Cymbidium sinense. In the present work, we found 64 C. sinense WRKY (CsWRKY) TFs, and they were further divided into eight subgroups. Chromosomal distribution of CsWRKYs revealed that the majority of these genes were localized on 16 chromosomes, especially on Chromosome 2. Syntenic analysis implied that 13 (20.31%) genes were derived from segmental duplication events, and 17 orthologous gene pairs were identified between Arabidopsis thaliana WRKY (AtWRKY) and CsWRKY genes. Moreover, 55 of the 64 CsWRKYs were detectable in different plant tissues in response to exposure to plant hormones. Among them, Group III members were strongly induced in response to various hormone treatments, indicating their potential essential roles in hormone signaling. We subsequently analyzed the function of CsWRKY18 in Group III. The CsWRKY18 was localized in the nucleus. The constitutive expression of CsWRKY18 in Arabidopsis led to enhanced sensitivity to ABA-mediated seed germination and root growth and elevated plant tolerance to abiotic stress within the ABA-dependent pathway. Overall, our study represented the first genome-wide characterization and functional analysis of WRKY TFs in C. sinense, which could provide useful clues about the evolution and functional description of CsWRKY genes.Transcription factors (TFs) of the WRKY family play pivotal roles in defense responses and secondary metabolism of plants. Although WRKY TFs are well documented in numerous plant species, no study has performed a genome-wide investigation of the WRKY gene family in  As one of the most prominent flowering plant families, Orchidaceae include 801 genera and more than 30,000 specie . In the Ipomoea batatas; The transcription factors (TFs) are critical in controlling plant development and stress response. WRKY, one of the largest families of higher plant TFs, plays essential roles in pathogen defense, abiotic cues, phytohormone signaling, and management of plant development and secondary metabolism. The WRKY domain (WD) is the essential characteristic of the WRKY protein, which is ~60 amino acids in length and consists of the highly conserved signature WRKYGQK, followed by a C2H2-or C2HC-type of zinc-finger motif. The high binding affinity of WRKY TFs to the consensus W-box cis-elements requires both heptapeptide sequence and zinc-finger motif . Given tCymbidium sinense, a very famous traditional orchid plant in China and Southeast Asia, and identified 64 CsWRKY proteins. Computational analysis was conducted to assess their physicochemical properties, including conserved domain, phylogenetic relationship, motif composition, functional annotation, and protein–protein interaction (PPI). In addition, the expression profiling of CsWRKY genes in various plant tissues in response to hormone treatments abscisic acid (ABA), gibberellic acid, salicylic acid, and methyl jasmonate (MeJA) indicated that most CsWRKY genes were responsive to various plant hormones, and Group III genes appeared to be more actively expressed to cope with the stress response. Collectively, our current findings provided valuable insights into the understanding of WRKY TFs in orchid plants and identified many candidate regulators involved in multiple hormone signaling.In the present study, we analyzed the genome sequences of C. sinense genomic database , and basic information of their encoded proteins, including length, molecular weight (MW), and isoelectric point (pI). CsWRKY38) to 676 (CsWRKY61) amino acids, and their average length was 317 residues. The pI ranged from 4.84 (CsWRKY46) to 10.00 (CsWRKY62), and the MW ranged from 5,929.61 Da (CsWRKY12) to 7,4025.70 Da (CsWRKY61).A total of 64 WRKY TFs were found through the database . Each WRCsWRKY proteins, 275 conserved WDs, including 114 OsWRKY proteins from rice (O. sativa japonica) and 85 AtWRKY proteins from Arabidopsis, were extracted to construct the evolutionary tree using the maximum likelihood (ML) method. As shown in the evolutionary tree , and Group II proteins were further sorted into five subgroups . The CsWRKY TFs had two standard motifs, including a WD and a zinc-finger-like domain. The former, typically the WRKYGQK sequence, could combine with the W box cis-element to trigger the expressions of their downstream genes. Besides the WRKYGQK sequence, three variants, WKKYGQK (CsWRKY47) in Group IIa, WRKYGKK  in Group IIb, and WRKYGRK  in Group IIc were also revealed, respectively. The latter, the zinc-finger-like domain, also had two types, namely C2H2 and C2HC.To explore the phylogenetic relationship among ary tree , the 64 CsWRKY proteins, 11 of which contained two WDs. The remaining CsWRKY63 had a single intact WD in its C terminus, and the other three members  had a single WD at the N terminus , Group IIb (four proteins), Group IIc (20 proteins), Group IId (two proteins), and Group IIe (seven proteins). The zinc-finger motifs of Group III members (11) belonged to the C2HC type, with the C-X7-C-X23–24-26-H-X1-C motif , followed by chromosome 8 (eight genes). With only one gene, the least number of CsWRKYs was found on Chromosome 10. Three chromosomes contained the members from all three groups, 10 chromosomes contained the members from two groups, and the other three only had the members from one group.A total of 64 candidate angement . ChromosCsWRKY gene family, we constructed two comparative syntenic maps between C. sinense and A. thaliana, O. sativa, V. vinifera, M. cuminata, or Z. mays at the genome-wide level. 27, 28, and 29 orthologous gene pairs between C. sinense and O. sativae, V. vinifera, or M. cuminata, respectively. However, only 12 and 19 orthologous gene pairs between C. sinense and Z. mays or A. thaliana, were identified, respectively. More details about these orthologous gene pairs are given in C. sinense and O. sativae than those between C. sinense and A. thaliana, which probably resulted from the nearer phylogenetic relationship between C. sinense and O. sativae.To further assess the origin and evolution of the CsWRKY gene pair. Typically, a Ka/Ks ratio of 1, >1, or <1 indicates neutral selection, adaptive evolution with positive selection, and negative or purifying selection, respectively  and harbored at least one motif. Besides, Motif 2 was composed of the C2H2 motif, while Motif 12 consisted of the C2HC motif. Furthermore, the conserved motifs were explicitly present in various groups. For instance, Group IIa members harbored four conserved motifs , Group IIb members contained five conserved motifs , and 14 Group IIc members contained four conserved motifs . Clearly, a few motifs existed in one or more groups and subgroups. For example, Motifs 4, 8, 7, and 12 existed in Group I members, and Motif 10 existed only in Group III, while Motif 15 was mainly present in Group IIb. This result suggested that these groups were endowed various functions during evolution. However, even if these members belong to the same clade, the functions of family members vary greatly  and seven hormone-related elements . CsWRKYs, with 61 genes containing these two elements. Box 4 and G box were two types of light-response elements, ARE was involved in anaerobic induction, and ABRE (ABA-responsive elements), CGTCA-motif, and TGACG-motif (MeJA-responsive elements) also appeared frequently in promoter regions of CsWRKY genes, which were found in 59, 49, 49, 47, 42, and 42 promoters, respectively. Many W-boxes were identified in 37 CsWRKY gene promoters, indicating that these genes were modulated by other WRKY TFs or themselves. LTR and MBS elements that respond to low temperature and drought stresses were identified in the promoters of 17 and 28 CsWRKY genes, respectively. Moreover, 25 promoters showed a TC-rich repeats element (cis-acting element participated in defense and stress response), and only 15 promoters had a GARE-motif (gibberellin-responsive element). Thus, the cis-element analysis showed that the expressions of CsWRKY genes in C. sinense might be related to various environmental factors.Cis-elements in promoter region are important for gene expression . To veriCsWRKY proteins was established using STRING 10.5 software based on the Arabidopsis association model. Our results showed that there were close interaction networks among 43 CsWRKYs , functioning as a regulator of pathogen defense responses  were not detected, showing that these genes might be pseudogenes or have particular temporal and spatial expression patterns. Most of the 55 CsWRKY transcripts investigated were expressed in all tissues with low expression levels, suggesting that these TFs worked with other proteins synergistically or interactively during plant growth and development. Moreover, many genes had preferential expression in various tissues. For example, 19 CsWRKY transcripts  exhibited high expression levels in root particularly. By contrast, 2, 10, 3, and 2 exhibited the highest transcript abundances in the stem, leaf, flower, and fruit, respectively. These genes might play special roles in specific tissues. Additionally, several gene pairs with a close relationship, such as CsWRKY03/63 and CsWRKY19/33, indicated similar expression profiles, suggesting that the functionality of these genes was redundant.Based on RNA-seq and expression profiling analysis, 55  tissues . HoweverCsWRKY genes, quantitative real-time PCR (qRT-PCR) was accomplished for 21 randomly chosen CsWRKY genes, including four Group I members, 11 Group II members, and six Group III members. We first optimized the treatment conditions, and the seedling leaves were sampled after exposure to 100 μmol GA, ABA, SA, IAA, JA, or ACC for 2 h.WRKY are widely involved in plant abiotic stress regulation . To furtCsWRKY01, CsWRKY18, CsWRKY20, CsWRKY25, CsWRKY30, CsWRKY31, CsWRKY37, and CsWRKY42 were significantly up-regulated by over 5-fold under the GA treatment  staining showed that CsWRKY was clearly localized to the nucleus  at Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences (China). For tissue/organ-specific expression analysis, various plant parts, such as roots, leaves, stems, and flowers, were harvested from a 2-year-old plant. For expression in fruit tissues, 1/2/3/cm, mature green fruits, breaker fruits, and 10-day breaker fruits were harvested. For phytohormone treatment, 2-year-old plants in flowerpots were used, and each group (n = 3) was sprayed with 100 μM ABA, gibberellin (GA3), auxin (IAA), and MeJA. The samples were collected at 0, 0.5, 1, 2, 3, and 4 h after exposure from nine individual plants. For different organ samples, roots, stems, and leaves were collected from three plants grown for 45 days. All materials