Schistosoma, and this agent is virtually the only drug of choice for the treatment of human schistosomiasis. Mass drug administration (MDA) with praziquantel has been shown to be effective in greatly reducing the prevalence and morbidity due to schistosomiasis worldwide. In addition to antischistosomal activity, a large number of experiential and clinical evidence has demonstrated the action of praziquantel against fibrosis caused by S. mansoni and S. japonicum infections through decreasing the expression of fibrotic biomarkers such as α-smooth muscle actin (α-SMA), collagen, matrix metalloproteinase (MMP), and tissue inhibitor of metalloproteinase (TIMP), and inhibiting the expression of proinflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β, as well as chemokines, and similar antifibrotic activity was observed in mouse models of fibrosis induced by carbon tetrachloride (CCl4) and concanavalin A (Con-A). In this review, we discuss the role of praziquantel in the prevention and treatment of fibrosis associated with schistosomiasis and the possible mechanisms. We call for randomized, controlled clinical trials to evaluate the efficacy and safety of praziquantel in the treatment of schistosomiasis-induced hepatic fibrosis, and further studies to investigate the potential of praziquantel against fibrosis associated with alcohol consumption, viruses, and toxins seem justified.Schistosomiasis remains a major global public health concern. Currently, the control of this neglected tropical disease still depends on chemotherapy to reduce the prevalence and intensity of the parasite infection. It has been widely accepted that praziquantel is highly effective against all species of  Schistosoma, remains a global public health concern that ranks second to malaria among human parasitic diseases in terms of the number of people infected and at risk of infection [Schistosomiasis, a neglected tropical disease caused by the blood fluke of the genus nfection . Worldwinfection . Althougnfection –5, the cnfection –8.β1, interferon (IFN)-γ), and microRNAs (miRNAs) have been found to contribute to the pathogenesis of schistosomiasis-induced hepatic fibrosis [Hepatosplenic schistosomiasis is characterized by typical pathological changes at chronic and advanced stages, including egg granulomas, fibrosis, and tissue damage within the liver and other host tissues . Hepaticfibrosis –17. Portfibrosis , and curfibrosis . Howeverfibrosis . A searcSchistosoma, notably, adult stages of the parasite [Since praziquantel, a broad-spectrum schistosomicide, was developed in 1970 s , it has parasite . As a coparasite . Mass drparasite –29. Furtparasite ,31. Thishttps://www.wanfangdata.com.cn/), CNKI (https://www.cnki.net) and VIP (https://qikan.cqvip.com/) using the terms “schistosomiasis,” “fibrosis,” and “praziquantel” to retrieve publications concerning the action of praziquantel against fibrosis associated with schistosomiasis during the period from January 1st, 1970 to December 31st, 2021. Inclusion criteria involved: (1) studies reporting the activity of praziquantel against fibrosis associated with schistosomiasis; and (2) animal studies or clinical reports. Publications that met the following exclusion criteria were: (1) review articles; or (2) the full text was unavailable.A joint search was performed in international and national electronic databases, including Web of Knowledge, PubMed, Scopus, Google Scholar, Wanfang Data /I collagen (Col1) ratio to normal limits [S. mansoni cercariae each, a significant reduction in hepatic and splenic granulomas, fibrosis, and circulating cathodic antigen (CCA), and circulating anodic antigen (CAA) was seen following praziquantel treatment [Col1, Col3, and Col4 gene expression were observed 6 and 12 months post-treatment, and 71.4% resorption of hepatic fibrous tissues was found 12 months post-treatment in a CBA/Jk mouse model of S. mansoni infections [S. mansoni, treatment with praziquantel resulted in elimination of egg granulomas or collagen fibrils, and reduced the expression of signal transducer and activator of transcription 1 (STAT1), STAT4, IFN-γ, TBET, IL-4, C–C motif chemokine ligand 12 (CCL12), and CCL22 [S. mansoni and fed either a low-protein diet (8% protein) or standard chow (22% protein) [In a murine model of reatment . In Swisl limits . In Syrireatment . Followifections . In Swisnd CCL22 . In addiprotein) . In a reprotein)  , Col1, and IL-13 expression [In addition, combined therapy of praziquantel with alpha lipoic acid , silymarfibrosis –44, the pression .S. japonicum, praziquantel treatment was found to reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations, the areas of egg granulomas, areas of collagen deposition and hepatic hydroxyproline contents, alleviate fibrotic proliferation, inflammatory infiltration, hepatocyte degeneration and necrosis, improve liver size and texture, decrease the levels of fibrosis-related parameters including α-SMA, SPET4, matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, Col1α1, and Col3α1 in relative to infected but untreated controls, and inhibit the expression of proinflammatory cytokines tumor necrosis factor (TNF)-α and TGF-β1[γ was reported to result in a greater decline in hepatic fibrosis score and area of collagen deposition, higher reduction of Col1 and Col3 expression, more downregulation of Smad2 expression and upregulation of Smad7 expression as compared to treatment with praziquantel alone [Amusium pleuronectes polysaccharide presented a more inhibitory effect on hepatic fibrosis than treatment with praziquantel or the polysaccharide alone in S. japonicum-infected mice, as revealed by lower Col1, Col2, and Col4 levels, lower parasite egg burdens and higher IL-12 and IFN-γ productions [S. japonicum-induced hepatic fibrosis [In animal models of fibrosis induced by nd TGF-β1, 46–52 (el alone , 54, andductions . In addifibrosis –58.S. mansoni or S. japonicum infection, which encourages the randomized, controlled clinical trials to confirm the antifibrotic activity of praziquantel.The results of these experimental studies demonstrate the effectiveness of praziquantel against hepatic fibrosis induced by S. mansoni-infected subjects, administration of praziquantel at a total dose of 40 or 50 mg/kg resulted in improvements in the ultrasonographic parameters of fibrosis [S. japonicum-infected patients, praziquantel treatment at a total dose of 40 or 60 mg/kg was found to improve the ultrasonographic and biochemical parameters in patients with mild fibrosis but not in patients with severe fibrosis [S. japonicum-infected Filipinos following one-year treatment of praziquantel at a total dose of 60 mg/kg given in a split dose [To date, there are no randomized, controlled trials to examine the role of praziquantel in prevention and control of fibrosis associated with schistosomiasis; however, available data have shown the antifibrotic value of praziquantel in treatment of patients with schistosomiasis-induced fibrosis , 59. In fibrosis –64 (Tablfibrosis , 65–68 (lit dose . Furtherα-SMA, collagen, MMP, and TIMP, and inhibiting the expression of proinflammatory cytokines such as IL-6, TNF-α, and TGF-β, as well as chemokines [Previous studies have demonstrated that praziquantel is active against schistosomiasis-induced hepatic fibrosis through decreasing the expression of fibrotic biomarkers including emokines , 47–52. emokines , inhibitemokines , and incemokines , which wCurrently, there are no effective treatments for hepatic fibrosis except liver transplantation . Early dS. japonicum and S. mansoni infection [4-induced mouse model of hepatic fibrosis [It has been proven that praziquantel is a highly effective and mildly toxic schistosomicide , and pranfection –52,61–68fibrosis . In the fibrosis . Since hfibrosis –84, and In summary, praziquantel, an old schistosomicide, is a promising antifibrotic drug. Although randomized, controlled clinical trials are required to validate the antifibrotic action, and there are still a large number of challenges ahead, the antifibrotic activity of praziquantel may benefit thousands of patients, due to its low cost, easy administration, and low toxicity. In addition, the timing, dosing, optimal regimens, and mechanisms of action of praziquantel for treatment of fibrosis require further investigation.


