.37, 29.14, 27.73, 25.15, 22.70, 21.11, 19.17, 14.12, 11.61. Other spectra such as COSY, HMBC and HSQC are shown in supplementary file S2, S3 and S4. FTIR analysis: 3026.2:C-H (Aromatic), 2922.2 C-H (Alkane), 2855.1: C-H (Aromatic); 1729.5: C = O (Saturated); 670: C-H (Aromatic); 1729: C = O; 1237: C-O (Alkyl ester); 1461: C = C (Aromatic); 1371: O–H . LCMS (ESI +): m/z detected for C23H34O3 was 357.0 g/mol. IUPAC name of the compound: propyl 3-hydroxy-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylate. Structure of isolated compound (A-1) has been shown in Fig. Compound was obtained as light green colored, solid substance about 37 mg approximately. Grewia tiliaefolia methanol extract had shown concentration dependant increase in inhibition potential. The inhibition potential was observed in the order G. tiliaefolia methanol extract < A-1 < Gallic acid. The IC50 values of Gallic acid, A-1 and G. tiliaefolia methanol extract were found to be 22.31 µg/ml, 36.08 µg/ml and 50.8 µg/ml respectively , Glu AMPA receptor (PDB code: 5YBF), and AChE enzyme (PDB code: 1EVE), and their binding energies were compared to those of their co-crystallized ligands, Diazepam, HBT-1, and Donepezil, respectively. After docking the poses, we calculated their binding energies and evaluated their docked poses, as depicted in Fig. To tackle the limitations of molecular docking i.e., no flexibility of protein structure, the whole protein ligand complex was simulated for 50 ns and time dependent stability of complex was studied as shown in Fig. https://preadmet.webservice.bmdrc.org/) and all the ADME related parameters were calculated. Results were summarized in Table 2D6 enzyme, therefore there is no drug-gene interaction. Moreover, on studying the Lipinski’s parameters as shown in Table Structure of isolated molecule was uploaded to PreADMET website . These results have showed that the rGrewia tiliaefolia. A-1 has shown significantly higher antioxidant activity in DPPH and reducing power assay and higher AChE inhibitory activity as compared to plant extract. Also, the docking study have revealed that compound interacts with the proteins involved in the pathophysiology of epilepsy and other neurodegenerative disorders.Propyl 3-hydroxy-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-yclopenta[a]phenanthrene-17-carboxylate, a cholesterol like moiety, designated as A-1 was isolated from 26. For characterization and structure elucidation, different spectral techniques were used such as NMR, FTIR and LCMS. NMR (proton and Carbon) which indicates the number of carbon and hydrogen atoms present in the compound HSQC NMR spectra informs about the proton-carbon single bond correlations, where the protons lie along the observed F2 (X) axis and the carbons are along the F1 (Y) axis27. COSY NMR spectra appraises about the proton-proton bonds28. HMBC NMR spectra gives detail about 1H/13C multiple-bond connectivity29. FTIR analysis implicates the different functional groups present in the compound30. LCMS informs about the mass of compound31. Collectively, the whole information is gathered to elucidate the structure. G. tiliaefolia methanol extract was subjected to isolation using column chromatography. Compound was isolated and characterized using spectral analysis, such as NMR, FTIR and LCMS. Gathering all the information structure was elucidated. IUPAC name of the compound is Propyl 3-hydroxy-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylate.Isolation of compounds is of prime importance in the field of drug research and development32. Such a pathogenic condition which develops due to this imbalance between the antioxidant defence system and reactive oxygen species generation, is fundamental in the development of neurological illnesses as well as a number of other disorders33. Antioxidant analysis is a preliminary step in the pharmacological evaluation of drugs or extracts34. Antioxidant activity of extract and