were immediately frozen in liquid nitrogen and preserved at −80°C for further analysis of WRKY genes. Considering the sensitive response of Group III to hormone signals, we randomly selected CsWRKY18 for further functional analysis. We generated transgenic A. thaliana plants overexpressing the CsWRKY18 gene under the CaMV 35S promoter, according to the general method . The incorrect and redundant predicted sequences were manually removed, and then all putative CsWRKY genes were further verified using NCBI’s Conserved Domain Database.A. thaliana and O. sativa were downloaded from TAIRThe genome sequences of  our lab , and theMultiple sequence alignment of the WDs and full-length proteins was conducted by CLUSTAL . The syntenic block map was established by MCscan with cscore = 0.7. Each orthologous gene pair was then further investigated with PAL2NALThe alignment was performed by LASTZ using CDS sequences of CsWRKYs, the expression patterns of CsWRKY genes in five different tissues were identified, such as roots, stems, leaves, flowers, and fruits. The transcriptome data of all tissues were came from our previous report , and then 1 μg RNA was used to synthesize cDNA in a 20-μl reaction system according to the instructions in PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA). qRT-PCR was carried out on a Bio-Rad iCycler Real-Time PCR Detection System (United States) by TaKaRa SYBR Premix Ex Taq™ (Tli RNaseH Plus) with three biological replications. Briefly, the reaction was conducted in a 20-μl reaction system containing 10 μl SYBR Premix (2×), 1 μl cDNA, 1 μl sense and anti-sense primer (10 μM), and 7 μl ddH primers . The β-aT method .A functional protein association network was constructed in the STRING program based on the Arabidopsis association model with the confidence parameter of 0.15 and the number of interactions of 5.Giardia lamblia) and soil-living amoeba-like slime mold . Possibly since the green alga Chlamydomonas reinhardtii contains only one Group I WRKY gene  except for CsWRKY03 and CsWRKY63 in Group I. The WD sequence variation may influence the normal interactions and binding specificities with downstream target genes (CitWRKY28 (Group I) and CitNAC029 can promote the accumulation of cuticular wax in Arabidopsis leaves  is strongly affected by salt stress (CsWRKY25 (Group I) is up-regulated in Penicillium digitatum-infected citrus peels (PcWRKY11 (Group II WRKY from Polygonum cuspidatum) significantly increases the tolerance to salt stress in transgenic A. thaliana . These findings suggested the functional conservation of WRKY genes during orchid evolution.Compared with Group I and II WRKY TFs, Group III TFs alter C2H2 to C2HC zinc-finger motif C-X7-C-X26-HTC. It has been considered the most adaptable and most advanced in monocot evolution . In addibidopsis , 28 in rbidopsis , 35 in mbidopsis , 62 in Datenatum , 57 in PCsWRKY18 and indicated its role in the ABA pathway and abiotic stress. Different abiotic stresses can induce AtWRKY30. Meanwhile, the overexpression of AtWRKY30 greatly enhances the resistance of Arabidopsis in response to salt stress  is an essential positive player in the resistance against the rice blast fungus M. oryzae (AtWRKY30 (Group IIIb). Meanwhile, the overexpression of AtWRKY30 significantly improves the resistance of Arabidopsis in response to salt stress , grants from the National Key R&D Program (2018YFD1000404 and 2019YFD1001003), the National Natural Science Foundation of China (31902065), the Science and Technology Project of Guangdong Province (2019B030316033), Guangzhou Science and Technology Project (201707010307 and 202002030016), Innovation Team of Modern Agriculture Industry Technology System in Guangdong Province (2021KJ121), and Guangdong Academy of Agricultural Sciences Discipline Team Construction Project (202127TD and BZ202006).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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We identified eight EpCAM+ lung epithelial cell types, among which AT2 cells were the most abundant. HDM challenge altered the relative abundance of epithelial cell types and resulted in cell type-specific transcriptomic changes. Bulk and cell type-specific analysis also showed that loss of Tet1 led to the altered expression of genes linked to augmented HDM-induced lung inflammation, including alarms, detoxification enzymes, oxidative stress response genes, and tissue repair genes. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Collectively, our data showed that lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, which may promote allergen-induced lung inflammation.Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. In order to explore the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation, we established a model of HDM-induced lung inflammation in Tet1 knockout and littermate wild-type mice, then studied EpCAM Asthma is one of the most common pulmonary disorders with high heterogeneity . Previou+ cells together and in each cell type, and the effects of Tet1 loss in the presence of HDM was also evaluated. We observed similar effects of Tet1 deficiency and HDM on the transcription of alarmins and detoxification enzymes, as well as of the down-regulation of oxidative stress pathways and the upregulation of markers of epithelial injury following Tet1 knockout in the presence of HDM. These data suggest possible pathways through which Tet1 protects against HDM-induced lung inflammation. Given the use of RNA from total mouse lung tissues in these previous studies, critical questions remained regarding the cellular origin(s) of inflammatory mediators and molecules  following allergen challenges, and the role that Tet1 plays in regulating these mediators/molecules in individual cell types in vivo. In this study, our primary goal was to identify epithelial cell types that were influenced by HDM treatment and Tet1 knockout in vivo using single-cell RNA-seq analysis. We hypothesized that Tet1 regulates genes in proinflammatory and oxidative stress response pathways in specific lung epithelial cells, and that the loss of Tet1 has similar effects on these pathways compared to HDM treatment. The transcriptomic effects of Tet1 loss and HDM challenges were compared in all EpCAM−/− mice (knockout/KO mice) and their littermate Tet1+/+ mice (wild-type/WT mice), as described in a previous study [+ and Mg2+ and razor blades. The finely minced tissues were incubated in 6mL of digestion solution at 37 °C for 40 min, with gentle inversion for 30 s at 5 min intervals. After incubation, samples were washed with a DPBS/1% BSA wash buffer and passed through a 70 μm filter strainer. After spinning down cells and removing the supernatant, cells were treated with AKC lysis buffer to remove red blood cells, and washed once more to remove dead cells and excess RNA in the buffer. CD45+ cells (immune cells) and CD31+  were then depleted using mouse CD45 and CD31 MicroBeads (Miltenyi Biotec), and the remaining cells were counted and incubated with anti-CD326-APC (eBioscience), anti-CD31-PE-cy7 (eBioscience), and anti-CD45-PE (Biolegend). CD326+ CD31− CD45− cells were collected on a MoFlo sorter (Beckman Coulter/MoFlo XDP) with >99% purity at the Research Flow Cytometry Core at Cincinnati Children’s Hospital Medical Center  pipeline for library preparation. After cDNA preparation and GEM generation and clean-up, cDNA was quality checked and quantified on an Agilent Bioanalyzer DNA High-Sensitivity chip at a 1:10 dilution. Successful traces showed a fragment size range of approximately 1000–2000 bp. A total of 12 indexing cycles for the library were used. The quality of the indexed libraries was checked on an Agilent Bioanalyzer DNA High-Sensitivity chip at a 1:10 dilution. Successful libraries had fragment size distributions with peaks around 400bp. Libraries were sequenced using 4 Illumina Hiseq lanes (Paired-end 75 bp).We used Cell Ranger v3.1.0  for inithttps://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/) (accessed between 1 March 2021 and 1 April 2022).We used the FindClusters function from Seurat to cluster the cells, using a wide range of resolutions, from 0.01 to 2, to identify the best resolution for our dataset. FindClusters is a shared nearest-neighbor modularity optimization-based clustering algorithm. Our eventual selection was a resolution of 1, yielding 11 initial cell clusters. We then used markers from the single-cell mouse cell atlas  and anotWe used DoRothEA ,21,22 toq < 1 × 10−4).In order to assess the differentiation of AP and BAS cells to AT2 cells, we performed single-cell trajectory analysis using Monocle . First, + CD45− CD31− (referred to as EpCAM+) lung epithelial cells from animals in an established HDM-induced allergic airway inflammation model [To identify cell types that are important in responses to HDM challenges and that are subject to Tet1 regulation, we isolated EpCAMon model  and charon model , airway Epcam and Scgb1a1 , regardless of cell type (n = 732), followed by AP cells (n = 431)  between WT-HDM and WT-Sal cells in our analysis of all EpCAMell type . Based oell type . We furtell type . All celell type . AT2 celn = 431) . Many ge1)  [Gstp1, Gstm1, and Gpx2) were only found in ciliated cells  were doalarmin) ,51 was ued cells . Hmgb1 wed cells , includiIl33 (AT2 cells) and Hmgb1 (AT2 and ciliated cells), (2) the upregulation of genes that promote the proliferation and migration of airway smooth muscle cells and tissue remodeling  and Pfn1 (six cell types)), and (3) the reduced expression of detoxification enzymes  involved in AhR signaling in ciliated cells. Together, this gene dysregulation following Tet1 loss may have resulted in a pro-inflammatory transcriptomic state in AT2 and ciliated cells.In summary, we found that Tet1 loss in saline-treated mice led to (1) increased expression of alarmins that promote type 2 inflammation, particularly Il33, Hmgb1, Ager, Chia1, Ifitm3, Igfbp7, Pfn1, Retnla) and five downregulated genes   in AP cells and one gene (Nnat) in BAS cells were consistently changed in a similar fashion. Combining all these genes with the same directions of changes in EpCAM+ cells, AT2 cells, AP, BAS, and ciliated cells, there was significant enrichment for NRF2-mediated Oxidative Stress Response, HMGB1 Signaling and Airway Inflammation in Asthma  . Eight mSec14l3) . Of thesSec14l3) , includiand Gpx2 C,D. Addin Asthma . IPA anaT2 cells A,B, cellT2 cells C, and thT2 cells D. Collec+ cells  were downregulated in KO-Sal mice compared to WT-Sal mice.To further understand the effects of Tet1 loss in the presence of HDM, we studied the 