Ammonia-oxidizing archaea (AOA), key players in agricultural upland soil nitrification, convert soil ammonium to nitrite. The microbial oxidation of ammonia to nitrite is an important part of the global biogeochemical nitrogen cycle. In the present study, we recovered six novel AOA metagenome-assembled genomes (MAGs) containing genes for carbon (C) fixation and nitrogen (N) metabolism by using a deep shotgun metagenomic sequencing strategy. We also found that these AOA MAGs possessed cobalamin synthesis genes, suggesting that AOA are vitamin suppliers in agricultural upland soil. Collectively, the present results deepen our understanding of the metabolic potential and phylogeny of AOA in agroecosystems. Soil total DNA was extracted from 0.5 g soil using an Omega Mag-Bind Soil DNA Kit (Omega Biotek) following the user’s manual. DNA libraries were prepared using the KAPA HyperPlus Kit (Roche Sequencing). A deep shotgun metagenomic sequencing strategy (an average of 47 Gb for each sample) was used to recover metagenome-assembled genomes (MAGs) by using MetaBAT2 (v2.10.2) runMetaBat.sh parameters . A maximi.e., approximately 58–81%, were predicted using the eggNOG database  recovered from assembly data were assigned to AOA groups. ThauHEB1 and ThauHEB2 were obtained from Harbin, ThauQZ2 from Quzhou, ThauCW1 from Changwu, ThauSQ3 from Shangqiu, and ThauSY3 from Shenyang. The completeness of MAGs ranged from between 52.05 and 96.76% and contamination from between 0.97 and 8.58%. The completeness of ThauQZ2 and ThauHEB2 were 96.76 and 93.05%, respectively, while the contamination of these two MAGs were 1.94 and 2.51%, respectively, which suggested that ThauQZ2 and ThauHEB2 represent nearly complete AOA genomes. The genome sizes of our six MAGs varied from 0.985 to 2.573 Mb and the GC contents varied from 28.75 to 48.85%. The GC content of the nucleic acid sequence is a fundamental molecular trait that may be used as an indicator to reflect the evolutionary characteristics of AOA, possibly representing different adaptation traits for responses to environmental heterogeneity. The estimated numbers of CDSs (coding sequences) were 1,499 for ThauHEB1, 3,170 for ThauHEB2, 3,119 for ThauQZ2, 2,237 for ThauCW1, 1,447 for ThauSQ3, and 1,899 for ThauSY3. The percentages of CDSs with functions, database . The numNitrosotalea, and species belonging to the Nitrosotalea lineage were generally associated with nitrification activity in acidic environments  with the reference species of four MAGs  varied from between 77.05 and 92.47%, and ThauHEB2 and ThauSY3 MAGs had no species that were assigned in the GTDB database (R95)  cycle  cycle a, which AOA MAGs a, indicaamoA, amoB, and amoC genes; ThauQZ2 and ThauHEB2 had amoA and amoB genes; and ThauSQ3, ThauSY3, and ThauCW1 had amoA gene. In addition, ThauHEB1, ThauHEB2, ThauQZ2, and ThauCW1 possessed the nirK gene. Although previous studies reported structural variations between archaeal and bacterial copper-containing nitrite reductase (NirK) enzymatic properties  involved in adenosyl cobyrinate a,c-diamide transformation to adenosyl cobyrinate hexaamide, adenosylcobinamide, adenosine-GDP-cobinamide, and Vitamin B12 were detected in ThauHEB1, ThauHEB2, ThauSY3, ThauQZ2, and ThauCW1; and other genes (cbiC/cbiD/cbiE/cbiF/cbiG/cbiH/cbiL/cbiT) involved in intermediate transformation, such as Factor, Co-Factor, and Co-Precorrin, were also found in these AOA MAGs. In addition, all six AOA MAGs had genes involved in assimilatory sulfate reduction, which suggests that the activity of AOA is accompanied by sulfur metabolism. Sulfur assimilation to synthesize cysteine molecules is apparently essential for all forms of life, including archaea, bacteria, and eukaryotes. Specifically, ThauHEB1 and ThauQZ2 had the full set of genes responsible for the sulfate reduction pathway, successively converting sulfate to adenosine 5′-phosphosulfate (APS), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), sulfite, and sulfide, including sat encoding sulfate adenylyltransferase, cysC encoding adenylylsulfate kinase or related kinase sulfates, cysH encoding phosphoadenosine phosphosulfate reductase, and sir encoding sulfite reductase. However, the remaining MAGs had a truncated sulfate reduction pathway; for example, ThauHEB2 had sat, cysH, and sir; ThauSY3 had sat, cysC, and cysH; ThauSQ2 had sat and sir; and ThauCW1 had cysC, cysH, and sir.Previous studies reported that Thaumarchaeota play an important role in cobalamin biosynthesis (vitamin B12) in aquatic environments  under BioProject PRJNA792913, with the accession numbers SRR17477666, SRR17477667, SRR17477668, SRR17477669, and SRR17477670. Theaccession numbers of the metagenome assembled genomes areJAKEIF000000000, JAKEIG000000000, JAKEIH000000000,JAKEII000000000, JAKEIJ000000000, and JAKEIK000000000.Microbes Environ 37: ME22035.Zhao, H., and Zhang, L. (2022) Metagenome-assembled Genomes of Six Novel Ammonia-oxidizing Archaea (AOA) from Agricultural Upland Soil. https://doi.org/10.1264/jsme2.ME22035Supplementary Material


The zebrafish intestine and liver, as in other vertebrates, are derived from the endoderm. Great effort has been devoted to deciphering the molecular mechanisms controlling the specification and development of the zebrafish intestine and liver; however, genome-wide comparison of the transcriptomes between these two organs at the larval stage remains unexplored. There is a lack of extensive identification of feature genes marking specific cell types in the zebrafish intestine and liver at 5 days post-fertilization, when the larval fish starts food intake. In this report, through RNA sequencing and single-cell RNA sequencing of intestines and livers separately dissected from wild-type zebrafish larvae at 5 days post-fertilization, together with the experimental validation of 47 genes through RNA whole-mount in situ hybridization, we identified not only distinctive transcriptomes for the larval intestine and liver, but also a considerable number of feature genes for marking the intestinal bulb, mid-intestine and hindgut, and for marking hepatocytes and cholangiocytes. Meanwhile, we identified 135 intestine- and 97 liver-enriched transcription factor genes in zebrafish larvae at 5 days post-fertilization. Our findings provide rich molecular and cellular resources for studying cell patterning and specification during the early development of the zebrafish intestine and liver. The digestive tract and accessory organs (liver and pancreas) form the digestive system, which is specified and developed from distinct regions of the endoderm  ,2,3. In +ada, + rbp2a+fabp2, + slc10a2+fabp6, +lamp2 and caudal intestine [Previous studies have shown that intestinal cells from the intestinal bulb toward the proctodeum express regionalized specific genes to execute different physiological functions, such as food digestion and nutrient absorption ,20,21,22ntestine . Recentlntestine , were coIn this report, we micro-dissected the intestinal tube and liver buds, respectively, from zebrafish larvae at 5 dpf and then obtained satisfactory RNA sequencing (RNA-seq) data from these samples. The RNA-seq data allowed us to identify not only the organ-specific functional genes, but also the corresponding spectrum of TF genes expressed in the intestine and liver at 5 dpf. We also obtained the RNA-seq data for three segments of the intestine  at 5 dpf, which allowed us to acquire a more elaborate molecular view of the functional specialization of these regions at 5 dpf. Next, we acquired the scRNA-seq data from the micro-dissected intestine and liver at 5 dpf and annotated 10 cell types in the intestine and 8 cell types in the liver. For the intestine, 3 of the 10 cell types represented the regionalized enterocytes along the intestine at 5 dpf. In addition, mapping the 385 intestine-specific genes identified by our RNA-seq analysis (intestine vs. liver: fold-change > 4 and transcripts per million (TPM) > 50) to the 10 intestine cell types by a feature plot unraveled their cellular specificity. In total, the expression patterns of 47 genes were validated by whole-mount in situ hybridization (WISH). These findings not only extend our understanding of the molecular and cellular features in the larval intestine and liver, but also provide a rich resource for studying the process of organogenesis and development of the intestine and liver.zju1pparda mutant was generated using the CRISPR-Cas9 method with gRNA (5′-GAACGGGCTGGTCTGGAACC-3′) specifically targeting pparda.The AB wild-type (WT) zebrafish strain was used in this study. Fertilized eggs were placed in a petri dish containing egg water and allowed to grow to 5 dpf at 28 °C. All animal procedures were adopted in full accordance with the requirement by the ‘Regulation for the Use of Experimental Animals in Zhejiang Province’. This work was approved by the Animal Ethics Committee in the School of Medicine, Zhejiang University (ETHICS CODE Permit NO. ZJU2011-1-11-009Y). The Embryos were fixed in 4% PFA (PBS) for 12 h at 4 °C. WISH probes were labeled with digoxigenin  according to the manufacturer’s instructions. WISH was performed as previously described . To geneThe intestine tissue or liver bud were dissected from 5 dpf-old zebrafish embryos. Intestine tissues from ~30 embryos or liver buds from ~50 embryos were pooled and then treated with TRIZOL for total RNA extraction. The micro-dissected intestine was also cut into three fragments corresponding to the anterior, middle and posterior portions of the intestine, and the fragments were pooled for total RNA extraction. The obtained total RNA (~200–400 ng/sample) was used for library construction via the Smart-seq2 protocol as described in . LibraryAnalysis of the differentially expressed genes (DEGs) was conducted with DESeq2 v1.28.1) and gene ontology (GO) set overrepresentation analysis was performed with ClusterProfiler (v3.16.1) .1 and ge. TFBS anhttps://dropest.readthedocs.io/en/v0.8.5/dropest.html). Further analyses were performed with the R package Seurat  (https://mran.microsoft.com/snapshot/2020-05-30/bin/windows/base/). A threshold of unique counts over 2500 or less than 200 was set to filter cell doublets. Low-quality cells that had >20% mitochondrial counts were filtered.The scRNA-seq of the intestine tissue and liver bud was performed following the microwell-Seq approach in this study . For eachttp://ftp.ensembl.org/pub/release-107/, accessed on 14 May 2022) showed that 58.4–63.4% and 80–81.9% of the total clean reads from the three intestine and three liver samples, respectively, were mapped to the zebrafish genome  from the WT zebrafish embryos at 5 dpf and obtained three independent samples by pooling intestine tissue from ~30 embryos in each sample A. Similah genome . 