A-1 was analyzed by DPPH and RPA assay. In DPPH assay, A-1 exhibited higher activity as compared to extract. In RPA similar pattern was observed. A-1 was found to be stronger antioxidant as compared to plant extract.Reactive oxygen species (ROS) that are produced as a result of oxidative stress in the biological system can cause damage to biological macromolecules, which can make the pathological condition at the cellular level more apparent12. ACh plays a significant part in brain processes which all gets affected after its level decline35. Low levels of ACh have been discovered to be associated with neurological disorders that eventually cause cognitive decline35. Natural cholinesterase inhibitors are commonly used for the treatment of neurological disorders so as to maintain the ACh level in brain14. Keeping this in mind AChE inhibitory activity of isolated compound was evaluated. It was observed that compound has shown significantly higher activity as compared to plant extract.AChE is responsible for the breakdown of ACh in the synaptic area which inhibits the nerve impulses37. In silico study was conducted to envisage the interaction of isolated compound with selected target proteins which play important role in the pathogenesis of the neurological disorders such as epilepsy and Alzheimer’s disease. The results have shown that compound was found to interact with all the target proteins but has shown highest binding energy with AChE. The order of interaction of compound with target proteins is AChE > GABA receptor > Glu AMPA receptor.Pharmaceutical industry now has a great opportunity to uncover new prospective drug targets thanks to sophisticated in silico methods, which has a direct impact on the efficiency and duration of clinical trials for finding novel drug targetsAlthough docking results revealed that compound showed maximum binding energy with AChE, but alone docking study is not sufficient to predict the success of molecule as a drug. Hence molecular dynamic simulation and Lipinski and ADME studies were performed to further evaluate its dynamics within the body. Results from MD simulations studies indicated that the protein and ligand complex are stable for 50 ns and low values of RMSD and RMSF implicates that the complex will be stable for much longer time span.38. It is important to stimulate the cholinergic receptors in the CNS or enhance the prolonged production of ACh in the synaptic cleft with the help of such active constituents that could retard the activities of AChE enzyme in the neuronal system. Compound can be considered as strong inhibitor, if inhibition of enzyme by that compound is 60% or more39. In our study, it was observed that A-1 has shown significantly higher AChE inhibitory activity and also molecular dynamic simulation studies and Lipinski/ADME studies further revealed that molecule showed good results in all parameters and can serve as Anti-Alzheimer drug, although appropriate in vitro and in vivo model are required to confirm its success at preclinical level.The research in the field of phytochemicals has focused on investigation of natural compounds responsible for antioxidative and neuroprotective properties that can also be useful for neurodegenerative disorders such as Alzheimer’s diseasesThe A-1 was novel compound. A-1 had shown significantly higher antioxidant activity as well as AChE inhibitory activity and stronger interaction with proteins involved in epilepsy and other neurodegenerative disorders, especially AChE enzyme. Further, this compound formed stable complex with AChE and remained stable for longer period, capable of crossing the BBB and obeys all drug like characteristics. This study provides the scientific basis for its possible pharmacological role as neuroprotective agent. Further studies are required to confirm the efficacy of A-1 against the epilepsy and Alzheimer’s disease at the preclinical and clinical level.Supplementary Information.