72 genes that were differentially expressed in KO-HDM compared with WT-HDM in all EpCAM+ cells . Among t+ cells , includils  and 16 were downregulated  in these 3 cell types following Tet1 deletion  showed reduced expression in KO-HDM  were hial cells ) and Rreogenesis ,72) in cd repair ,74,75. Foxa1 , Nfe2l1 (encoding Nrf1) and their binding partner Mafk , Sftpa1 (a marker for AT2 and AP cells), and Sftpb (a marker for only AT2 cells) varied significantly along all trajectories, other than in the saline-only comparison  and detoxification enzymes (Gsto1 and Aldh1a1) in total lung RNA. Furthermore, our previous results indicated that 59 pathways, including NRF2-mediated oxidative stress response, AhR signaling, and the interferon (IFN) signaling pathway, were significantly altered. However, the effects of Tet1 loss in specific lung cell types and the contribution of individual cell types in HDM-induced transcriptomic alterations remained unknown. Data from our current analyses showed that while AT2, alveolar progenitor cells, broncho alveolar stem cells, and ciliated cells underwent transcriptional changes following HDM challenges, AT2 and ciliated cells were the cell types most affected by Tet1 loss. Following HDM treatment, the upregulation of the alarms IL33 and Hmgb1 mainly occurred in AT2 cells, while the downregulation of detoxification enzymes mainly occurred in ciliated cells. Notably, Tet1 knockout had similar effects on alarms in AT2 cells and detoxification enzymes in ciliated cells. Further, in the presence of HDM, we found that genes involved in stress response pathways  and markers for tissue damage response/remodeling  were upregulated. Based on these observations, a model linking Tet1 loss to increased airway inflammation was proposed  that arIl33 . Although no significant pathological alterations in alveoli were observed in either asthmatic patients or animal models, several studies ,86,89 haIl33 A, Hmgb1,b1, Ager , and Ret  were consistently downregulated in ciliated cells, but not in AT2 cells (Ciliated cells are another major cell type within airway epithelium. Ciliated cells are essential for mucociliary clearance through their motile cilia’s coordinated directional movement, and can sense and respond to allergens, air pollution particles, and pathogens ,93. Asthallenges , which sed cells C,D. ThesT2 cells , implyinT2 cells .Gstp1, Gstm1, and Gpx2 in KO-Sal, compared to WT-Sal, as the Nrf2-mediated pathway is an alternative to the AhR pathway that regulates the expression of antioxidant enzymes [Our data also suggest that HDM and/or Tet1 KO in mice lungs led to cell type-specific changes in TF activity levels . Our pri enzymes . In addi enzymes  and barr enzymes , was inc enzymes ,98. TakeIl33, Hmgb1, Retnla, and Ager were identified, and a cluster of novel allergy-associated molecules, such as Chia1, Nnat, and Nr4a1 were found in different lung epithelial cells. Future studies should attempt to elucidate the role of these novel targets in asthma. Our results suggest that AT2 cells are essential for Th2 inflammation through stress responses, alarms, tissue injury and repair in allergen-induced lung inflammation, and that these genes are regulated by Tet1. In ciliated cells, Tet1 promotes the Xenobiotic Metabolism AhR signaling pathway, especially the detoxification enzymes, which may protect the lung from allergen-induced inflammation. As chronic airway inflammation induced by allergens plays a critical role in the pathophysiology of asthma, these novel findings support that Tet1 is a potential therapeutic target of asthma.Collectively, our results revealed possible mechanisms underlying the protective role of Tet1 in HDM-induced lung inflammation at the single-cell level. Different epithelial cells played common and unique roles in allergic lung responses. The major cellular contributors of multiple asthma-associated inflammatory mediators, such as


SiSAD (stearoyl-acyl carrier protein (ACP)-desaturase) coding genes with known SADs helped identify two SiSAD family members that may be palmitoyl-ACP-specific. Based on homology with lysophosphatidic acid acyltransferase (LPAAT) sequences, an uncharacterized gene has been identified as SiLPAAT1. Identified key regulatory genes associated with high oil content were also validated using publicly available transcriptome datasets of genotypes contrasting for oil content at different developmental stages. Our study provides evidence that a longer duration of active oil biosynthesis is crucial for high oil accumulation during seed development. This underscores the importance of early onset of oil biosynthesis in developing seeds. Up-regulating, identified key regulatory genes of oil biosynthesis during early onset of seed development, should help increase oil yields.Sesame, one of the ancient oil crops, is an important oilseed due to its nutritionally rich seeds with high protein content. Genomic scale information for sesame has become available in the public databases in recent years. The genes and their families involved in oil biosynthesis in sesame are less studied than in other oilseed crops. Therefore, we retrieved a total of 69 genes and their translated amino acid sequences, associated with gene families linked to the oil biosynthetic pathway. Genome-wide in silico mining helped identify key regulatory genes for oil biosynthesis, though the findings require functional validation. Comparing sequences of the  Plants accumulate oil primarily in the form of triacylglycerols (TAG) . TriacylaccC for biotin carboxylase (BC), accB for biotin carboxyl carrier protein (BCCP), accA for α-carboxyl transferase, and accD for β-carboxyl transferase [accD is chloroplast-encoded, while the others are nuclear encoded [FabD, is one of the key regulators in FA biosynthesis [β-ketoacyl-ACP synthase III  catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP to form 3-ketobutyryl-ACP. β-ketoacyl-ACP synthase I  elongates 3-ketobutyryl-ACP to palmitoyl-ACP in six condensation cycles. In the final elongation step, β-ketoacyl-ACP synthase II  converts palmitoyl-ACP to stearoyl-ACP in the plastid [FatA, FatB) catalyze the FA chain termination through hydrolysis of the thioester bond of acyl–ACP to release the free FAs [The first committed enzyme is acetyl-CoA carboxylase (ACCase), which acts as a control point over the carbon flux into FAs . In plannsferase . Of thes encoded . The malynthesis . It aidsynthesis . Subsequ plastid . The acyfree FAs . These free FAs are reactivated by a long-chain acyl-CoA synthetase (LACS) to form acyl-CoA esters and are exported to the ER . FA desaSesamum indicum L., 2n = 2x = 26) is an ancient oilseed crop of the Pedaliaceae family. Sesame seeds are nutritionally rich and possess 50–60% oil, 25% protein, and antioxidants such as sesamolin and sesamin [https://www.fao.org/faostat/en/#data; accessed on 28 September 2022). Understanding the genetic basis of oil biosynthesis and developing stable high oil yielding sesame varieties have been key objectives in sesame breeding in recent years [Sesame  and 1004 (110.47 kDa) for SiPAH2 , the number of transmembrane domains among proteins exhibited variability between a total of five transmembrane domains in SiFAD2-1 and only one in SiFAD7 . Lastly, the fourth subclade grouped all FAD6 proteins  , while sSiFabD as Gln112, Ser197, Arg222, His310, and Gln359 were highly conserved in AhFabD as Gln89, Ser174, Arg199, His287, and Gln336 [SiKASIII proteins  have the same amino acid residues at catalytic sites as AtFAB2 and RcSAD1 (except that SiSAD3 and SiSAD5 have isoleucine instead of leucine at the 142nd position), while SiSAD6 and SiSAD7 showed divergent residues in the aforesaid catalytic sites, suggesting that they might possess 16:0-ACP substrate specificity  essential for LPAAT activity [2+-dependent PA phosphatases, was identified in all PAH proteins  amino acid sequences recurring in fixed-length patterns called motifs. A motif may represent important biological features, such as protein-binding or targeting a particular subcellular location. Conserved motifs in all studied genes  were ideBased on the homology relationships for genes associated with oil biosynthesis in model plants and genotypes exhibiting higher oil yield, we have identified 12 genes in sesame that could be potential targets for enhancement in the sesame oil yield . The upsDGAT, LPAAT, FAD, SAD, KCS (β-ketoacyl-CoA synthase), Oleosin, nsLTP (non-specific lipid transfer protein), and DIR1 (defective in induced resistance1) genes were selected manually.The key regulatory genes associated with oil biosynthesis  were ideSAD gene showed higher expression at the early stage (10 DPA) in the high oil yielding genotype while its higher expression was found during middle stages (20 and 25 DPA) in the low oil yielding genotypes  and is decreased at 30 DPA in the high oil yielding genotype, while, in case of low oil yielding genotypes, its expression level increased only during the middle developmental stage (20 DPA)  in both high and low oil yielding genotypes. However, the expression level of these genes decreased towards later developmental stages (25 and 30 DPA) in low oil yielding genotypes while the expression pattern in high oil yielding genotypes is sustained at comparatively higher levels than the low oil yielding genotypes. Genes for lipid transfer proteins include nsLTP-encoding genes and DIR1 [KCS catalyzes the first step of the very long chain FA biosynthesis [nsLTP, DIR1, and KCS, tended to show higher expression in the high oil content sesames genotype when compared to the low oil yielding genotypes  . We alsoand DIR1 ,62. The ynthesis ,64. Thesenotypes J–L. Of tgenotype M–O. ThesWith the availability of high-throughput technologies and the sesame genome datasets, various genes involved in oil biosynthesis have been mined from the publicly available sesame genome and RNA-seq datasets and were studied using various bioinformatics tools. The present report focused on studies pertaining to conserved sites, catalytic sites, domains and motifs, physicochemical properties, subcellular location, and their homology with genes of other oilseed crop and model plants. TAG biosynthesis in oilseeds can be summarized in three major steps : (i) bioGossypium species [BCCP gene modulates the oil content in seeds of Arabidopsis and cotton [accD gene through chloroplast transformation resulted in increased ACCase levels that led to higher oil production in transgenic tobacco [Plant de novo FA synthesis occurs in the stroma of the plastid in a series of reactions: initiation, elongation, and