2 (fold-change)| > 2 and p < 0.01 in intestine or liver), we found that a total of 2110 genes were more abundant in the intestine (termed as intestine-enriched), while 1637 genes were enriched in the liver (termed as liver-enriched)  > 1) were identified . Throughnriched) .cdx1b, fabp2, fabp6, etc.) or liver  [http://zfin.org) . WISH confirmed the specific expression of the well-known intestinal genes ldha, ifi30, alpi.2, chia.1, slc34a2a and stk25  to the 2110 intestine- and 1637 liver-enriched genes. In total, 385 intestine-enriched and 308 liver-enriched genes passed the criteria and were arbitrarily classified as the embryonic intestine-specific and liver-specific genes, respectively . The maja, etc.) ,33, whernd stk25 D and theat 5 dpf E. Meanwhspecific D and CR6ic genes E.slc5a1, slc15a1b, slc18b1, slc25a24, etc.     and ‘actin filament organization’  were also among the top 10 categories  among the top 10 categories suggested that the intestine exhibited high antioxidant activities  and molecular function (GO_MF) terms resulted in, though in different orders, similar top 10 categories A. We ana24, etc. . Others o, etc.) , ‘lipid b, etc.)  and ‘car1, etc.) . Interestegories , reflecttivities . The enrmvda, acaca, lipca, fasn, cpt1ab, etc.)      , ‘negati1, etc.) , ‘blood 0, etc.)  and ‘comb, etc.) . Notablyb, etc.) A. The GOb, etc.) . Notablya, etc.) . The enrWe noticed that, although ‘lipid metabolic process’ under the GO_BP term and ‘oxidoreductase activity’ under the GO_MF term were among the top 10 categories in both the intestine and liver compare A, the excdx1b, hnf4a and gata5 are known to regulate the development of the gut epithelium; atoh1, ets2, her9 and family members of klf and elf are known to regulate the differentiation of absorptive and secretory intestinal epithelial cells; and xbp1, smad5, cebpa and cebpb are known to regulate the various metabolic pathways  and hnf1b are known to regulate the cholangiocyte differentiation; and family members of cebp, ppar, hmgb and klf are known to regulate genes involved in the hepatic metabolic pathways  | < 2), including pan-endodermal TFs, such as foxa1, gata6, hnf4a and hnf4b  . WISH us with WT . In addind hnf4b A.cdx1b, osr2, pdx1, hoxb13a and si:ch211-202h22.9 in the intestine  for the purpose of identifying TFs responsible for regulating the expression of these genes. This program was built based on human data, so it first needed to match the zebrafish target genes to their corresponding human orthologues and then search for their human TF regulators. In total, 1438 out of the 2110 genes had a match in the database, and 27 human TFs (FDR < 0.01) were identified to regulate their expression. Each TF was found to regulate multiple target genes. and each target gene could be regulated by different TFs   . To our 2 (fold-change)| > 1 and p < 0.05) showed that 452 genes were enriched in S1, 309 in S2 and 2449 in S3 , middle (S2) and posterior (S3) segments A. S1, S249 in S3 .zgc:112146, neu3.3 and stom13b to be enriched in S1   , suggesta, etc.) , suggesta, etc.) , suggestpdx1, sox19a and foxi3a were enriched in S1; osr2 was enriched in S2; and cdx4, foxd2 and many hoxa and hoxb family members were enriched in S3 , enteroendocrine cells (+pyyb) and smooth muscle cells (+tagln), respectively, along the intestine (UMAP of the scRNA-seq data of the 1229 cells grouped the cells into 10 clusters (cluster 0–9) A, left. ntestine C.gsto2 revealed its higher expression in the intestinal bulb  appeared to be restricted in the cluster 0 cells, while some others were shared with cluster 4  or with cluster 7   . Based ond fabp6 D, we sugThe cluster 1 feature genes were shared by the goblet, enteroendocrine, hindgut and Best4/Otop2 cells . Considefdps and hmgcs1 were highly enriched in the cluster 4 hepatocytes | > 2) and more abundancy (TPM > 50) between the intestine and liver , cluster 2 , cluster 3 , cluster 4 , cluster 5 , cluster 6 , cluster 7 , cluster 8  and cluster 9  were among the 385 intestine-specific genes (compare To identify more feature genes for each cell type in the intestine and liver at 5 dpf, we examined the expression of the 385 intestine-specific and 308 liver-specific genes in each individual cell acquired by the intestine and liver scRNA-seq, respectively. The scenario was that these genes were obtained from RNA-seq analysis by applying stringent criteria for higher specificity (|lognd liver . We firsnd liver , furthernd liver A,B. Notand liver . Detaile compare . TherefoNext, we examined the expression of 308 liver-specific genes at the single-cell level . Thirteeefna2a and CT990561.1 related to Ephrin signaling, which is essential for the establishment of the intestinal lumen, were identified [bmpr2b related to Bmp signaling, fgf10b related to Fgf signaling and dkk3a related to Wnt signaling, which are known to be essential for liver development and function, were identified [Notch1b and jag1a, related to Notch signaling during the development of the biliary system, were also identified among the liver-enriched genes [hhex and prox1a, two well-known genes for liver development, as well as stat5a, were identified [pdx1 and cdx1b, for intestinal development and functioning, were identified among the intestine-enriched genes [By 5 dpf, the zebrafish digestive system has been well established to accomplish its physiological function and meanwhile continues their development. In this report, we obtained the gene expression profiles in the zebrafish intestine and liver bud at 5 dpf. Our data are complementary to previous reports ,20,21,41entified . Among tentified ,46,47. Ned genes ,49. The entified ,14,50. Oed genes ,52,53.neu3.3- and pdx1-positive cells were located at the anterior region of the intestinal bulb. Because the hepatopancreatic duct was fused with the intestine at the anterior region of the intestinal bulb, these cells might function to collect the secreted products from the liver and pancreas [tmsb1-positive cells were located at the posterior region of the intestinal bulb, while osr2-positive cells were at the mid-intestine region. The hindgut was marked by slc10a2-positive cells, while the proctodeum was marked by stk25a- and ctsl.1-positive cells. Based on this new knowledge, future studies will focus on how these cells are specialized, especially regarding the genetic and signaling network formed by specific signaling molecules and TFs.Based on morphology and molecular markers, previous studies have divided the zebrafish intestine into the intestinal bulb, mid-intestine, hindgut and proctodeum regions ,19. A repancreas ,19. Plot


Chilling injury is a major hindrance to cucumber fruit quality during cold storage.+-ATPase, Ca2+-ATPase, cytochrome c oxidase, and succinate dehydrogenase in cucumber fruits.In this study, we evaluated the effects of fucoidan on fruit quality, reactive oxygen species homeostasis, and energy metabolism in cucumbers during cold storage. The results showed that, compared with the control cucumber fruit, fucoidan-treated cucumber fruit exhibited a lower chilling injury index and less weight loss, as well as reduced electrolyte leakage and malondialdehyde content. The most pronounced effects were observed following treatment with fucoidan at 15 g/L, which resulted in increased 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical scavenging rates and reduced superoxide anion production rate and hydrogen peroxide content. The expression and activity levels of peroxidase, catalase, and superoxide dismutase were enhanced by fucoidan treatment. Further, fucoidan treatment maintained high levels of ascorbic acid and glutathione, and high ratios of ascorbic acid/dehydroascorbate and glutathione/oxidized glutathione. Moreover, fucoidan treatment increased the activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase and their gene expression. Fucoidan treatment significantly delayed the decrease in ATP and ADP, while preventing an increase in AMP content. Finally, fucoidan treatment delayed the decrease of energy charge and the activities and gene expression of HAltogether, our findings indicate that fucoidan can effectively enhance antioxidant capacity and maintain energy metabolism, thereby improving cucumber cold resistance during cold storage. Cold storage is a common method of preserving horticultural products after harvest. However, for cold-sensitive horticultural products, such as cucumber , peach , and kiw2O2) to maintain H2O2 at a low level, minimize the production of hydroxyl radicals (-OH), and protect cell membrane structure (2O2 from plant tissues. The AsA-GSH cycle promotes the production of two non-enzymatic antioxidants (AsA and GSH) to maintain the balance of redox substances in plants (Cold stress induces the accumulation of reactive oxygen species (ROS) in plant