This study aimed to explore the clinical values of the percutaneous spinal endoscopy “isolation zone” technique for discogenic low back pain (DLBP).This retrospective case series study enrolled patients with intervertebral DLBP treated with the percutaneous spinal endoscopy “isolation zone” technique in the department of Orthopedics, Cangzhou central Hospital affiliated to TianJin Medical University between September 2017 and September 2020.Forty-five patients with DLBP were enrolled. The mean operation time was 94.7 ± 17.7 min. The visual analogue scale (VAS) score of lumbosacral pain was 6.95 ± 1.02 before operation, 2.64 ± 0.71, 1.80 ± 0.54, 1.42 ± 0.50, and 1.27 ± 0.45 at 1, 3, 6, and 12 months after operation, respectively. The Oswestry disability index (ODI) score of low back pain was 72.84 ± 5.95 before operation, 35.1 ± 5.30, 25.22 ± 4.85, 16.78 ± 4.63, and 10.91 ± 2.36 at 1, 3, 6, and 12 months after operation, respectively. At final follow-up, the treatment effect based on modified MacNab criteria was excellent in 24 cases, good in 13 cases, and fair in 8 cases. The excellent/good rate was 82.2%.The percutaneous spinal endoscopic “isolation zone” technique seems to be a promising surgical alternative for DLBP. Discogenic low back pain (DLBP) is a common type of spinal degenerative disease. DLBP results from multifactorial changes due to intervertebral disc degeneration that interact with the nervous system to induce pain . The paiThe traditional treatment of DLBP is mainly conservative such as rest, drug treatment, or physiotherapy, but this often cannot fundamentally solve the problem. For example, in cases of an intervertebral disc annulus fibrosus tear . For patMinimally invasive methods have been developed to improve the surgical outcomes of lumber disc surgery, such as the Yeung endoscopic spine system (YESS) technique , 9, intrTherefore, this study aimed to evaluate the clinical values of the percutaneous spinal endoscopy “isolation zone” technique for DLBP.This case series study enrolled patients with DLBP treated by spinal surgery in the department of Orthopedics, Cangzhou central Hospital affiliated to TianJin medical University between September 2017 and September 2020. The Ethics Committee of the Cangzhou central Hospital affiliated to TianJin medical University approved this study (No. 20210205). All patients provided written informed consent.The inclusion criteria were as follows: 1) patients with low back and lumbosacral pain symptoms, without typical sciatic nerve pain. Prolonged sitting, standing, bending, and physical labor could cause pain and discomfort in the lumbar back, hip, greater trochanter, and groin, and it was generally a dull pain; (2) preoperative X-ray, computed tomography (CT), magnetic resonance imaging (MRI) and/or other imaging data showed single segment disc lesions, mostly mild herniation of the intervertebral disc, no stenosis of bilateral lateral recess, and no obvious collapse of intervertebral space; (3) there was a high signal area of annulus fibrosus tear behind the responsible segment disc, and the MRI image showed a hiz (high intensity zone)  score of low back pain and pain around the buttocks were measured before operation and 1, 3, 6, and 12 months after operation. The treatment effect was evaluated by modified MacNab criteria at the last follow-up, alongside lumbar MRI imaging, and reporting of any complications.The modified MacNab criteria  evaluateAll patients were operated by a single senior physician skilled in spinal endoscopy in our research center. Regular oral nonsteroidal anti-inflammatory analgesics (such as diclofenac sodium) and central skeletal muscle relaxants (such as tizanidine hydrochloride) were administered 48 h before operation. All patients underwent the operation in the lateral position under the guidance of a G arm X-ray machine.Local anesthesia  was used in all patients. The anesthesia levels were skin and subcutaneous, lumbar dorsal fascia, articular process joint, and disc surface.All enrolled patients were treated with the “isolation zone” technique under percutaneous spinal endoscopy. The specific method involved puncture of the tip of the articular process on the target intervertebral space with an 18G puncture needle. The tip of the needle was located on the posterior edge of the intervertebral joint, and the anterior edge of the intervertebral joint. The guide wire was placed, the skin was cut about 7 mm, to place 2, 3.5, 4.5, and 5.5 mm catheters step by step to expand the soft tissue. The Tom Shidi needle was then placed along the guide wire, and the tip of the Tom Shidi needle was fixed at the tip of the facet joint under fluoroscopy, and pointed to the protruding target of the intervertebral disc. This was then used to gently hammer the bone penetrating the facet joint, while appropriately adjusting the depth of the