termination . The het species , and pea species . Overexpd cotton . Additio tobacco .SiaccB-2, SiaccC, SiaccD, SiKASIII, SiSAD, and FATB (Brassica napus (7.8 to 9.5) showed alkaline nature [An evaluation of the physicochemical properties of the amino acids of FA synthesis genes highlighted that, on an isoelectric point (pI) basis, most of the proteins were alkaline in nature except and FATB . Similare nature ,67. The e nature . The alie nature . The GRAe nature . Most stSiKASIII proteins [SiFatA; D282, N284, H286, and E320 in SiFatB-1, and D319, N321, H323, and E357 in SiFATB1-2)  .B. juncea, and 8 in black mustard [, B. juncea, B. rapa, B. nigra, olive, and camelina, and possess 3, 12, 7, 8, 3, and 3 SAD genes, respectively [AtSAD members, SSI2/FAB was characterized as a typical 18:0-ACP-specific acyl-ACP Δ9 desaturase [AtSAD, AtAAD2, and AtAAD3 catalyze 16:0-ACPΔ9 desaturation [SiSADs (SiSAD1-5) might be of 18:0-ACP specificity and two SiSADs (SiSAD6 and 7) possibly exhibit 16:0-ACP specificity , which is similar to AtAAD2 (F224), AtAAD3 (F216), and MuPAD (W151), but not in SiSAD7. This observation favors SiSAD6 as a better candidate for PAD than SiSAD7. Since the orthologous genes from different species tend to group together rather than with the paralogous genes within a species, homology relationships  as BrSAD6 with AtAAD2 or BrSAD7 with AtAAD3 (GhA-SAD6 and GhD-SAD8) and Arabidopsis (AtAAD2 and 3) for the preferential expression (of the respective gene copies) in the endosperm and aleurone tissues [The FAD gene family is well characterized in oilseed crops, including 31 in peanut , 68 in r mustard . SAD enzectively . Among sturation . The keyturation . Based ocificity . Of the ionships  indicateh AtAAD3 . As repo SiSAD6 W8, which  tissues ,76,77, tSiFAD2-1 from 705 accessions detected a mutation causing an amino acid change (R142H), which probably affects the desaturase activity, with mutant accessions accumulating extremely high levels of oleic acid (48%) in seed oil [SiFAD2-2, was retrieved from the sesame genome (Membrane-bound FADs include omega-6-FADs (FAD2 and FAD6), omega-3-FADs , and palmitate desaturase (FAD4) . FAD2 anseed oil . In the e genome . The omee genome .SiSAD and SiFAD families were hydrophilic in nature. SiFAD proteins were predicted as alkaline proteins while SiSADs were slightly acidic  led to the identification of twelve genes consisting of four KASIII, six KASI, and two KASII [KAS genes are reported to affect oil production. KASI-deficient mutant Arabidopsis seeds have been reported to produce significantly lower oil content [NtKASI-1 significantly enhanced oil accumulation in tobacco [Jatropha curcas demonstrated that silencing of JcKASII significantly altered FA composition and TAG biosynthesis [KASI (SiFabB-2) and SiDGAT2, were reported to determine the unsaturated to saturated fat ratio [SiFabB-2 at sequence level was found among the 705 sesame accessions studied [β-ketoacyl-acyl carrier synthases (KAS) catalyze chain-initiation, -elongation, and -condensation steps and are classified as KASIII, KASI, and KASII . A totaln sesame . In siliwo KASII . KAS gen content . Overexp tobacco . Virus-iynthesis . Statistat ratio . No vari studied .FatB1 from Umbellularia californica  and MlFatB from Madhuca longifolia in Brassica juncea increased the laurate levels by over 50% and stearate levels by 16-fold, respectively [SiFatB, SiFatA, KASII, KASI, and SAD [SiFatB1-1, SiFatA, SiFabF-1, SiFabB-2, and SiSAD-1, respectively. Availability of the sequence information is much helpful in identifying, on a homology basis, the actual gene copy (among the gene copies in a genome) being transcriptionally regulated with tissue (space) and developmental stage (time) specificity. Hence, the evolutionary or comparative genomics tools are helpful in establishing a homology relationship and, thereby, predict the actual gene copy being specifically expressed with reference to space and time in an organism. Unraveling such biological information on a homology-basis leads to novel insights due to the exceptional conservation of synteny among ortholog blocks [Acyl-ACP thioesterases play an essential role and exhibit two distinct classes (FatA and FatB) . FatA haectively ,88. A ge and SAD . Based og blocks ,92,93,94Arabidopsis contains one of the largest known LACS families, having nine LACS genes [LACS genes. In comparison to Arabidopsis, three LACS types, LACS3, LACS5, and LACS7, were absent in sesame , GPATs that catalyze esterification of fatty acyl moiety from acyl-CoA (or -ACP) to the Sn-1 position of the G3P, and sn-2-GPAT (AtGPAT1-8), which esterifies the Sn-2 position of the G3P [AtGPATs are classified into three different groups, cutin , suberin (AtGPAT5 and 7), and mitochondria-localized (AtGPAT1-3) [AtGPAT1 was reported to be involved in tapetum differentiation and male fertility [AtGPAT2 and GPAT3 are yet unknown [AtATS1 and ER-localized AtGPAT9 contribute to glycerolipid biosynthesis but not extracellular lipids [Brassica are well studied [Gossypium species, 28, 27, 16, and 14 GPAT homologs were identified from G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively [SiGPAT10-SiGPAT17 represent the cutin group   . AtGPAT1ertility , while t unknown . Plastidr lipids . GPAT fa studied . Similarectively . In sesain group , while t and -7) . SiGPAT1 AtGPAT9 . TherefoArabidopsis, five LPAATs have been reported, one plastidial (AtLPAAT1) and four (AtLPAAT2–5) microsomal targeted [SiLPAAT1) was identified to be plastid localized in this study and exhibits similarity with Arabidopsis AtLPAAT1 (SiLPAATB because it was found to be homologous with class-B LPAAT of Ricinus communis (RcLPAATB)  in Arabidopsis, soybean, and rapeseed resulted in an 8–48% increase in seed oil content [LPAAT incorporates FA at the sn-2 position of PA and is a crucial enzyme in membrane phospholipid and storage lipids biosynthesis . In plantargeted . Class-Atargeted , whereastargeted . Among eAtLPAAT1 . We havecLPAATB) . The micAhLPAAT2 , BnLPAATB. napus , and AtLbidopsis   is affected in phosphatidylcholine production at the ER [The dephosphorylation of PA to produce DAG catalyzed by PAPs is a committed step in TAG biosynthesis . Transie with PA , which ia TtPah1 . A doublt the ER . These tt the ER .DGATs are studied for enhancing oil production as they are one of the rate-limiting factors in plant storage lipid accumulation. Three structurally unrelated classes of DGATs were identified in plants: DGAT1, DGAT2, and DGAT3. DGAT1 and DGAT2 are associated predominantly with membranes, while DGAT3 is cytosolic ,116. ThrSiDGAT1 (–YYHDL) and in other plant DGAT1s [SiDGAT2 was found to have five conserved domains (SiDGAT1 is a membrane-bound O-transferase (MBOAT) protein with nine transmembrane domains, consistent with the DGAT1 proteins of Arabidopsis, B. napus, castor, peanut, and soybean, as reported earlier [SiDGAT2, two transmembrane domains at the N-terminal of the protein were predicted  of 12 samples representing different stage of seed development  for 1 high (ZZM4728) oil- and 2 low (ZZM3495 and ZZM2161) oil-yielding genotypes were downloaded from the GenBank SRA database [Using the available sesame genome sequence information ,130, thedatabase  for valihttp://www.cbs.dtu.dk/services/; accessed on 28 September 2022).Retrieved translated amino acid sequences were analyzed using the ExPASy ProtParam tool to estimate the molecular weight (MW), isoelectric point (pI), grand average of hydropathy (GRAVY), aliphatic index, and instability index . The subhttp://smart.embl.de; accessed on 28 September 2022), an annotation resource [The conserved domains for the retrieved proteins were also identified from the data reported in the NCBI conserved domain database . Domain resource . Multiplresource  with defresource  with a bresource  server wcis-acting regulatory elements (CAREs) using the PlantCare tool . Function annotation of identified CAREs was retrieved from the PlantCare database .The retrieved candidate promoter regions (3 kb upstream) were used to predict the potential http://www.dna.affrc.go.jp/PLACE/; accessed on 28 September 2022) and PlantPAN 3.0 [http://plantpan.itps.ncku.edu.tw/index.html; accessed on 28 September 2022).A total of 11 TFs were identified to be involved in regulating seed oil accumulation ,40,138. tPAN 3.0   9-desaturases, two members were predicted to be possibly 16:0-ACP specific . Similarbidopsis . MoreoveRcLPAATB . The genRcLPAATB . Validat


Citri Reticulatae Pericarpium (CRP) is one of the most commonly used food supplements and folk medicines worldwide, and possesses cardiovascular, digestive, and respiratory protective effects partially through its antioxidant and anti-inflammatory functions. The unique aromatic flavor and mild side effects make CRP a promising candidate for the development of anti-inflammatory functional food. However, recent studies show that the crude alcoholic extract and some isolated compounds of CRP show compromised anti-inflammatory activity, which became the main factor hindering its further development. To identify the bioactive compounds with anti-inflammatory potential, and improve the anti-inflammatory effects of the extract, a bioinformatics-guided extraction protocol was employed in this study. The potential bioactive candidates were identified by combing network pharmacology analysis, molecular docking, principal components analysis, k-means clustering, and in vitro testing of reference compounds. Our results demonstrated that 66 compounds in CRP could be grouped into four clusters according to their docking score profile against 24 receptors, while the cluster containing flavonoids and phenols might possess a more promising anti-inflammatory function. In addition, in vitro anti-inflammatory tests of the seven reference compounds demonstrated that hesperitin, naringenin, and gardenin B, which were grouped into a cluster containing flavonoids and phenols, significantly decreased LPS-induced NO, TNF-α, and IL-6 production of macrophages. While the compounds outside of that cluster, such as neohesperidin, naringin, hesperidin, and sinensetin showed little effect on alleviating LPS-induced NO and proinflammatory cytokine production. Based on the chemical properties of selected compounds, ethyl acetate (EtOAc) was selected as the solvent for extraction, because of its promising solubility of flavonoids and phenols. Furthermore, the ethanol alcoholic extract was used as a reference. The chemical profiling of EtOAc and crude alcoholic extract by HPLC/MS/MS also demonstrated the decreased abundance of flavonoid glycosides in EtOAc