tissues, leading to a series of oxidative damage events, such as membrane lipid peroxidation, and eventually inducing cell death . The platructure . The ascn plants . The ROSn plants , bell pen plants , okra , and succinate dehydrogenase (SDH) are key enzymes involved in energy synthesis in the mitochondrial membrane  were collected during the commercial harvest period (approximately 10 d after flowering) from a farm in Nanchang, Jiangxi, China. Fruits were washed with distilled water and dried naturally. Fruits of uniform size and without any disease or defects were randomly separated into four groups and coated with 0 (control), 5, 10, or 15 g/L fucoidan . Treatments were performed in triplicates comprising 300 fruits for each replicate. Treated fruits were packed in 0.01 mm thick polyethylene plastic bags and stored for 12 days at 4°C and 90% relative humidity. For each replicate, 20 fruits were collected every three days, 15 of which were placed at 20°C for two days to observe CI, and the pulp  of the remaining 5 fruits was collected at random and stored at -80°C after quick freezing under liquid nitrogen.Cucumbers  from five cucumber fruits were prepared for the measurement of electrolyte leakage. Electrolyte leakage, and malondialdehyde (MDA) content of the experimental cucumber fruits were measured as previously described .For weight loss: five cucumber fruits were weighed using an electronic scale at a time, and the measurements were repeated three times. Weight loss was calculated as follows:2O2 content using a DPPH scavenging rate detection kit , -OH scavenging rate detection kit , 2O2 content detection kit , respectively. The unit percentage (%) represents the DPPH and -OH scavenging rates. The 2O2 content are expressed as μmol s-1 kg-1 and mmol kg-1, respectively.One gram of pulp from control and fucoidan-treated cucumber fruits was used for the measurement of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging rate, -OH scavenging rate, -1. One U of POD and CAT activities represents an absorbance change of 0.01 per gram of sample per minute at 470 nm and 240 nm, respectively. In turn, 1 U of SOD activity represents the amount of SOD per gram of sample in 1 mL of reaction solution when SOD inhibition reached 50%.To quantify POD, SOD, and CAT activities, 1.0 g of pulp from control and fucoidan-treated cucumber fruits was homogenized with 5 ml of 50 mM pre-cooled sodium phosphate buffer (pH 7.5) containing 5 mM dithiothreitol and 5% polyvinylpyrrolidone. The mixture was then centrifuged at 12,000 g for 15 min at 4°C and the supernatant was collected. POD, SOD, and CAT activities were determined as previously described , and exp-1, where 1 U represents 1 μmol of enzyme oxidized per gram of sample per minute.The activities of APX, MDHAR, DHAR, and GR were determined using the appropriate test kits  with absorbance measured at 290, 340, 412, and 340 nm, respectively. Enzyme activities are expressed as U kg-1) was determined as previously described (One gram of homogenized cucumber fruit was mixed with 15 mL of pre-cooled 5% (w/v) trichloroacetic acid and centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected and AsA and DHA content (mg kgescribed . DHA con-1) were detected using the appropriate test kits  with absorbance measured at 412 nm.GSH and GSSG contents  were determined by high-performance liquid chromatography  using a C18 column  as previously described  with absorbance measured at 660 nm. One gram of pulp from control and fucoidan-treated cucumber fruits was used for measurement of CCO and SDH activities as previously described . Pairwise comparisons were made using the single factor method in SPSS26.0, and significant differences were analyzed using the least significant difference test and Duncan’s method (P< 0.05 or P< 0.01). Graphs were drawn using GraphPad Prism 8.0.Water-soaked depressions of the epidermis are the most obvious symptom of CI in cucumbers. After 12 days of storage at 4°C, the CI index was 0.65. Compared with controls, coating with 5, 10, or 15 g/L fucoidan decreased the CI index of cold-stored cucumbers (The DPPH and -OH scavenging rates in cucumbers initially increased and then decreased during cold storage. The DPPH scavenging rate peaked on day 6 of storage, while the -OH scavenging rate peaked on day 9. The DPPH and -OH scavenging rates of fucoidan-treated cucumber fruits were higher than those of control cucumbers (2O2 content, which peaked on day 6 of storage and subsequently declined slightly. In contrast, fucoidan treatment significantly reduced the accumulation of H2O2 in cold-stored cucumber fruits (The rate of CsPOD, CsCAT2, and CsSOD (The activities of the POD and CAT enzymes initially increased in control cucumbers upon cold storage, reaching a maximum on day 6 of storage, and gradually declining thereafter. Conversely, POD and CAT activities increased by 65% and 39%, respectively, following fucoidan treatment, compared to the control group on day 6 of cold storage, and the subsequent decline in these activities was considerably less pronounced than that in the controls (The AsA content of cucumber fruits decreased gradually during cold storage in both treated and control fruit groups, but remained higher in the fucoidan-treated group than in the control group on day 6. On day 12, the AsA content of fucoidan-treated fruit was 12% higher than that in control group (CsAPX, CsMDHAR, and CsDHAR were consistent with the trends described for enzyme activity, with fucoidan treatment inducing the expression of these genes. Finally, the expression level of CsGR in the fucoidan-treated group was significantly higher than that in the control group on days 3 and 6 (APX activity initially increased and then decreased during cold storage, reaching a maximum on day 6. The APX activity of fruits in the fucoidan-treated group was significantly higher than that in the control group throughout the storage period (The ATP and ADP contents of cucumber fruits declined continuously during cold storage. However, ATP and ADP were significantly higher in fucoidan-treated cucumbers than in control ones throughout the storage period. On day 12, ATP and ADP contents in the treatment group were 14% and 35% higher than those in the control group, respectively (+-ATPase initially increased and then decreased during cold storage. The activity of H+-ATPase in fucoidan-treated cucumbers peaked on day 6 and was 33% higher than that of the control group on day 12 (2+-ATPase activity remained stable during cold storage, and fucoidan-treated fruit maintained a higher level of activity than control ones (+-ATPaseCsH, 2+-ATPaseCsCa, CsCCO, and CsSDH corresponded with these trends in enzyme activity. Indeed, we found that fucoidan treatment significantly induced the expression of all four genes, thereby regulating the enhancement of their corresponding enzyme activities (Activity of HAstragalus polysaccharides delayed the CI and attenuated postharvest peel browning of banana fruit (CI easily occurs in cold-stored cucumber fruits after harvest and leads to a decrease in fruit quality and commodity value . Polysacna fruit . In vegena fruit . Chitosana fruit . Fucoidana fruit ., which na fruit . Fucoidana fruit . In this2O2 and 2O2 and O2-, thus maintaining its integrity and minimizing CI (- production rate, and maintained high CAT and POD activities, thereby inhibiting the accumulation of H2O2. Similarly, previous studies have reported that fucoidan treatment enhances antioxidant activity and improves the cold tolerance of strawberries (Cold stress-induced plant damage typically involves an imbalance between ROS production and depletion ; excessit system . The enzt system . These tizing CI . In thiswberries . Thus, w2O2 scavenging system in plants. AsA and GSH are important non-enzymatic antioxidants in this cycle, which can scavenge excessive ROS produced by plant cells in response to low-temperature stress injury (2O2 in cells and convert it into H2O and O2- (2O2scavenging, reducing the degree of membrane lipid peroxidation, and delaying the occurrence of CI in cucumber fruits.The AsA-GSH cycle is an important Hs injury . The AsAs injury . In thiss injury , and bels injury . GSH/GSSs injury . Hot wats injury . APX, MD and O2- . Enhance and O2- , peaches and O2- , okra (L and O2- , and ban and O2- . These r+-ATPase, and Ca2+-ATPase are key respiratory enzymes located in the inner mitochondrial membrane, and their activities reflect the energy production status of mitochondria (+-ATPase catalyzes ATP synthesis by establishing an H+ electrochemical gradient on both sides of the cell membrane to generate transmembrane proton propulsion (2+-ATPase is a Ca2+ pump responsible for transporting Ca2+ into the mitochondria, and a decrease in Ca2+-ATPase activity leads to the accumulation of Ca2+ in the cytoplasm, leading to the destruction of membrane structural integrity (+ and ATP (Energy molecules, such as ATP, play an important role in maintaining the integrity of cell membranes . Energy chondria . H+-ATPaopulsion . Ca2+-ATntegrity . SDH cat and ATP . CCO pro and ATP . Cold st and ATP . Altoget2O2 and Fucoidan treatment effectively reduced CI in cucumber fruits by modulating ROS scavenging and energy metabolism. Fucoidan treatment can reduce the accumulation of HThe original contributions presented in the study are included in the article/DL: Conceptualization, methodology, software, writing—original draft preparation. RY: validation, formal analysis, data curation, writing—original draft preparation. MX: investigation, resources. SL: visualization, data curation. JC: writing—review and editing, supervision. ZG: writing—review and editing, project administration, funding acquisition. All authors contributed to the article and approved the submitted version.