Tom Shidi needle into the spinal canal according to the protruding position of the intervertebral disc. The guide wire was replaced after the positioning was confirmed by frontal and lateral fluoroscopy. Bone drills with diameters of 4.5, 6, 7, 8, and 9 mm were successively inserted along the guide wire for intervertebral foramen plasty. The guide wire was replaced again and the expansion guide rod was inserted and the 7.5-mm working channel was inserted along the guide rod. It was confirmed that the working channel reached the target through frontal and lateral fluoroscopy  exercise, and a lumbar spine health care program daily.t‐test or one‐way analysis of variance (ANOVA). Two-tailed P-values < 0.05 were considered significant.The data were analyzed by SPSS 22.0 statistical software . Continuous variables with a normal distribution were expressed by mean ± standard deviation (SD), and categorical variables were expressed as numbers (percentages). Comparisons for continuous data were performed using Student’s P < 0.05). At the last follow-up, the treatment effect according to the modified MacNab criteria was excellent in 24 cases, good in 13 cases, and fair in 8 cases. The excellent and good rate was 82.2%  years old were included. 29 cases were L4/5, 16 cases were L5/S1. Among the 45 patients, there was no forced interruption due to intolerable pain of local anesthesia. The mean operation time was 94.7 ± 17.7 (range: 65–125) min. The VAS score of lumbosacral pain was 6.95 ± 1.02 before operation, 2.64 ± 0.71, 1.80 ± 0.54, 1.42 ± 0.50, and 1.27 ± 0.45 at 1, 3, 6, and 12 months after operation, respectively. The ODI score of low back pain was 72.84 ± 5.95 before operation, 35.1 ± 5.30, 25.22 ± 4.85, 16.78 ± 4.63, and 10.91 ± 2.36 at 1, 3, 6, and 12 months after operation, respectively. There was significant improvement compared with that before operation  in 52 patients . In thatThe endoscopic methods above also improved spinal function as indicated by ODI. In this study, the ODI decreased from 72.84 ± 5.95 preoperatively to 10.91 ± 2.36 at 12 months postoperatively. The studies described above also showed the ODI score dropped from the preoperative level of 22.3 to 8.8 at 12 months , 35.8 ± DLBP is one of the common spinal degenerative diseases in clinic. It mainly manifests as atypical low back pain and lower limb pain. The pain is located mostly in the lower waist, hip and groin areas, posterolateral thigh, and knee joints. The positioning is inaccurate. The symptoms worsen when standing, sitting, or bending for long periods , 18. TheFor DLBP, it is very necessary to determine the responsible segment. In addition to the lumbar MRI showing signal changes of the intervertebral disc with hiz and modic signs and Schmorl nodules on the posterolateral side, discography, induction test, and transforaminal nerve block are important diagnostic techniques . The angAlthough there are many treatment schemes for DLBP, such as lumbar fusion surgery, intradiscal intervention technologies such as intradiscal electrotherapy, intradiscal injection of platelet rich plasma or hepatocytes, or intradiscal ozone technology the above technologies are controversial at present, and the treatment results are often uncertain , 29. ComOur experience suggests that the “isolation zone” spinal endoscopy technology used in this study has the following advantages in the treatment of DLBP: (1) nerve block through the intervertebral foramen can accurately determine the responsible segment of DLBP, and avoid the damage to the intervertebral disc caused by the traditional intradiscal injection of contrast agent and the false-positive test; (2) endoscopic treatment of the responsible segment through the intervertebral foramen not only has less damage to the stable structure of the lumbar spine, but also preserves the spinal motor unit. It has advantages over the traditional fusion surgery in preventing the adjacent segment lesions after lumbar surgery; (3) under local anesthesia, the patients can autonomously reflect the nerve function of the lower limbs during the operation. The risk of nerve injury is small, and the postoperative recovery is fast. Patients can exercise early; (4) the pathogenic factors of the responsible segments are comprehensively treated to achieve the purpose of treating DLBP.There were several limitations. Firstly, this was a single center study, and the sample size is limited. As a retrospective analysis there may have been some bias in the patients selection. There was no comparison group, so no clear conclusions can be made on the effectiveness of the treatment compared to standard treatment.The percutaneous spinal endoscopic “isolation zone” technique provided satisfactory clinical results for DLBP. The percutaneous spinal endoscopic “isolation zone” technique seems to be a promising surgical alternative for DLBP.