extract but increased abundance of phenols, phenolic acid, and aglycones. In accordance with the prediction, the EtOAc extract of CRP, but not the crude alcoholic extract, significantly decreased the NO, IL-6, and TNF-α production. Taken together, the results suggested selective extraction of phenols and flavonoids rich extract was able to increase the anti-inflammatory potential of CRP partially because of the synergistic effects between flavonoids, phenols, and enriched polymethoxyflavones. Our study might pave the road for the development of ethyl acetate extract of CRP as a novel functional food with anti-inflammatory function. Inflammation, especially chronic inflammation, is commonly viewed as the driving factor in the initiation and progression of various diseases, such as cancer, autoimmune diseases, cardiac vascular diseases, neurodegenerative diseases, and diabetes ,2, whichA variety of effective anti-inflammatory agents are available or under development nowadays, including nonsteroidal anti-inflammatories, antibodies against proinflammatory cytokine, small molecules that block the activity of kinases, histone deacetylase inhibitors, PPAR agonists, and even small RNAs . HoweverCitri Reticulatae Pericarpium (CRP) is a commonly used food complement and functional food and folk medicine  which poTo solve these problems, a systems pharmacology-based compound identification protocol was employed in the current study ). According to the chemical scaffold, 66 compounds were subdivided into eight different categories, such as flavonoids and flavones (4), polymethoxyflavones (6), flavonoids glycoside (4), phenols and phenolic acid (11), sesquiterpenoids (5), monoterpenoids (12), fatty acids (18), and others (6). The predicted targets retrieved from the TCMSP database are listed in The compounds isolated from Citri Reticulatae Pericarpium and their potential targets were retrieved from the TCMSP database  , nuclear receptors (PPARγ), the signal transducer and activator of transcription family member (STAT1), and enzymes that participated in the inflammatory process (COX2), which possessed not only differing molecular functions but also varied in the size and geometry of active sites. For example, the nuclear receptors (such as PPARγ) and COX2 possessed a closed and hydrophobic pocket for the ligand, while the kinases catalytic domain showed a semi-closed pocket for the ligand. On the contrary, the STATs exhibited a shallow ligand binding pocket close to the surface. The results from ligand–receptor interaction analysis demonstrated that the driving force in stabilizing the predicted binding pose differed remarkably among the reference compounds.The polar interactions between the polar group or the saccharide motif of flavonoid glycosides were demonstrated to be the main driving force in stabilizing their binding pose within selected receptors. As shown in The hydrophobic interactions played an essential role in stabilizing the predicted binding pose of flavonoids and polymethoxyflavones. The flavonoids and polymethoxylflavones penetrated the inner site of the semi-open binding pocket of kinases, as well as the closed binding pocket of COX-2 and PPARγ, and stabilized mainly through the hydrophobic interactions between surrounding hydrophobic residues . In addition, we found that the flavonoids fell into the open and shallow pocket of STAT1 and stabilized mainly through hydrophobic interactions with surrounding hydrophobic residues, such as Phe628, Val631, Tyr645, and Trp616, as well as the polar interactions between ligands with Ser606, Glu609, Asp638, and Asp632. The increased contribution of hydrophobic interaction in flavonoids and polymethoxylflavones may not only compensate for the decreased stability caused by decreased polar interactions (presented in flavonoid glycosides) but also increase the stability and selectivity of ligands towards the receptors with more hydrophobic binding pockets (such as JNK). The increased binding affinity towards kinases and enzymes that participated in the pro-inflammatory process may provide a plausible explanation for the observed increased anti-inflammatory capacity of hesperitin and naringenin.To selectively enrich the compounds with anti-inflammatory functions, the chemical and physical properties, such as oral bioavailability, molecular weight, logP, and caco2 cell permeability were analyzed . BecauseThe chemical profiles of crude ethanol extract and the EtOAc extract were analyzed by HPLC/MS/MS, and the retention time, accurate mass, mass defect, formation of negative/positive ion, and fragmentation patterns, as well as literature matching, allowed putative identification, i.e., identification without standards, of compounds within two extracts. For example, the secondary metabolites with some degree of hydrophilicity, such as flavonoids and glycosides, were eluted after 1–5 min, while the secondary metabolites, such as flavonoids, polymethoxyflavones, possessed an increased degree of hydrophobicity and eluted in the middle part of the elution gradient after 7–25 min. As expected, the EtOAc extract showed an increased abundance of elution after 10–25 min , Shanghai, China), hesperidin was purchased from J&K Chemical , while neohesperidin, gardenin B, and sinensetin were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. . Lipopolysaccharides (LPSs) from Escherichia coli O111:B4 was purchased from Sigma-Aldrich Chemical Co. . Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco Life Technologies . The Griess assay kit was obtained from Beyotime Biotechnology . Mouse ELISA kits to detect TNF-α, IL-6, and IL-10 were purchased from Neobioscience Technology Co., Ltd. .Hesperitin was purchased from ACMEC, naringenin was purchased from Macklin, naringin was purchased from GuoHua Reagent Co., Ltd. , and the crude alcoholic extract or EtOAc extract was prepared as follows: CPR samples were crushed into 200 mesh size and extracted with 100 mL 70% ethanol alcohol or extracted with 100 mL EtOAC for 30 min  under ultrasonic. Ethanol alcohol and EtOAc of analytical grade were supplied from DaMao Reagent . The extraction process was repeated twice, and the combined supernatant was filtered, concentrated in an evaporator under reduced pressure, and further dried to solid extract under a vacuum. The final extract was dissolved in methanol or DMSO and filtered with the 0.22 μm membrane filter for LC/MS/MS analysis or in vitro test.https://tcmsp-e.com/tcmsp.php (accessed on 7 May 2022)) and used to construct the compound–target network. The target names were converted to gene symbols by searching the DrugBank database. A total of 135 proteins that were highly relevant to macrophage activation and polarization [The bioactive ingredients and their potential targets of Citri Reticulatae Pericarpium were retrieved from the TCM systems pharmacology database and analysis platform (TCMSP) database  , PI3KPDBID: 3L54), JNK(PDBID: 3ELJ), PDE3A(PDBID: 7L29), NOS2(PDBID: 4NOS), NOS1(PDBID: 6CIC), SYK(PDBID: 1XBC), STAT1(PDBID: 1BF5), STAT3(PDBID: 1BG1), STAT5(PDBID: 1Y1U), cSRC(PDBID: 2BDF), PPARG(PDBID: 3GZ9), TLR4(PDBID: 3VQ1), MAPK14(PDBID: 1A9U), MTOR(PDBID: 4JT6), ITGAM(PDBID: 1IDO), JAK1(PDBID: 3EYG), JAK2(PDBID: 4D0X), JAK3(PDBID: 3LXK), IKK PDBID: (4KIK), RAC1(PDBID: 5N6O), TYK2(PDBID: 4GIH), CSF1R(PDBID: 3LCO), Tubulin(PDBID: 1SA0), COX2(PDBID: 6COX) according to literature [, JNK was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology , and maintained in DMEM high-glucose medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. After removing the old medium, the macrophages were treated with compounds or extract solution (dissolved in fresh medium) with indicated concentration for 24 h in the presence of 100 ng/mL LPS. The 0.1% DMSO was used as the vehicle control. After treatment, the NO, IL-6, and TNF-α secreted by macrophage RAW 264.7 cells were determined by Griess reaction, or ELISA kit according to the manufacturer’s instructions, respectively. Cell viability was routinely tested by MTT assay, and the selected compounds, as well as the extracts, did not affect the viability of macrophages up to 100 μM and 50 μg/mL, respectively (data not shown).The effects of compounds or extracts on NO and pro-inflammatory cytokine secretion of macrophages were determined by Griess reaction and ELISA, respectively. RAW 264.7 cells  were seeded in 96-well plates and incubated under 37 °C and 5% COm/z 50–1200. The compounds were identified by molecular weight and fragments.Chemical characterizations of Citri Reticulatae Pericarpium extract and its EtOAc extract were carried out by high-performance liquid chromatography coupled with mass spectrometry . For separation, a Waters ACQUITY UPLC BEH C18 Colum, 1.7 μm, 50 × 2.1 mm column was employed. The flow rate was set to 300 μL/min and the injection volume was 5 μL. The mobile phase consisted of eluent A, MilliQ water, and eluent B, acetonitrile, and both of them were acidified with 0.1% formic acid. The elution gradient was set to 0–8 min, 10–30% of B; 8–22 min 30–45% of B; 22–27 min, 45–95% of B; 27–31 min, 95% of B, 31–34 min, 10% of B. The MS analysis was performed in the positive and negative electrospray ionization mode, at a resolving power of 40,000 over a full scan range of p < 0.05, ** p < 0.005, *** p < 0.001. The analyses were performed using GraphPad Prism® version 7.0 .One-way ANOVA with Tukey post hoc test was used for the statistical analysis. Significant difference was considered when * In summary, combining network pharmacology analysis, molecular docking, principal component analysis, and k-means clustering, the flavonoids, phenols, and certain polymethxyflavones, but not flavonoid glycosides, were predicted to possess the potential for anti-inflammatory activity. Furthermore, our model also predicted that the EtOAc extract of CRP, which contained an increased abundance of bioactive ingredients and decreased abundance of flavonoid glycosides, might exhibit more promising anti-inflammatory potential than crude alcoholic extract. The hypothesis was proved by HPLC/MS/MS-based chemical profiling and in vitro tests. Unlike previous studies that considered the increased anti-inflammatory potential of flavonoids and polyphenols merely because of their increased lipophilicity and better bioactivity, the results of our study indicated the synergistic effects between flavonoids, phenols, and polymethoxyflavones might contribute to increased anti-inflammatory efficacy. As a result, the EtOAc extract of CRP held the potential to act as a natural immunomodulatory ingredient or functional food.