Alzheimer’s disease (AD) is a devastating progressive neurodegenerative disease characterized by neuronal dysfunction, and decreased memory and cognitive function. Iron is critical for neuronal activity, neurotransmitter biosynthesis, and energy homeostasis. Iron accumulation occurs in AD and results in neuronal dysfunction through activation of multifactorial mechanisms. Mitochondria generate energy and iron is a key co-factor required for: (1) ATP production by the electron transport chain, (2) heme protein biosynthesis and (3) iron-sulfur cluster formation. Disruptions in iron homeostasis result in mitochondrial dysfunction and energetic failure. Ferroptosis, a non-apoptotic iron-dependent form of cell death mediated by uncontrolled accumulation of reactive oxygen species and lipid peroxidation, is associated with AD and other neurodegenerative diseases. AD pathogenesis is complex with multiple diverse interacting players including Aβ-plaque formation, phosphorylated tau, and redox stress. Unfortunately, clinical trials in AD based on targeting these canonical hallmarks have been largely unsuccessful. Here, we review evidence linking iron dysregulation to AD and the potential for targeting ferroptosis as a therapeutic intervention for AD. Alzheimer’s disease (AD) is a progressive age-dependent neurodegenerative disease for which there is currently no cure ,2. AD isIron is important for neuronal activity and it plays a critical role in myelination, neurotransmitter biosynthesis and energy metabolism ,13. BecaDamaged mitochondria can increase production of ROS. ROS, when produced in large quantities, can result in oxidative stress and produce oxidative damage to proteins, DNA, and lipids ,21,22. I2+ and Fe3+, which enables iron to act in electron transfer reactions ,tive HO• .ROS generated within the proximity of membrane lipids can cause oxidative damage through the initiation of peroxidation of polyunsaturated fatty acids (PUFA), resulting in lipid hydroperoxide. Lipid hydroperoxide can be converted to a non-harmful alcohol product by GPx4 . HoweverIn humans, iron content is stable with an estimated 1–2 mg being ingested and a similar amount eliminated daily ,14,16. T3+ atoms to circulating transferrin (TF) to form a Fe3+-TF complex (3+-TF complex binds to the transferrin receptor (TFR), the ternary Fe3+-TF-TFR complex is transported into the cell, and subsequently, to the endosome [3+ in the endosome is coupled with the influx of a proton to reduce the endosomal pH [3+ to Fe2+. Fe2+ is then transported into the cytosol by DMT1 coupled to extrusion of proton, restoring the endosome back to its original neutral condition. While in the cytosol, Fe2+ is transferred to mitochondria for use in heme biosynthesis, formation of iron-sulfur clusters, and as co-factors for mitochondrial enzymes [Cellular iron uptake starts with the binding of two Fe complex . When thendosome ,14,16,75 enzymes ,77.2+ is stored in a non-toxic form as Fe3+ in ferritin. Ferritin has two isoforms: heavy (H) and light (L). Heavy (H) ferritin possess ferroxidase activity and is involved in rapid iron uptake and reutilization. Light (L) ferritin is thought to play role in the nucleation of iron for long term storage. Iron when not stored as ferritin is exported extracellular through ferroportin [In addition, Feroportin ,16.2+ out of the cell by ferroportin requires ferroxidase activities of hephaestin and ceruloplasmin, a multicopper ferroxidase enzyme that oxidizes Fe2+ to Fe3+ [3+ into the circulating system. However not all tissue have ceruloplasmin. For example, in the brain, amyloid precursor protein (APP), an integral membrane protein that acts as a cell surface receptor [The export of Fe to Fe3+ . Thus, creceptor , is utilreceptor ,80,81.Cellular iron levels are regulated by the activity of two iron-regulatory protein (IRP) factors that sense the availability of iron ,15. Upon3+) and decreased transverse relaxation rate (R2) to measure tissue damage, progressive development of AD was shown to be associated with increased brain iron levels [3+ levels and significant tissue damage in hippocampus compared to the thalamus, consistent with the hippocampus of AD patients being highly susceptible to iron accumulation and damage. Importantly, these results demonstrate that iron accumulation in the brain can be region specific. As a result, some regions of the brain  are more prone to iron accumulation, and thus damage, than other regions . In line with this observation, brain iron burden correlates well with a decline in cognitive function in AD [Iron concentrations in the brain increase with age ,84,85,86on in AD ,88,89. Fon in AD ,90,91. Ton in AD . From 11on in AD . Others on in AD .2+ and/or Fe3+) that contributes to cognitive dysfunction in AD is unknown. To partially address this issue, an in vitro study used a multi-disciplinary approach, including X-ray micro-spectroscopy, X-ray absorption spectroscopy, electron microscopy and spectrophotometric Fe2+ quantification techniques, to interrogate the interaction between Aβ1-42 and Fe3+ [3+ and Aβ1-42 form aggregates that promote the reduction of Fe3+ to Fe2+ [2+ is a redox active form of iron that reacts with H2O2 to produced HO•, which drives oxidative damage of DNA, proteins, and lipids  How does an increase in iron levels in specific brain regions promote AD progression? (2) What are the underlying molecular mechanisms by which elevated brain iron toxicity correlate with increases in amyloid plaque? (3) Does iron-induced dysfunction in AD result from cellular or intracellular iron dysregulation?The brain accounts for about 20% of whole body oxygen consumption under resting conditions, despite comprising only about 2% of the mammalian body mass. This indicates that the brain is a highly energetic organ, and thus, is exquisitely sensitive to changes in energy status. Energy dysregulation is among one of the earliest signs of neurodegeneration in AD and is largely attributed to impaired mitochondrial function. Several components of the mitochondrion are altered during neuronal energy crises. Below, we focus on dysfunction in mitochondrial bioenergetics , mitocho2•− or H2O2 [2O2, is a primary mechanism for Aβ1-42 related hypometabolism [Mitochondrial bioenergetics is directly impaired by iron dysregulation through the inhibition of the ETC and indirectly through increased generation of ROS (via mitochondria and NADPH oxidase). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are membrane bound proteins that transfer electron from NADPH to oxygen to generate O or H2O2 ,101,102. or H2O2 ,104,105. or H2O2 ,106,107. or H2O2 . Consist or H2O2 . This de or H2O2 . RNA-seq or H2O2 . Finallytabolism . Specifitabolism . Taken t3+ promotes mitochondrial fragmentation through calcinuerin-mediated dephosphorylation of DRP1 (at ser637) in mouse HT-22 hippocampal neurons [2+ and mitochondrial Ca2+ overload, which then initiates mitochondrial fragmentation [3+ reduced expression of mitochondrial fusion protein OPA1, leading increased mitochondrial fragmentation which resulted in fragmented mitochondria characterized by, mitochondrial number, and total mitochondrial area [Mitochondria are dynamic organelles that constantly change their location, length, number, shape and size to meet cellular energy demands ,113,114. neurons . The mecentation ,119,120.ial area ,121. Indial area ,125,126.ial area ,125,126.+/NADPH ratio (iNAP) [Currently there is a gap in our understanding of how dysregulation in iron alters mitochondrial function in AD. Prior studies have associated iron dysregulation with progression and advance form of AD in both in-vitro and in-vivo ,92,93,94o (iNAP)  could beFerroptosis is a non-apoptotic mechanism of cell death that is driven by iron dependent lipid peroxidation ,27,28,29GPx4 activity strongly correlates with ferroptosis. For example, pharmacologic and genetic inhibition of GPx4 activity exacerbates ferroptosis, while GPx4 overexpression ameliorates ferroptosis-induced cell death ,137,138.Ferroptosis is linked to many diseases including cancer, ischemia-perfusion injury and neurodegeneration ,157,158.Reduced GPx4 activity is a hallmark of ferroptosis and plays critical role in AD pathogenesis . AblatioTransgenic overexpression of GPx4 in 5xFAD mice protects 5xFAD mice from developing an AD phenotype . SpecifiSeveral additional studies employed small molecules as a tool to study neurological damage in AD ,163,164.Overall, based on the above studies, mice that lack Fpn and GPx4 exhibit hallmarks of ferroptosis similar to that associated with AD. Moreover, mouse models of AD are characterized by biomarkers of ferroptosis including impaired mitochondrial function and high levels of lipid peroxidation.AD is a devastating progressive neurodegenerative disease, with patients experiencing signs of impaired memory loss and deterioration in cognitive ability due to progressive neuronal loss ,2,3,4. TIn this review, we highlight the need for a paradigm shift in AD research. Specifically, we suggest that the most effect interventions will utilize an integrative approach that combats the complex heterogenous nature of AD. For example, interventions designed to prevent a cellular energy crisis by targeting ferroptosis may provide a novel mechanism to protect against AD. Targeting ferroptosis will unearth previously unknown mechanisms and could serve as a potential therapeutic intervention against AD.