The most common affected sites were the axilla (60.2%), buttocks (59.0%), and the inguinal and femoral regions (47.0%). Mean abscess and inflammatory nodule count was 13.0 . Among patients with a comorbidity (57.8%), the most common were diabetes mellitus, hypertension, and chronic kidney disease. No patient reported a serious infection or any safety event of special interest. One patient died from a serious adverse event of cardiac failure unrelated to adalimumab. At week 12, 57.4% of patients achieved HiSCR, and significant reductions from baseline in skin pain, DLQI (both p < 0.0001), and CRP (p = 0.0029) were observed. These results support the administration of adalimumab as a well‐tolerated and effective treatment for Japanese patients with HS in real‐world clinical practice.Hidradenitis suppurativa (HS) is a painful chronic skin disease characterized by abscesses, nodules, and tunnels in the skin. Adalimumab, a monoclonal antibody against tumor necrosis factor‐α, is approved for the treatment of HS in Europe, the USA, and Japan. This multicenter, open‐label, post‐marketing, observational study  evaluated the safety and effectiveness of adalimumab in routine clinical practice in Japan (March 2019–May 2021). Patients with HS were treated with s.c. doses of adalimumab according to the dosage described in the package insert. The primary end‐point was safety . Secondary end‐points assessed effectiveness, including HS Clinical Response (HiSCR), skin pain, Dermatology Life Quality Index (DLQI), and C‐reactive protein (CRP). Here, we report 12‐week interim effectiveness results. A total of 84 eligible patients from 65 sites were enrolled; 83 patients were included in this analysis. Mean age was 42.0 years, mean body mass index was 26.9 kg/m Lesions predominantly develop in regions rich in apocrine glands, including the axilla, groin and anogenital region, and buttocks, and in the interbreast area in females.®; AbbVie) is a human monoclonal antibody that binds to TNF‐α and has high affinity and selectivity for human TNF‐α. Two multicenter, randomized, double‐blind, placebo‐controlled, phase 3 studies (PIONEER I and PIONEER II) evaluated the safety and efficacy of adalimumab treatment for patients (predominantly from Western countries) with moderate to severe HS.p = 0.003; PIONEER II: 58.9% vs. 27.6%, p < 0.001), and rates of serious adverse events (AE) were similar across treatment groups.Hidradenitis suppurativa is histopathologically characterized by severe inflammatory cell infiltration by neutrophils, lymphocytes, and histiocytes. Inflammatory cytokines including tumor necrosis factor‐α (TNF‐α), as well as activated neutrophils and lymphocytes, are considered to be involved in the onset of HS.The objective of this post‐marketing surveillance was to evaluate the long‐term safety and effectiveness of adalimumab in real‐world clinical practice in Japanese patients with HS. We report the interim analysis of safety up to the date of data cut‐off, and effectiveness up to 12 weeks, for patients with HS registered in 2019 and 2020 in Japan.22.1This was a multicenter, open‐label, post‐marketing, observational study. The enrollment period was 23 April 2019 to 14 February 2020, and the study period was 11 March 2019 to 14 May 2021. Data cut‐off for interim analysis was 31 December 2020. Patients were registered for the study by a central registration method at medical institutions that signed a written contract for the study. Patient data were collected by the physicians in charge of the study using case report forms and were submitted to the study sponsor at the end of the observation period using an internet‐based electronic data capture system. All patients were required to provide consent to participate in the study. This study was conducted in accordance with the GPSP Ordinance . Institutional review board and ethics committee approvals, although not required according to the GPSP Ordinance, were obtained if requested by the medical institution. The study was registered at ClinicalTrials.gov (identifier: NCT03894956).2.2Patients who were prescribed adalimumab for the first time by their treating physician were eligible for inclusion in the study. Per routine clinical practice, patients were eligible for adalimumab treatment only after all existing treatment options  had been proven to be ineffective for the treatment of HS. Patients previously treated with adalimumab and patients who did not