Myocardial inflammation has been consistently associated with genetic arrhythmogenic cardiomyopathy (ACM) and it has been hypothesized that episodes mimicking acute myocarditis (AM) could represent early inflammatory phases of the disease.We evaluated the temporal association between recurrent acute myocarditis (RAM) episodes and the later diagnosis of a genetic ACM.Between January 2012 and December 2021, patients with RAM and no previous cardiomyopathy were included . A follow-up visit including clinical evaluation, resting and stress electrocardiogram, cardiac magnetic resonance imaging, and genetic testing was carried out. Endpoints of the study was the incidence of both ACM diagnosis criteria and ACM genetic mutation at the end of follow-up.Twenty-one patients with RAM were included and follow-up was completed in 19/21 patients (90%). At the end of follow-up, 3.3 ± 2.9 years after the last AM episode, 14/21 (67%) patients with an ACM phenotype  underwent genetic testing. A pathogenic or likely pathogenic mutation was found in 8/14 patients (57%), 5/8 in the Desmoplakin gene, 2/8 in the Plakophillin-2 gene, and 1/8 in the Titin gene. Family history of cardiomyopathy or early sudden cardiac death had a positive predictive value of 88% for the presence of an underlying genetic mutation in patients with RAM.RAM is a rare entity associated with the latter diagnosis of an ACM genetic mutation in more than a third of the cases. In those patients, RAM episodes represent early inflammatory phases of the disease. Including RAM episodes in ACM diagnosis criteria might allow early diagnosis and potential therapeutic interventions. Genetic ACM is the consequence of pathogenic variants in genes encoding for desmosomal and cytoskeleton proteins. Originally described as a right ventricular disease , genetic ACM is increasingly recognized as a left or biventricular entity (Arrhythmogenic cardiomyopathy (ACM) is a genetically-determined or acquired . No endomyocardial biopsy was routinely performed in our center.Between January 2012 and December 2021, we screened consecutive patients admitted to our tertiary university center for clinically-suspected acute myocarditis (AM), defined by at least one clinical presentation and one diagnostic criterion from the European Society of Cardiology diagnostic criteria . We perfPatients were included in the present study in case of RAM, defined by more than one episode of AM requiring hospital admission. Index AM was defined as the first episode of AM. Patients were excluded in case of pre-existing cardiomyopathy or angiographically detectable coronary artery stenosis (≥50%). A follow-up program was carried out in patients with RAM, the last follow-up visit being at least 6 months after last RAM episode. Last follow-up visit included clinical examination, resting 12-lead ECG, 24-hour Holter monitoring, exercise test and CMRi. CMRi was performed on a 1.5-Tesla scanner . All patients underwent a dedicated study protocol for myocarditis, including balanced steady-state free precession sequences cine images for morpho-functional evaluation, T1-weighted turbo spin-echo sequences for detection of myocardial fat infiltration, triple inversion recovery sequences for detection of myocardial oedema and early and late gadolinium enhancement (LGE) detection, respectively 3 min and 15 min after contrast agent intravenous administration.The primary endpoint of this study was the incidence of ACM diagnosis criteria at the last follow-up visit, as defined by the Padua criteria for right, left, and biventricular ACM . The secIn patients with at least one diagnosis criteria of ACM at the end of follow-up, a genetic testing was performed using Next Generation Sequencing (NGS) method and analyzed with Genodiag. Sanger method was then used to validate the variant by NGS. The panel of genes analyzed was: ABCC9, ACAD9, ACTC1, ACTN2, ALPK3, ANKRD1, BAG3, CALR3, CAV3, CRYAB, CSRP3, CTNNA3, DES, DSC2, DSG2, DSP, DTNA, EMD, EYA4, FBN1, FHL1, FLNC, GAA, GATA4, GLA, HCN4, HEY2, JPH2, KRAS, LAMA4, LAMP2, LDB3 (ZASP), LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOM1, MYOZ2, MYPN, NEBL, NEXN, NKX2-5, PDLIM3, PKP2, PLN, PRDM16, PRKAG2, PTPN11, RAF1, RBM20, RYR2, SCN5A, SDHA, SGCD, SLC25A4, SOS1, SYNPO2, TAZ, TCAP, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TTN, TTR, VCL. Pathogenicity of variants was classified according to current guidelines (grade IV being likely pathogenic and grade V being pathogenic).t-test or a Mann-Whitney U test, as appropriate, was performed for comparison between two groups. The Chi-square test was used for analysis of categorical variables. A p < 0.05 denoted statistical significance. Analyses and figures were conducted using IBM SPSS Statistics for Macintosh .Data are presented as mean ± standard deviation or median (quartile 1–quartile 3) as appropriate, and categorical variables are given as number of patients with the attribute (percentage). For continuous variables, Student’s The study protocol was compliant with the STROBE statement and registered as a clinical trial (Recurrent Acute Myocarditis Registry NCT04589156). Approval for this study was obtained from the local Ethics Committee and was in accordance with the declaration of Helsinki.Between January 2012 and December 2021, a total of 21 patients  with RAM were included. Characteristics of patients, index AM, and first RAM are detailed in Follow-up was completed in 19/21 patients (90%), 3.3 ± 2.9 years after the last episode of AM. Clinical, resting, and stress ECG and CMRi data at the end of follow-up are detailed in According to study protocol, 14 patients with an ACM phenotype at the end of follow-up underwent genetic testing. A pathogenic  or likely pathogenic  mutation was found in 8/14 (57%) patients. Gene mutations were most frequently found in the Desmoplakin (DSP) gene , followed by the Plakophilin-2 (PKP2) gene , and the Titin (TTN) gene : DSP p.Glu1343Asnfs*, DSPc.3082_3084 + 13del, DSP C.3403C > T, DSP c.2588T > C, DSP c.5028_5031del, PKP2 c.1132C > T , and TTN c.14372-2A > G. In patient #12, an additional mutation of unknown significance was found in BCL2-associated athanogene-3 (BAG3) gene.Considering the 14 patients with RAM and ACM phenotype at the end of follow-up, family history had a sensitivity of 88% and a positive predictive value of 100% for the presence of an underlying genetic mutation. An illustrative example of a patient (number 5) with RAM, ACM phenotype, and DSP mutation is illustrated in The main findings of this study can be summarized as follow: (1) collecting data from a unique and large cohort of patients with RAM and no previous history of cardiomyopathy, we found that an ACM phenotype develops in more than 2/3rd of cases during follow-up; (2) in those patients, we found a high prevalence of previously unknown genetic mutation, illustrating the fact that that RAM represents the early phenotypic expression of a genetic ACM related to desmosomal or cytoskeleton gene mutations.In the vast majority of patients, AM is an acquired cardiac disease related to a viral infection and/or an autoimmune process. It is a major cause of sudden cardiac death in young adults  but alsoi.e., ARVC) using post-mortem histological analysis . Therefore, implementation of AM episode(s) in the current criteria for genetic ACM diagnosis . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.PO, PM, and FL elaborated the study protocol. PO, MA, and YC collected the study data and performed statistical analysis, and drafted the manuscript. All authors critically reviewed the manuscript.


Alcohol and several therapeutic drugs, including acetaminophen, are metabolized by cytochrome P450 2E1 (CYP2E1) into toxic compounds. At low levels, these compounds are not detrimental, but higher sustained levels of these compounds can lead to life-long problems such as cytotoxicity, organ damage, and cancer. Furthermore, CYP2E1 can facilitate or enhance the effects of alcohol-drug and drug-drug interactions. In this review, we discuss the role of CYP2E1 in the metabolism of alcohol and drugs (with emphasis on acetaminophen), mediating injury/toxicities, and drug-drug/alcohol-drug interactions. Next, we discuss various compounds and various nutraceuticals that can reduce or prevent alcohol/drug-induced toxicity. Additionally, we highlight experimental outcomes of alcohol/drug-induced toxicity and potential treatment strategies. Finally, we cover the role and implications of extracellular vesicles (EVs) containing CYP2E1 in hepatic and extrahepatic cells and provide perspectives on the clinical relevance of EVs containing CYP2E1 in intracellular and intercellular communications leading to drug-drug and alcohol-drug interactions. Furthermore, we provide our perspectives on CYP2E1 as a druggable target using nutraceuticals and the use of EVs for targeted drug delivery in extrahepatic and hepatic cells, especially to treat cellular toxicity. A recent report from the Centers for Disease Control and Prevention (CDC) showed approximately 100,306 drug overdose-related deaths in one year 2020–2021), which indicates an increase of 28.5% from the previous year 020–2021,. Both APCYP2E1, one of the major metabolizing enzymes in humans, is mainly expressed in the liver . To a leAlthough alcohol is primarily metabolized by alcohol dehydrogenase (ADH) to toxic acetaldehyde, it is the activation of the microsomal ethanol oxidizing system (MEOS), a secondary alcohol-metabolic pathway, that further causes cytotoxicity, organ damage, and cancer when CYP2E1 converts alcohol into acetaldehyde when ADH is saturated ,18,19. AIn the era of the COVID-19 pandemic, there have been increasing reports of alcohol consumption and self-medication ,22. SincIn this review article we first discuss the roles of CYP2E1 in the metabolism of alcohol and drugs, facilitating cellular injury/toxicities, and interactions involving drugs/alcohol metabolism. Next, we review nutraceuticals that have shown potential in decreasing or averting alcohol-/drug-induced toxicity, then we highlight provisional outcomes of alcohol/drug-induced toxicity and potential treatment strategies. Finally, we cover the role and implications of EVs containing CYP2E1 in hepatic and extrahepatic cells during drug/alcohol interactions. In this section, we review in vitro and in vivo animal studies that have investigated the causal effects of hepatic CYP2E1 mRNA, protein, and/or activity on alcohol- and drug-induced hepatoxicities and liver damage. The expression of CYP2E1 mRNA, protein, and/or enzyme activity are not always correlated with each other. Subsequently, we discuss various nutraceutical compounds tested to prevent or treat hepatotoxicity and liver damage. The studies with nutraceuticals are important because disulfiram, and DAS, 4-methylpyrazole are non-specific inhibitors of CYP2E1 inhibitors, and have other targets/effects. APAP is a commonly used analgesic found in mono and combination formulations and thus is easily overdosed, especially with improper use or duplicate therapy. This explains why it is a common cause of acute liver injury ,38,39. FA study conducted by Licong et al. found that fisetin (FST), a flavonoid, reverses APAP-induced toxicity and reduces ROS in human fetal hepatocytes (L-02 cells) and male C57 mice . The Jia2O2 