NAGLU gene encoding alfa-N-acetyl-glucosaminidase. Enzyme replacement therapy for neuronopathic MPS requires efficient enzyme delivery throughout the brain in order to normalize HS levels, prevent brain atrophy, and potentially delay cognitive decline.Sanfilippo type B is a mucopolysaccharidosis (MPS) with a major neuronopathic component characterized by heparan sulfate (HS) accumulation due to mutations in the n = 22) were treated with tralesinidase alfa administered i.c.v. The patients were monitored for drug exposure; total HS and HS nonreducing end (HS-NRE) levels in both cerebrospinal fluid (CSF) and plasma; anti-drug antibody response; brain, spleen, and liver volumes as measured by MRI; and cognitive development as measured by age-equivalent (AEq) scores.In this phase I/II open-label study, patients with MPS type IIIB  phase, a 300 mg dose of tralesinidase alfa was necessary to achieve normalization of HS and HS-NRE levels in the CSF and plasma. In Part 2, 300 mg tralesinidase alfa sustained HS and HS-NRE normalization in the CSF and stabilized cortical gray matter volume (CGMV) over 48 weeks of treatment. Resolution of hepatomegaly and a reduction in spleen volume were observed in most patients. Significant correlations were also established between the change in cognitive AEq score and plasma drug exposure, plasma HS-NRE levels, and CGMV.Administration of tralesinidase alfa i.c.v. effectively normalized HS and HS-NRE levels as a prerequisite for clinical efficacy. Peripheral drug exposure data suggest a role for the glymphatic system in altering tralesinidase alfa efficacy.Clinicaltrials.gov NCT02754076.BioMarin Pharmaceutical Inc. and Allievex Corporation. Lysosomal storage diseases (LSDs) are a family of genetic disorders affecting the breakdown and recycling of complex molecules within the lysosomes . The lysN-acetyl-glucosaminidase   is specifically due to mutations in a gene coding for alfa-.2.1.50) , it was .2.1.50) . Althoug.2.1.50) .Endogenous NAGLU contains mannose-6-phosphate (M6P) residues, which enable receptor-mediated endocytosis and targeting to the lysosome where HS is degraded . UnfortuThe objectives of this study were to evaluate the safety, tolerability, and efficacy of tralesinidase alfa administered via an implanted i.c.v. device to patients with MPS IIIB. Here, we report the pharmacokinetics (PK) and pharmacodynamics (PD) results of our clinical investigation of tralesinidase alfa in the treatment of children with MPS IIIB over a 48-week period. Overall, the current study highlights the ability of tralesinidase alfa to alter the natural course of MPS IIIB and suggests that this positive effect may lead to meaningful clinical benefits.Interventional Study 250-201 was divided into Part 1 and Part 2 . Part 1 Patient 9001 in Study 250-201 Part 1 received 28 weekly doses at 30 mg of tralesinidase alfa, followed by 10 doses at 100 mg, and then 11 doses at 300 mg before being recruited to Study 250-201 Part 2. Patient 9002 received 8, 10, and 19 doses of 30, 100, and 300 mg tralesinidase alfa, respectively, before being recruited to Study 250-201 Part 2, whereas patient 9003 received 20, 10, and 9 doses of 30, 100, and a 300 mg dose of tralesinidase alfa in Study 250-201 Part 1.Characteristics of the participants in Study 250-201 Part 2 are summarized in No deaths occurred during the course of the study. One participant discontinued the study following a serious adverse event (SAE) of subdural hygroma associated with increased intracranial pressure occurring after the seventh dose of tralesinidase alfa. This event was considered common terminology criteria for adverse events (CTCAE) grade 3 and was assessed as unrelated to the study drug or the device by the site’s principal investigator. Clinical symptoms  resolved within 24 hours with medical management. In total, this patient received only 8 doses of tralesinidase alfa. The most common treatment-emergent adverse events (TEAEs) included vomiting, pyrexia, upper respiratory tract infection, headache, and CSF pleocytosis. SAEs assessed by investigators to be related to the study drug were CSF pleocytosis, vomiting, angioedema, fluctuating consciousness, and pyrexia. SAEs assessed by investigators to be related to the i.c.v. device were infection, device malfunction, CSF leakage, and wound infection. Overall, these TEAEs and SAEs were consistent with known complications of enzyme replacement therapy (ERT), i.c.v. devices, and/or neurodegenerative disease in pediatric populations.Eight hypersensitivity events were reported for 5 patients, 5 events of rash, and 1 event each of angioedema, choking, and maculopapular rash. Blood samples for total IgE, C4, serum tryptase, and drug-specific IgE did not show evidence of anaphylactic reaction in any of these 5 patients for any of the hypersensitivity events observed.n = 2 participants, Study 250-201 Part 1), 100 mg , and 300 mg tralesinidase alfa . Analysis of the PK data is focused on the 300 mg dose in 17 participants from Study 250-201 Part 2, who were not previously exposed to tralesinidase alfa.0–last, achieved for patients 9002 and 9003 after a single dose of 30 mg tralesinidase alfa, were 1,130,000 and 1,620,000 ng/mL/h, for a maximum concentration (Cmax) of 403,000 and 470,000 ng/mL, measured 1 hour after drug administration, respectively. The average CSF AUC0–last and Cmax were 19,100,000 ng/mL/h and 3,440,000 ng/mL , respectively, achieved for patients (n = 17) receiving the first i.c.v. dose of tralesinidase alfa (300 mg) in Study 250-201 Part 2 (0–last) was 5,050,000 ng/mL/h and the highest was 49,800,000 ng/mL/h. The estimated average t1/2 and tlast were 5 hours and 68 hours, respectively, suggesting that tralesinidase alfa was efficiently distributed throughout the brain and quickly absorbed via the CI-MPR, as intended. The average AUC0–last and Cmax appeared to scale linearly with the dose, suggesting that the drug exposure achieved in CSF of patients with MPS IIIB was dose proportional  significantly decreased over time from a mean value of 18,800 ng/mL/h at week 1 to 10,900 ng/mL/h at week 5, to 9,700 ng/mL/h at week 12, and to 7,260 ng/mL/h at week 36 . At week 1, drug exposure was, on average, 1,000-fold lower in plasma compared with exposure values in CSF  and dropped to 22,000-fold lower by week 36 . Of note, decreases in drug exposure in plasma were not substantial from week 5 to week 12 or from week 12 to week 36. The estimated tmax in plasma was 7.5 hours at week 1 (n = 16) and remained approximately 10 hours for treated patients with quantifiable drug exposure in plasma at weeks 5, 12, and 36, i.e., an AUC0–last above 10,000 ng/mL/h. For the other participants, exposure was too low to estimate the Cmax and tmax.Plasma exposure  in CSF at weeks 36 and 48 (10 = 2.61) at week 4 to 1:3,645 (log10 = 3.56), 1:98,415 (log10 = 4.99), and 1:37,345 (log10 = 4.57) at weeks 12, 36, and 48, respectively , when the median ADA titer in serum was only 1:405 at week 4. In vitro cell-based studies suggested that antibodies detected in ADA-positive CSF samples were not able to neutralize the uptake of tralesinidase alfa .Total HS and HS-NRE concentrations in CSF were measured weekly over the course of Study 250-201 Part 1 . For refDuring the course of Study 250-201 Part 2, HS-NRE levels in the CSF were normalized after the first 4 weeks of treatment to levels below the 10 ng/mL higher-end-of-normal cutoff . Total HP < 0.0001). HS-NRE levels in plasma were significantly reduced to less than 40 ng/mL in participants after 4 weeks of administration of 300 mg tralesinidase alfa  at the baseline visit for Study 250-201 Parts 1 and 2  and cognitive score, and 2 were the patients who received only 17% and 57% of the expected doses of tralesinidase alfa. By week 24 of Study 250-201 Part 2, the mean liver volume was reduced to 0.82 L/m2 , and by week 48, the mean value was 0.87  when comparing measurements at week 48 versus those at baseline.Changes in liver and spleen volumes at weeks 1, 24, and 48 of Study 250-201 Part 2 are depicted in 2 , 13, the 1 and 2 . Of the 2 of BSA, for a mean value of 0.23 , whereas normal spleen volumes for 2- to 10-year-old children were estimated to be between 0.04 and 0.15 L/m2 of BSA , and this decrease was maintained through week 48  when comparing the volumes at week 48 with those at baseline.At Study 250-201 Part 1 or Part 2 baseline, spleen volumes in children with MPS IIIB ranged from 0.15 to 0.44 L/m2 of BSA . By weekCGMV is a measure of the region of the brain most affected by atrophy in patients with MPS IIIB within the age range included in Study 250-201 . PatientP < 0.0001, 2-tailed, paired t test in both cases). Every participant in Study 250-201, except 2, lost CGMV during the course of Study 250-201  of CGMV from baseline to week 24, but lost only 2 mL from week 24 to week 48, for a total loss of 60 mL  from week 1 to week 48 .Nineteen of 22 patients treated with tralesinidase alfa had a total cerebellar volume within normal range at baseline . Three p0–last, at weeks 5, 12, and 36 of Study 250-201 Part 2, HS-NRE biomarker levels, and CGMV over the 48-week study period. We noted a significant correlation between the change in cognitive AEq over the course of Study 250-201 Part 2 and average tralesinidase alfa exposure (AUC0–last) in plasma at weeks 5, 12, and 36 . Eight of the 12 patients with plasma drug exposure AUC0–last of less than 6 μg/mL/h had plasma HS-NRE cumulative concentrations above the normal range, i.e., between 360 and 540 ng/mL/week .We also observed an inverse correlation between cumulative plasma HS-NRE concentrations and a change in cognitive AEq score from week 1 to week 48 of Study 250-201 Part 2 . We noter = 0.59, P = 0.0082, n = 19). All 4 patients with change in CGMV of 0 mL or greater from week 1 to week 48 of Study 250-201 Part 2 had a change in AEq score of 0 or higher, while the patient who received only 17% of their intended dose of compound lost 54 mL in CGMV and had a loss of 21 months on the cognitive AEq scale.We observed a correlation between a change in the cognitive AEq score over the course of Study 250-201 Part 2 and a change in CGMV during the same period  administered weekly via i.c.v. infusion are in line with those for other ERTs, especially agents such as cerliponase alfa administered via i.c.v. dosing .Our study of 22 patients with MPS IIIB, aged 25–118 months at baseline, established an effective dose of 300 mg tralesinidase alfa administered i.c.v. as necessary to achieve and sustain normalization of both total HS and disease-specific HS-NRE levels. The normalization of total HS and HS-NRE levels in the CSF provides evidence that tralesinidase alfa can be potentially beneficial to patients with MPS IIIB. While HS-NRE normalization in CSF is critical and a prerequisite for potential efficacy, the concentration of both tralesinidase alfa and the greater dynamic range of measurement of HS-NRE levels in plasma might be indicative of the efficacy of tralesinidase alfa in the CNS . We hypoN-sulfoglucosamine sulfohydrolase resulted in no resolution of hepatosplenomegaly and no clear clinical benefits in a clinical study involving 6 patients with MPS IIIA. Although significant, the reduction in HS levels in the CSF of these patients was probably too slow and too little to translate into clinical benefits . Neutralizing antibody testing for positive ADA samples in the CSF were low; less than 1:100 was the highest titer measured (data not shown), suggesting no effect of ADAs on drug exposure or efficacy in the brain tissue, the primary target organ. The majority of tralesinidase alfa–treated patients  had normalized HS and HS-NRE levels in both the CSF and plasma during the course of Study 250-201 Part 2, regardless of their ADA titers , and stable-dose (Part 2) study involving males and females between 1 and less than 11 years of age  with a confirmed diagnosis of MPS IIIB. Three patients, i.e., patients 9001, 9002, and 9003, were enrolled in Study 250-201 Part 1 and received weekly escalating doses, i.e., 30, 100, and 300 mg of tralesinidase alfa via an i.c.v. device such as the Ommaya reservoir and a catheter implanted into the lateral ventricle. Twenty-three patients, 19 of whom were rolled over from our natural history study  evaluate the safety and tolerability of tralesinidase alfa administered to patients with MPS IIIB via an implanted i.c.v. reservoir and catheter, and (b) evaluate the impact of tralesinidase alfa on cognitive function as assessed by the DQ. Additional objectives were to (a) evaluate the