provide consent to participate in the study were excluded.2.3Adalimumab was administered s.c. according to the dosage and administration described in the package insert. The first dose was 160 mg, the second dose  was 80 mg, the third  and subsequent doses were 40 mg every week or 80 mg every 2 weeks. The observation period was 52 weeks from the start of adalimumab treatment; if treatment discontinued before 52 weeks, the occurrence of AE was observed up to 70 days after the last dose. Discontinuation was defined as the time when there was no possibility of re‐administration of adalimumab before 52 weeks from the start of treatment or when observation was no longer possible.2.4The following patient information was collected: age, sex, bodyweight, disease period, severity of HS (Hurley staging classification),2.5The primary end‐point was safety and included the number and percentage of patients who reported any serious infection, any adverse drug reaction (ADR), any infection, and any safety event of special interest  during the study. AE were defined as any unfavorable or unintended signs , symptoms, or illness, with or without a causal relationship with adalimumab. ADR were AE whose causal relationship with adalimumab, as determined by the physician, could not be ruled out. AE and ADR were coded using the Medical Dictionary for Regulatory Activities/Japanese edition (MedDRA/J) version 23.1. AE occurrence was investigated up to 70 days after discontinuation of adalimumab.Secondary end‐points were assessed at 12, 24, and 52 weeks, and at adalimumab discontinuation. For this interim analysis, results at 12 weeks only are reported. End‐points included the following: the proportion of patients achieving a HiSCR,2.6t‐tests were performed. Two‐sided p < 0.05 was considered statistically significant. Multiplicity in the hypothesis test was not considered, and imputation of missing data was not performed. Statistical analysis was conducted using SAS software version 9.3 (SAS Institute).A primary end‐point of this study was to determine the incidences of ADR in patients with HS. Infection is one of the risks of TNF‐α inhibitors. Sample size was based on the incidences of infections in two phase 3 clinical studies in patients with HS (PIONEER I and PIONEER II), which were 24.8% (38/153) and 25.2% (41/163), respectively.33.12, 42 (50.6%) patients had a history of smoking, six (7.2%) patients had a family history of HS, and comorbidities were reported for 48 (57.8%) patients. The most common comorbidities were diabetes mellitus , hypertension , and chronic kidney disease . One (2.1%) patient had latent tuberculosis, and no patients had inflammatory bowel disease (IBD). All comorbidities reported at baseline are listed in Table A total of 84 patients registered and provided consent for the study across 65 medical institutions. One patient who withdrew consent during the observation period was excluded from the safety and effectiveness analysis populations. The demographics and characteristics of the 83 patients included in the study are presented in Table 3.2Hidradenitis suppurativa disease characteristics prior to adalimumab treatment are reported in Table The majority of patients  had received prior medication for HS before starting adalimumab treatment Table . Non‐pha3.3The majority of patients  received adalimumab for a period ≥12 to <24 weeks  received concomitant medication for HS, with 37 of these patients (71.2%) receiving an oral antibacterial drug  patients, 5/12 (41.7%) patients, and 6/10 (60.0%) patients had discontinued treatment with minocycline hydrochloride, doxycycline hydrochloride hydrate, and roxithromycin treatment, respectively Table .3.5No patient reported a serious infection, six (7.2%) patients reported an ADR, six (7.2%) patients reported any infection, and no patients reported any safety event of special interest Table . Overall3.6p ≤ 0.0001) for all HS lesion types: inflammatory nodules, −3.9 (4.5); abscesses, −2.8 (4.7); and drainage fistulas, −3.1 (6.1). The proportion of patients to have an AN count of 0, 1, or 2 at baseline was 7.8%; this increased to 48.4% at 12 weeks of adalimumab treatment . The mean (SD) number of all HS lesions  was reduced at 12 weeks compared with baseline counts Table : baselinas, −3.1 .1. The pp = 0.0029).The proportion of patients who showed an overall improvement status (physician‐assessed) of “improved” at 12 weeks of adalimumab treatment was 93.8%; 