production are mainly due to poor coupling of CYP2E1 with NADPH-cytochrome P450 reductase (CPR) [The metabolism of alcohol by hepatic CYP2E1 leads to ROS production, consequently generating damaging toxic effects in the liver through mitochondrial dysfunction, DNA damage, and lipid peroxidation in a chronic-binge mice model . Enhancese (CPR) . ROS infse (CPR) . To prevse (CPR) . HepG2 cse (CPR) . In an ise (CPR) . In the se (CPR) . Cryptotse (CPR) . In mousse (CPR) ,58. In cse (CPR) .The N-terminal signal of CYP2E1 protein regulates its targeting to mitochondria . CYP2E1 Multi-drug combinations are given to increase the therapeutic efficacy and success of drugs in treatment regimens of different diseases and comorbidities. However, these same drug combinations can also lead to unfavorable outcomes such as toxicity, treatment failure, or adverse drug interactions ,64. DrugA study conducted by Liu et al. on rats, using APAP and roxithromycin in combination, showed the induction of CYP2E1 mRNA and protein, enhanced levels of NAD(P)H, quinone oxidoreductase 1 (NQO1), TNFα, malondialdehyde, and lower mRNA and protein expression of glutathione peroxidase and superoxide dismutase (SOD) . They alHeavy alcohol consumption with other drugs leads to liver damage stemming from the generation of superoxide anion radicals and hydrogen peroxide after CYP2E1 metabolism. . In alcoIn this section, we discuss the role of CYP2E1 in alcohol-induced and drug-induced toxicity in extrahepatic cells, and also organ damage, as well as drug-drug and alcohol-drug interactions in vitro and in vivo animal studies. Further, this section comprises a discussion on the nutraceuticals used in various studies to prevent or treat extra-hepatotoxicity and organ damage.In addition to liver damage, drug overdose and/or adverse drug interactions, such as with APAP, also cause kidney injury, especially nephrotoxicity . NephrotCheng et al., injected lipopolysaccharide (LPS) intraperitoneally into mice to induce acute kidney injury (AKI). AKI mice were then treated with romidepsin (FK228) or depsipeptide; both compounds downregulated the expression of CYP2E1 by blocking the binding of hepatocyte nuclear factor-1 alpha (HNF-1α) with CYP2E1 promoter, thus reducing nephrotoxicity . In addiBrain CYP enzymes cause an immediate impact on neurotoxicity and drug response through different metabolic mechanisms in the brain ,85,86. EU937 monocytic cell lines and primary cells derived from peripheral blood mononuclear cells (PBMCs) are model cells to study many diseases including HIV-related pathogenesis, especially in the presence of drug abuse including alcohol. Alcohol consumption is highly prevalent in the HIV population compared to the general population ,90. FurtHeterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo ,5 pyridiGenotype sequencing is a commonly used and helpful tool to predict potential drug interactions in clinical research and practice . PopularThe metabolic process of chlorzoxazone through CYP2E1 and the formation of 6-hydroxychlorzoxazone, (detectable in human plasma or urine) is used to assess the enzymatic activity of CYP2E1 . Bedada Wang et al. observed increased CYP2E1 activity in obese type-II diabetic patients with increased expression of CYP2E1 mRNA . Loizou EVs are small extracellular nanovesicles 30–200 nm) that are produced by many cell types and are secreted in biofluids such as plasma, cerebrospinal fluids, urine, saliva, and milk . EVs pla–200 nm tKumar’s group reported the presence of various relevant physiologically and pharmacologically active CYP enzymes in human plasma EVs, including CYP2E1, as well as in monocytic cell-derived EVs ,130,131.Intracellular signaling of EVs induced by the metabolic processing of alcohol leads to hepatitis, alcoholic-associated liver disease, and liver fibrosis . AdditioRahman et al. demonstrated that both human and murine plasma EVs cargos, specifically CYP2E1, play a role in exacerbating xenobiotic-induced toxicity in hepatic and extrahepatic cells ,36,135. CYP2E1 is a druggable target to reduce the incidence of alcohol- and APAP-induced toxicity and drug-drug and alcohol-drug interactions . HoweverThe role of plasma EVs in cell-cell communication is becoming extremely important to understand the various physiological effects in disease pathologies and drug-induced organ toxicities, especially upon alcohol and APAP exposures ,137,143.EVs, being similar to that liposomes and niosomes, are actively being investigated to develop novel drug delivery systems, especially across the BBB ,142. ZhuAlthough there is a potential for EVs in drug delivery, EVs-based therapy will not reach patients without regulatory approval. Since there have been unsatisfactory outcomes of therapeutics for cancer, liver and cardiovascular diseases, EV-based therapy might be eligible for facilitated regulatory pathways (FRPs) . Researc


Skin is the largest organ in the body comprised of three different layers including the epidermis, dermis, and hypodermis. The dermis is mainly composed of dermal fibroblasts and extracellular matrix (ECM), such as collagen and elastin, which are strongly related to skin elasticity and firmness. Skin is continuously exposed to different kinds of environmental stimuli. For example, ultraviolet (UV) radiation, air pollutants, or smoking aggravates skin aging. These external stimuli accelerate the aging process by reactive oxygen species (ROS)-mediated signaling pathways and even cause aging-related diseases. Skin aging is characterized by elasticity loss, wrinkle formation, a reduced dermal-epidermal junction, and delayed wound healing. Thus, many studies have shown that natural polyphenol compounds can delay the aging process by regulating age-related signaling pathways in aged dermal fibroblasts. This review first highlights the relationship between aging and its related molecular mechanisms. Then, we discuss the function and underlying mechanism of various polyphenols for improving skin aging. This study may provide essential insights for developing functional cosmetics and future clinical applications. The skin dermis is composed of the upper papillary layer and the lower reticular layer. The papillary layer includes abundant fibroblasts, blood vessels, and phagocytes, while the reticular layer includes mainly collagen fibers in the dermal matrix . The derMany studies have reported various harmful effects of UV on the dermis ,7. The pFor this reason, protection from extrinsic or intrinsic aging is an essential issue in the cosmeceutical and dermatological fields. Novel active ingredients are required to retard or prevent skin aging by suppressing the harmful effects of UV. Especially, many researchers have demonstrated that natural polyphenols worldwide can be identified as a potential active ingredient to improve the aged skin dermis ,11,12. P2) by activating iNOS and COX-2, respectively. The expression levels of pro-inflammatory cytokines including TNF-α and IL-6 are remarkably increased in UV-irradiated human dermal fibroblasts (HDFs) [N-terminal kinase (JNK), and p38, which then affects the phosphorylation of nuclear transcription factor AP-1 , which contributes to express MMPs [External stimuli such as UV irradiation or air pollutants can generate ROS, which causes an imbalance between ROS production and antioxidant mechanisms, leading to causing oxidative stress ,6. This s (HDFs) ,6,16. Thess MMPs . This paess MMPs   is another harmful stimulus to accelerates the aging process in dermal fibroblasts [2O2-damaged dermal fibroblasts [Hydrogen peroxide -damaged dermal fibroblasts, kaempferol inhibited the phosphorylation of NF-κB, which is important for the IL-1β secretion and the expression of cleaved caspase-3 . KaempfeA tumor necrosis factor-α (TNF-α)-induced damaged fibroblast could also be alleviated by several polyphenols ,49,50,51Transforming growth factor-β (TGF-β) is a key regulator of ECM biosynthesis . EspeciaTGF-β binds to the TGF-β receptor, which enhances the phosphorylation level of Smad2/3. The Smad2/3 binds with Smad 4 and then moves to the nucleus. This pathway contributes to increasing collagen fibers. In the diagram, polyphenol chemicals in each box are organized into the classification system of polyphenols.2O2/UVB-induced decrease in cell viability, the impairment of TGFβ/Smad signaling in H2O2/UVB-treated Hs68 cells, and dermal aging in UVB-induced C57BL/6J nude mice [2O2-induced expression of hsa-miR-4535, which is a candidate miRNA for targeting Smad4 and led to activating the Smad4 complex in HDFs. Topical application of galangin to the dorsal skin of C57BL/6J nude mice remarkably reduced UVB-induced skin photodamage by accelerating TGF-β/Smad collagen synthesis signaling, diminishing epidermal hyperplasia, and wrinkling. Interestingly, galangin also remarkably decreased the expressions of type I collagen, type III collagen, and TGF-β1, whereas it increased the expression of Smad7 in the HS rabbit ear model [Various polyphenols have been studied to activate this TGF-β/Smad signaling pathway in aged dermal fibroblasts, which is important for the production of ECM . Apigeniude mice . Interesar model . Genistear model . Interesar model . The main characteristics of senescent cells contain oxidative DNA damage, double-strand DNA breaks, and the impairment of DNA repair mechanisms. Compared to young cells, senescent cells exhibit a reduction in the extracellular matrix. The senescent cells exhibit increased cell-cycle inhibitors p21 and p16 and increased β-galactosidase activity, loss of nuclear high mobility group box 1 (HMGB1), and decreased lamin B1 ,64. ThesUV irradiation is one of the key stimuli causing fibroblast senescence in vitro and in vivo ,69. Chro2O2/UVB-induced cell viability loss in HDFs [Cip1/WAF1, p16INK4A, and senescence-associated β-galactosidase in H2O2-damaged Hs68 cells.Polyphenols such as flavonoids may prevent dermal fibroblasts from the aging process by targeting cellular pathways important for modulating cellular senescence and the secretion levels of SASPs . Apigeni in HDFs . The knoSenescence signals trigger DNA damage. Senescent cells are characterized by a DNA damage response, including chronic Ataxia Telangiectasia-mutated (ATM) and Ataxia Telangiectasia and Rad3-related (ATR) kinase signaling, which ultimately induces cell cycle arrest and senescence by activation of the p53/p21 and p16 pathways. In the diagram, polyphenol chemicals in each box are organized into the classification system of polyphenols.Fisetin exhibited potent senolytic properties in vitro and in vivo. Administration of fisetin to old wild-type mice decreased the expression levels of p16 and p21, down-regulated the SASPs, and recovered tissue homeostasis by suppressing the PI3K/AKT/mTOR . KaempfeAutophagy is one of the conserved cellular processes that degrades damaged organelles or abnormal macromolecules to maintain cell survival and adaptation during starvation and oxidative stress . AutophaAutophagy function and activity are reduced in aged human dermal fibroblasts because of the impaired degradation of autophagy . Tashiro2. C3G recovered the levels of Atg5 and LC3-II in UVA-induced HDFs. To confirm this phenomenon, HDFs were pretreated with C3G and then treated with the autophagy inhibitor, 3-methyladenine (3-MA), after UVA irradiation of 12 J/cm2. 