impact of tralesinidase alfa on cognitive function as assessed by AEq score; (b) characterize the PK of tralesinidase alfa in CSF and plasma; (c) characterize the immunogenicity of tralesinidase alfa in CSF and serum; (d) evaluate the impact of tralesinidase alfa treatment on CSF, serum, and urine GAGs; and (e) evaluate the impact of tralesinidase alfa treatment on brain structure, liver size, and spleen size as assessed by MRI.NAGLU gene mutations confirmed by genetic testing, and all presented with signs and symptoms consistent with MPS IIIB. Individuals were excluded from the study if they (a) had another neurological illness that may have caused cognitive decline; (b) received stem cell transplantation, gene therapy, or ERT for MPS IIIB; (c) received any investigational medication within 30 days prior to the baseline visit or were scheduled to receive any investigational drug during the course of the study; (d) presented with a medical condition or extenuating circumstance that, in the opinion of the investigator, might compromise the patient’s ability to comply with protocol requirements, the patient’s wellbeing or safety, or the interpretability of the patient’s clinical data; (e) participated in another natural history study; (f) had contraindications for neurosurgery; (g) had contraindications for MRI scans; (h) had a history of a poorly controlled seizure disorder; (i) were prone to complications from intraventricular drug administration, including patients with hydrocephalus or ventricular shunts; or (j) required ventilation support, except for noninvasive support at night.All study participants had deficient NAGLU enzyme activity and Cognitive function was measured using the nonverbal scales of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III)  or the KThe BSID-III is a validated and standardized developmental tool comprising 5 domains  intended to assess developmental function in children ages 1 to 42 months . CognitiThe KABC-II is a validated and standardized clinical psychological diagnostic test for assessing cognitive development . The subBSID-III or the KABC-II assessments were performed every 12 weeks in Study 250-201. Baseline, week 24, and week 48 visits involved 2 days of testing, with the cognitive, adaptive, and behavioral testing occurring on the first day (morning preferred), and MRI, lumbar puncture, and laboratory testing occurring on the second day to avoid confounding effects of sedation or general anesthetic administration.Head and abdominal MRIs were used to assess regional brain, liver, and spleen volumes at baseline, week 24, and week 48 of Study 250-201. Brain volumetric analysis was performed on 3D T1-weighted images using the FreeSurfer Image Analysis Suite, version 5.3   as descrBrain volumes are noted without normalization, similarly to previous publications , 34. RegLiver and spleen volumes were calculated by ICON Laboratory Services. Because liver and spleen volumes increased linearly compared with both age and BSA, these volumes were normalized to BSA, calculated using the Mosteller formula to allow for comparison among patients ranging from 2–9 years of age . Normal Tralesinidase alfa quantification in CSF and K2EDTA plasma was based on an electrochemiluminescence immunoassay (ECLA) technique using standard-bind, 96-well Meso Scale Discovery (MSD) streptavidin-coated plates. Samples were collected before dosing and 0.5, 4, 10, 24, 48, 72, 96, and 168 hours after drug administration at weeks 1 (baseline), 5, 12, and 36 of Study 250-201 Part 2. CSF samples were collected from the lateral ventricle through the i.c.v. port. Methods were validated, and sample analysis was conducted by Charles River laboratories.ADAs were measured in the CSF and serum using an ECLA method with MSD technology. Samples were diluted in a Master Mix with an equal concentration of ruthenylated (sulfo-tagged) tralesinidase alfa and biotinylated tralesinidase in the wells of polypropylene plates. Detection of ADAs is based on the bivalent characteristics of the antibodies; ADAs bind to both sulfo-tagged and biotinylated molecules to form an antibody complex bridge. Samples are dispensed onto streptavidin-coated MSD assay plates to allow binding of biotinylated tralesinidase alfa to the streptavidin in the wells. Only the samples that contain ADAs bound to both the biotinylated and sulfo-tagged tralesinidase alfa generate an ECL signal. In the presence of tripropylamine, ruthenium produces a chemiluminescent signal that is triggered when voltage is applied. The signal produced is proportional to the amount of ADAs present. Endpoint titer determination estimates the relative levels of ADAs for those positive samples. ADA assays were validated and samples were run by ICON Laboratory Services.Neutralizing antibodies were quantified in CSF using a cell-based assay that measured the ability of ADAs to interfere on CI-MPR–mediated uptake of tralesinidase alfa by the human Jurkat cell line. Tralesinidase alfa conjugated to Alexa 647 was monitored and quantified by flow cytometry using median fluorescence intensity (MFI) as the readout. The method was validated and samples were analyzed by Eurofins Pharma Bioanalytics Services US.d-galactoside, a β-gal fluorogenic substrate. The fluorescence measured in the eluate is directly proportional to the concentration of drug-specific IgE in the patient sample. The method was validated and samples were analyzed by Viracor Eurofins Clinical Diagnostics.Levels of tralesinidase alfa–specific IgE were quantified using tralesinidase alfa covalently coupled to ImmunoCAP. After washing away nonspecific IgE, a β-gal–labeled mouse monoclonal antibody against human IgE was added to form a complex. After incubation, unbound enzyme anti-IgE was washed away, and the bound complex was incubated with the ImmunoCAP Development Solution containing 4-methylumbelliferyl-β-n = 60) and plasma (n = 91) samples were established at 148 and 323 ng/mL for total HS and 10 and 15 ng/mL for HS-NRE in CSF and plasma, respectively. NAGLU genotyping and enzyme activity testing were performed by the Greenwood Genetic Center.Total HS and HS-NRE measurements in CSF and plasma were performed by ARUP Laboratories using a previously described method . The lowt test and linear correlation analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software). P values of less than 0.05 were considered significant.No corrections or carry-forwards were made for missing values. A 2-tailed, paired Written informed consent from a parent or legal guardian and assent from the participant, if required, was obtained prior to conducting any study-specific assessments. This study was approved by the IRB affiliated with each individual clinical site: Children’s Hospital Oakland, Oakland, California, USA; Mackay Memorial Hospital-Taipei Branch, Taipei City, Taiwan; Great Ormond Street Hospital for Children NHS Foundation Trust – Great Ormond Street Hospital, London, United Kingdom; Universitätsklinikum Hamburg-Eppendorf (UKE), Hamburg, Germany; Fundación Cardio Infantil – Instituto de Cardiología, Bogotá, Colombia; Complexo Hospitalario Universitario de Santiago (CHUS) – Hospital Clínico Universitario, Santiago de Compostela, Spain; and Gazi Üniversitesi Tıp Fakültesi, Cocuk Sagligi ve Hastaliklari AD, Cocuk Beslenme ve Metabolizma ve Çocuk Genetik Hastaliklari BD, Gazi, Ankara, Turkey.AJS and SMM designed the clinical protocol. NM, AK, KVC, IO, FE, PH, MJDCL, MLC, SPL, SB, MC, and MS recruited participants and performed and supervised clinical practices. IN designed and performed the brain MRI analysis. B Kuca supervised the clinical study execution and was responsible for regulatory submissions. EZ, JK, and B Kaufman performed the data analysis. EZ and JK designed the data analysis and wrote the original draft of the manuscript. All authors reviewed and edited the manuscript.


Ailanthus altissima, or “tree of heaven”. The molecular studies involving ailanthone’s effect on plant growth are limited. In the current study, we combined whole-transcriptome and physiology analysis of three Arabidopsis thaliana ecotypes treated with ailanthone to identify the effect of this allelopathic chemical on genes and plant growth. Our physiology results showed 50% reduced root growth, high proline accumulation, and high reactive-oxygen-species accumulation in response to ailanthone stress. Deep transcriptome analysis revealed 528, 473, and 482 statistically significant differentially expressed genes for Col-0, Cvi-0, and U112-3 under ailanthone stress, including 131 genes shared among the three accessions. The common genes included 82 upregulated and 42 downregulated genes and varied in expression at least twofold. The study also revealed that 34 of the 131 genes had a similar expression pattern when Arabidopsis seedlings were subjected to other herbicides. Differentially expressed genes significantly induced in response to ailanthone included DTXL1, DTX1, ABCC3, NDB4, UGT74E2, and AZI1. Pathways of stress, development and hormone metabolism were significantly altered under ailanthone stress. These results suggest that ailanthone triggers a significant stress response in multiple pathways similar to other herbicides.Many plants naturally synthesize and secrete secondary metabolites that exert an allelopathic effect, offering compelling alternatives to chemical herbicides. These natural herbicides are highly important for sustainable agricultural practices. Ailanthone is the chemical responsible for the herbicidal effect of  Ailanthus altissima, commonly known as the tree of heaven. This chemical is a quassinoid that suppresses the growth and germination of other plants around the tree by secretion through various plant organs. The highest concentration is in the roots and the bark, but the chemical can be found in all plant parts, including the leaves and fruit [Allelopathy refers to the chemical inhibition of one plant by another via the secretion of a chemical into the environment, which acts as a growth inhibitor. The chemicals responsible for this inhibition are known as allelochemicals. Ailanthone is one such allelochemical produced by nd fruit . The treSynthetic herbicides are the dominant means of controlling weed pests in agriculture today. These herbicides become less effective over time because they have a particular mode of action that allows the weed pests to quickly build up resistance . This reA. altissima, commonly known as the tree of heaven, is a deciduous tree species in the family Simaroubaceae native to Southeast Asia. It is a highly invasive species, widespread in many countries, especially in Europe and North America, where the tree was introduced as an ornamental species. The tree of heaven reproduces rapidly from both seeds and clonally via root shoots, with high seed production of up 325,000 samaras in an eight-year-old tree. Stem growth is highly rapid, and A. altissima is believed to be one of the fastest-growing trees in North America. As an invasive