4.9% reported an overall improvement status of “not improved” Figure . Mean S. The meap < 0.0001). The proportion of patients to achieve an NRS30 at 12 weeks was 76.3% .The mean (SD) NRS of skin pain decreased from 5.3 (3.2) at baseline to 2.0 (2.3) at 12 weeks of adalimumab treatment Figure . The mea% Figure . Mean  was lower than expected when compared with the incidence observed at 12 weeks in the phase 3, open‐label, placebo‐controlled trials of adalimumab treatment in patients (weekly dosing) with moderate to severe HS .The clinical response (HiSCR) observed in this study at 12 weeks was similar to that observed in PIONEER II at 12 weeks, both of which were greater than that observed in PIONEER I at 12 weeks .2, and comorbidities ; most patients had a disease duration of ≥10 years. Marzano et al.p = 0.0016). Given that 40% of patients in this Japanese study had a disease duration of ≥10 years, treatment response to adalimumab may have been affected. The difference in effectiveness by disease duration was not examined in this report, and further subanalyses, such as responders versus non‐responders, may be important.Racial differences in HS rates have been reported. In particular, in East Asia, the prevalence of HS is higher in men compared with women, but in Western countries, the prevalence is higher in women.In this study of Japanese patients with HS, the predominant HS‐affected areas were the axilla (60.2%) and buttocks (59.0%), confirming previous reports of an increased involvement of the gluteal region in patients from East Asia compared with patients in Western countries.Comorbidities reported to be associated with HS include diabetes mellitus, metabolic syndrome, dyslipidemia, obesity, hypertension, polycystic ovarian syndrome, smoking, IBD, and spondyloarthropathy.Current guidelines recommend a holistic approach to manage HS and associated comorbidities; in general, a combination of pharmacological and non‐pharmacological therapy is recommended.The strengths of this study are that the results are real‐world data that included a Japanese population of >80 patients with HS, and the study included a variety of outcome measures to evaluate the effectiveness of adalimumab in clinical practice. The study was limited by the open‐label design, with no control or comparator group, and the variable data capture outside of the controlled environment of a clinical trial. Patients who were previously prescribed adalimumab for HS and other diseases were excluded from this study; this may explain why no comorbidity of IBD was observed in this patient cohort. This study is still ongoing, and the real‐world, long‐term (up to 52 weeks) effectiveness of adalimumab in Japanese patients with HS is still to be determined.In conclusion, the safety and effectiveness of adalimumab were demonstrated in Japanese patients in the largest observational trial for HS patients. The safety and effectiveness data reported here are consistent with the data from previous clinical trials, and no new safety concerns were observed in patients with HS in real‐world clinical practice. The study also documented the characteristics of Japanese patients prescribed adalimumab for HS.N.H. has received consultancy fees and speaker honoraria from AbbVie GK and Maruho. K.H. reports personal fees from Janssen Pharmaceutical and Meiji Seika Pharma; grants and personal fees from AbbVie GK, Boehringer Ingelheim, Eisai, Kaken Pharmaceutical, Kyowa Kirin, Maruho, Mitsubishi Tanabe Pharma, Novartis Pharma, Sanofi, and Taiho Pharmaceutical; and grants from Nihon Pharmaceutical and Sun Pharma Japan. K.T. has served as a paid speaker for and/or participated in clinical trials sponsored by companies that manufacture drugs used for the treatment of psoriasis, including AbbVie GK, Boehringer Ingelheim, Celgene (Bristol‐Myers Squibb), Eisai, Eli Lilly Japan, Janssen Pharmaceutical, Kyowa Kirin, LEO Pharma, Maruho, Mitsubishi Tanabe Pharma, Novartis Pharma, Taiho Pharmaceutical, and Torii Pharmaceutical. I.K. has no conflict of interest. M.O., T.K., and E.I. are employees of AbbVie GK. T.T. has received research funds from Maruho and honoraria for serving as a speaker, consultant, and advisory board member from AbbVie GK, Boehringer Ingelheim, Celgene (Bristol‐Myers Squibb), Eli Lilly Japan, Janssen Pharmaceutical, Kyowa Kirin, LEO Pharma, Mitsubishi Tanabe Pharma, Novartis Pharma, and Sanofi.Table S1‐S5Click here for additional data file.