species, the tree of heaven has many adverse effects on the native biodiversity because of altered soil properties, rapid growth and reproduction, and its very unpleasant smell and allergenic properties. Its allelopathic effects can inhibit growth and kill plants around the tree base. However, A. altissima does have some ecological benefits, such as its use in land reclamation and reforestation, in paper production, as timber, and for its medicinal properties [A. altissima to quickly take over and out-compete many native plant ecosystems by inhibiting the growth of other plants. Allelopathy can also have a negative effect on the plants that produce them; examples include corn, sugarcane, and rice [and rice . AilanthArabidopsis thaliana. We studied the effect of ailanthone in three different ecotypes of A. thaliana: Col-0, Cvi-0, and U112-3.RNA sequencing is a high-throughput sequencing method that can decode the expression profile of all the RNA populations in a cell. Hence, in the present study, we used physiological and transcriptomic approaches to study the effect of ailanthone in a model plant, A. thaliana seedlings. To determine the effective dose of ailanthone on Arabidopsis root growth, we treated 8-day-old Col-0 seedlings with ailanthone concentrations ranging from 50 nM to 1 µM. Higher levels of ailanthone significantly inhibited the PRL  seeds in the presence of different ailanthone concentrations . Col-0 seeds showed 100% germination in all treatments when observed on the MS agar media plates; however, higher levels of ailanthone  completely inhibited seedling growth  and were recorded by using the WinRhizo software. The difference in growth phenotypes between control and treatment plates of Col-0, U112-3, and Cvi-0 seedlings is shown in The overall primary root length was ~7.5 cm for the Col-0 control but ~4 cm for ailanthone-treated Col-0 plants; Cvi-0 showed similar results to Col-0 in both control and treatment conditions A. U112-3Proline biosynthesis has been linked to the oxidative pentose phosphate (OPP) pathway and glutamate-glutamine metabolism . SeveralSimilar to proline accumulation, ROS is also strongly induced by biotic and abiotic stresses. We further aimed to check the ailanthone effect on ROS accumulation in Col-0, Cvi-0, and U112-3 by DAB staining. Rosettes that were 18 days old and treated with 0.5 and 2 µM ailanthone were analyzed with DAB. Col-0, Cvi-0, and U112-3 leaves stained more intensively when plants were grown on 0.5 and 2 µM ailanthone as compared with control leaves . These rA. thaliana reference genome (TAIR10) using the STAR universal RNA-seq alignment tool with default parameters. A total of 166,058,758, (95.93%), 174,167,848 (92.53%) and 165,868,158 (93.67%) quality-filtered reads were mapped to the reference genomes for Col-0, Cvi-0, and U112-3, respectively; ~8% of the reads remained unmapped  and log2 fold change. It includes 231, 266, 178 upregulated and 297, 207, and 304 downregulated genes. The Venn diagram comparing Col-0, Cvi-0, and U112-3 to identify DEGs that were shared between the three ecotypes is presented in In total, 528, 473, and 482 statistically significant differentially expressed genes (DEGs) were identified for Col-0, Cvi-0, and U112-3, respectively. The volcano plot in n = 43), response to abiotic or biotic stimulus (n = 38) and plant-developmental process (n = 20). Transcription factor activity was enriched with 15 DEGs under the molecular function. GO enrichment also revealed that chloroplasts carried 31 DEGs and mitochondria carried 28 DEGs under the cellular component category  by using the STAR universal RNA-seq alignment tool with default parameters. A total of 166,058,758, (95.93%), 174,167,848 (92.53%), and 165,868,158 (93.67%) quality-filtered reads were mapped to the reference genomes for Col-0, Cvi-0, and U112-3, respectively; ~8% of the reads remained unmapped. Overall, 528, 473, and 482 statistically significant DEGs were identified for Col-0, Cvi-0, and U112-3, respectively: 231, 266, and 178 upregulated and 297, 207, and 304 downregulated, respectively. To obtain more information on the ailanthone effect on plant growth and development, we treated Col-0, Cvi-0, and U112-3 seedlings with 0.5 µM ailanthone along with mock treatment and performed global transcriptome analysis by RNA-seq. A total of 178,639,612, 187,148,924, and 178,521,414 raw reads were generated for Col-0, Cvi-0, and U112-3, respectively. The Q30 percentage of reads in each library was ≥88%. The reads from the 3 ecotypes were aligned to the The three accessions shared 131 genes, which were differentially expressed with ailanthone treatment in comparison to controls : 82 wereDTXL1 and DTX1, belonging to the multidrug and toxic compound extrusion or multi-antimicrobial extrusion (MATE) family [DTXL1 and DTX1) upon ailanthone treatment further strengthens the idea that ailanthone has a herbicidal activity in plants by modulating the primary and secondary metabolic pathways that are generally disturbed by stress and toxic compound treatment.In corroboration with the ailanthone herbicide effect, two of the DEGs, ) family , were up) family ,25. The The ATP-dependent multidrug-resistant transporter ABCC3, also called multidrug resistance-associated protein 3 (MRP3) ,27, was NDB4) gene expression was upregulated >12-fold with ailanthone treatment in the three accessions. NDB4 is one of the components of the non-phosphorylating alternative respiratory electron transport chain pathway in plants [NDB4 were found to exhibit lower ROS formation, and plants showed altered leaf area and root:shoot ratios. NDB4 RNAi lines performed better with salinity stress than did control plants, thus implicating an NDB4 role in the stress response [NDB2, the homolog of NDB4, also showed better tolerance to salinity stress, whereas overexpressing NDB2 plants were sensitive to salinity stress, which confirms that the NDB group of genes plays a key role in the stress response. NDB and alternative oxidase (AOX) genes genetically interact with and work in a similar pathway [NDB2 alone did not induce the NADH oxidation, but when combined with AOX overexpression, NADH oxidation was increased in isolated mitochondria. AOX expression was also increased 4.6-fold in ailanthone-treated Col-0, Cvi-0, and U112-3 accessions, which suggests that indeed ailanthone induces the NDB-mediated stress response in plants. These observations suggest that to cope with the stress response, plants trigger NDB4 activity and promote alternative respiratory electron transport. Because ailanthone treatment induces NDB4 and AOX expression, ailanthone may promote plant stress in a similar way as with other stresses.NA(P)H dehydrogenase B4  and azelaic-acid-induced 1 (AZI1), were upregulated 6.0- and 4.4-fold, respectively, with ailanthone treatment. Moreover, these genes were also induced with imazapyr, primisulfuron-methyl, cloransulam-methyl, and glyphosate herbicide treatment and their function has been implicated in defense mechanisms ,32. UGT7aic acid ,33,34. Aaic acid , which pA. thaliana accessions  were ordered from the Arabidopsis Biological Resources Center (ABRC) at Ohio State University. All A. thaliana plants were grown under constant conditions (25 °C) in a Percival grow chamber. The seeds were sown on square Petri plates on Murashige and Skoog (MS) medium. The seeds were sown, sealed, and incubated for 72 h at 4 °C. The plants were grown for 1 week on control media, then transferred onto treatment and control plates using 50 mL MS media and adding the treatment (0.5 µM ailanthone). The plants were marked where the root growth stopped and then grown on control and treatment plates for an additional 7 days. The plants grown on control and treatment plates were used for the physiological studies and transcriptome analysis. Root-trait data such as overall root length, number of root hairs, tips, crossing, and forks were measured by using WinRhizo .A collection of 2O2 was detected in mature Arabidopsis rosette leaves and root tissue by staining with 3, 3′-diaminobenzidine (DAB) by vacuum infiltration. DAB is oxidized by H2O2 in the presence of heme-containing proteins, such as peroxidases, to generate a dark brown precipitate. This precipitate was used as a stain to detect the presence and distribution of H2O2 in plant cells [2O2 in leaf and root tissue.ROS accumulate when plants are under various biotic and abiotic stresses. Excessive ROS levels cause oxidative damage to proteins, DNA, and lipids. ROS also act as signaling molecules to regulate development and stress responses . H2O2 want cells . The darA. thaliana ecotypes, Col-0, Cvi-0, and U112-3, using the E.Z.N.A. Plant RNA Kit . The quality of the total RNA was assessed using agarose gel electrophoresis and a bioanalyzer . The quantity of the RNA was estimated by using a qubit fluorimeter . An RNA-seq library was prepared for each of the ecotypes separately by using NEBNext Ultra II RNA Library Prep Kit for Illumina with NEBNext Poly (A) mRNA Magnetic Isolation Module . The RNA-seq library quality and quantity were assessed by using a Bioanalyzer  and qubit fluorimeter . The library was subjected to paired-end sequencing (2 × 75 bp) with the Illumina NextSeq 500 platform .A whole-plant sample was collected from the control and treatment plates with three biological replicates for each condition. Total RNA was isolated from these whole plant tissues of the https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 12 September 2022). The sequencing adapters and low-quality reads (Phred score QV < 30) were removed by using the read trimming tool Trimmomatic [A. thaliana reference genome TAIR 10  using the STAR RNA-Seq aligner [p < 0.05. Gene annotations were obtained by using Omicsbox . Gene ontology (GO) enrichment analysis was performed with ShinyGO. Pathway analysis of DEGs was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database with ShinyGO . Gene Investigator  was used to identify the gene expression studies involving the herbicides that were previously reported.The generated image files in the BCL format from NextSeq 500 were converted to FASTQ with 2 × 75 bp reads by using the bcl2fastq tool . The quality of raw reads was analyzed by checking the adapter content, GC content distribution, and average base quality score of the reads by using the fastqc tool (mmomatic . The qua aligner  to gener aligner . The dif aligner . The DEGWe analyzed the physiological and transcriptomic response of ailanthone in three ecotypes of Arabidopsis. We identified 131 common DEGs that were induced in common in all three ecotypes. These DEGs included 34 genes whose activity was directly correlated with the herbicide response previously reported by four independent gene-expression studies. We identified several important genes and biological pathways that were altered in response to ailanthone in Arabidopsis. These results suggest that ailanthone triggers a stress response in multiple pathways similar to other herbicides.