3-MA significantly decreased the inhibitory effects of C3G on morphological changes, oxidative damage, and apoptosis in UVA-damaged HDFs. The topical application of isoorientin ameliorated the UVB-damaged skin of mice by activating autophagy [Thus, several polyphenols have been reported to protect against photoaging by activation of autophagy . Cyanidiutophagy .DNA damage has been considered to be the primary cause of aging for a long time . Many stApigenin improved this UVB-induced loss of NER proteins in HDFs, meaning its protective effect against CPDs formation . InteresMost scientific studies have focused on identifying natural polyphenols with various beneficial effects such as blocking ultraviolet rays, removing harmful oxygen, collagen synthesis, and preventing skin wrinkles. In this review, we provided information on several key molecular mechanisms in aged dermal fibroblasts and discussed natural polyphenols including many flavonoids, which have anti-aging effects and their molecular mechanisms. As described previously, the evaluation of polyphenol efficacy for aged dermal fibroblast has mainly focused on ROS, inflammation, and the TGF-β/Smad pathway. Recently, many studies on the effects of polyphenols on molecular mechanisms such as autophagy ,95,96,97Most polyphenol compounds are usually stable and bioactive in plants. However, after the extraction from plants, these polyphenols are generally degraded because they are very sensitive to light or heat ,102. TheThus, the research and development of these natural polyphenols in aged dermal fibroblasts should proceed as follows. First, the studies of polyphenols on autophagy, senescence, and the DNA repair system in aged dermal fibroblasts should be further progressed. Second, new aging biomarkers should be identified to understand dermal aging. Third, adequate formulations for the topical application of these effective natural polyphenols should be investigated and optimized regarding skin delivery improvement such as novel liposome technology. Fourth, clinical trials to maximize anti-aging efficacy by a combination of effective polyphenols or alone should be carried out. These studies may contribute to reducing oxidative stress, inflammation, and cellular damage in the aged dermis and can be used as an effective agent of cosmeceuticals for improving skin health. Until 4 April 2022, we searched PubMed for published articles that investigated the effects of polyphenols on aged dermal fibroblasts. To reflect the latest research, the search timeframe was limited from 2012 year to the present (within 10 years). The search combined the keywords “polyphenol”, “flavonoid”, “lignan”, “tannic acid“, “aging”, “autophagy”, “senescence”, and “dermal fibroblast”. We also contained “liposome technology for the application of cosmetics”. Records were chosen by title and/or abstract to exclude studies that did not help answer the question in this review. Inclusion criteria: (1) published in English; (2) intervention included a flavonoid or polyphenol; (3) TGFβ/Smad, autophagy, senescence, or senolytic, or DNA repair.Data were extracted from selected studies  as follo


Cellular activities, such as attachment, spreading, proliferation, migration, and differentiation are indispensable for the success of bone tissue engineering. Mesenchymal stromal cells (MSCs) are the key precursor cells to regenerate bone. Bioactive compounds from natural products had shown bone regenerative potential. Notoginsenoside R1 (NGR1) is a primary bioactive natural compound that regulates various biological activities, including cardiovascular protection, neuro-protection, and anti-cancer effects. However, the effect of NGR1 on migration, adhesion, spreading, and osteogenic differentiation of MSCs required for bone tissue engineering application has not been tested properly. In this study, we aimed to analyze the effect of NGR1 on the cellular activities of MSCs. Since human adipose-derived stromal cells (hASCs) are commonly used MSCs for bone tissue engineering, we used hASCs as a model of MSCs. The optimal concentration of 0.05 μg/mL NGR1 was biocompatible and promoted migration and osteogenic differentiation of hASCs. Pro-angiogenic factor VEGF expression was upregulated in NGR1-treated hASCs. NGR1 enhanced the adhesion and spreading of hASCs on the bio-inert glass surface. NGR1 robustly promoted hASCs adhesion and survival in 3D-printed TCP scaffold both in vitro and in vivo. NGR1 mitigated LPS-induced expression of inflammatory markers IL-1β, IL-6, and TNF-α in hASCs as well as inhibited the RANKL/OPG expression ratio. In conclusion, the biocompatible NGR1 promoted the migration, adhesion, spreading, osteogenic differentiation, and anti-inflammatory properties of hASCs. With the prolongation of life span and the aging of the population, diseases such as tumors and osteoporosis are increasing rapidly. In addition, cases of bone defects caused by trauma, tumors, infections, congenital dysplasia, or developmental malformations are also increasing ,2,3. OstPanax notoginseng is a traditional Chinese medicine with a long history, which is used as a tonic and hemostatic drug ) was analyzed using the Living Image software, 4.5.All animal experiments obtained approval from the Animal Care Committee of PLA General Hospital of Southern Theatre Command, Guangzhou, China. PKH26-labeled hASCs were seeded on 3D-printed β-TCP scaffolds and treated with or without 0.05 µg/mL NGR1 for 3 h. Male nude mice  were anesthetized by intraperitoneal injection of 1% pentobarbital. Cell-scaffold constructs were carefully implanted in each of the two dorsal subcutaneous pockets. After implantation, the skin was closed using black non-resorbable 5-0 Mersilk sutures . Fluorescent images were acquired by the Living Image software with the IVIS Lumina LT Series III system . The autofluorescence background signal intensity was subtracted. The fluorescence intensity was represented by a multicolor scale ranging from red (least intense) to yellow (most intense). Signal intensity images were superimposed over grayscale reference photographs for anatomical representations. In all experiments, signals were collected from a defined ROI using the contour ROI tool, and total radiant efficiency  for n ≥ 3 (number of experiments). Data were analyzed using GraphPad Prism . Significance differences were determined by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test, two-way ANOVA with Tukey’s multiple comparisons test, or the unpaired In summary, NGR1 enhances adhesion, spreading, migration, immunomodulation, and osteogenic differentiation of hASCs. In addition, NGR1 also enhanced the survival rate of hASCs on cell-scaffold constructs in vivo. This study explored the influence of NGR1 on hASCs’ function from different aspects required for effective bone regeneration.


Lymph node metastasis is an important route of lung cancer metastasis and can significantly affect the survival of lung cancer.  All the analysis was conducted out in the R software. Expression profile and clinical information of lung adenocarcinoma (LUAD) patients were downloaded from The Cancer Genome Atlas database. CRHR2, ITIH1, PRSS48, MAS1L, CYP4Z1, LMO1, TCP10L2, KRT78, IGFBP1, and PITX3. Next, we performed univariate Cox regression, LASSO regression, and multivariate Cox regression sequentially to construct a prognosis model based on MAS1L, TCP10L2, and CRHR2, which had a good prognosis prediction efficiency in both training and validation cohorts. Univariate and multivariate analysis indicated that our model is a risk factor independent of other clinical features. Pathway enrichment analysis showed that in the high-risk patients, the pathway of MYC target, unfolded protein response, interferon alpha response, DNA repair, reactive oxygen species pathway, and glycolysis were significantly enriched. Among three model genes, MAS1L aroused our interest and therefore was selected for further analysis. KM survival curves showed that the patients with higher MAS1L might have better disease-free survival and progression-free survival. Further, pathway enrichment, genomic instability, immune infiltration, and drug sensitivity analysis were performed to in-deep explore the role of MAS1L in LUAD.  In our study, we firstly identified the characteristic genes of lymph node metastasis in LUAD through two machine learning algorithms, least absolute shrinkage and selection operator (LASSO) logistic regression, and SVM-RFE algorithms. Ten characteristic genes were finally identified, including MAS1L, TCP10L2, and CRHR2 is a useful tool to predict prognosis and lung cancer lymph node metastasis. Results showed that the signature based on  Lung cancer is a leading cause of cancer-related deaths all over the world, in which non-small-cell lung cancer (NSCLC) is the most common pathological subtype and accounts for approximately 85% of cases . Many faCCL19 expression had poorer outcomes and more LN metastasis, as well as impaired CD8+ T cell function [CXCR4 and VEGF-C are correlated with lymph node metastasis and might synergistically promote lung cancer progression [Lymph node metastasis is an important feature of lung cancer and is associated with poor prognosis . In the function . Moreovegression . Thus, eMAS1L, TCP10L2, and CRHR2 was then established, which had a good prognosis prediction efficiency in both training and validation cohorts. Next, pathway enrichment analysis was performed to explore the underlying biological differences between high- and low-risk patients. Among three model genes, MAS1L aroused our interest and therefore was selected for further analysis. KM survival curves showed that the patients with higher MAS1L might have better disease-free survival and progression-free survival. Further, pathway enrichment, genomic instability, immune infiltration, and drug sensitivity analysis were performed to in-deep explore the role of MAS1L in LUAD.In our study, we firstly identified the characteristic genes of lymph node metastasis in LUAD through least absolute shrinkage and selection operator (LASSO) logistic regression and SVM-RFE algorithms. A prognosis model based on https://portal.gdc.cancer.gov/; 522 patients, age: 65.33 ± 10.02) database. The original expression profile files were “STAR-counts” form and further sorted using the author's R code (tpm_unstranded). Clinical information was collated using Perl code. Genomic reference file GRCh38.gtf was used for probe annotation. The baseline information of patients was shown in The open-accessed transcriptional profiling data and clinical information of lung adenocarcinoma (LUAD) were downloaded from The Cancer Genome Atlas  algorithms were used for characteristic genes screening. LASSO logistic regression was performed based on the glmnet package . SVM is P < 0.05. Next, LASSO regression was used for dimensionality reduction [∗Coef A + Gene B ∗ Coef B + … + Gene N ∗ Coef N”. Each patient would be assigned a riskscore according to the above formula [Patients were randomly assigned to training and validation cohorts with a 1 : 1 ratio. Based on the characteristic genes identified by LASSO logistic regression and SVM-RFE algorithms, univariate Cox regression analysis was firstly performed to determine prognosis-related genes with the threshold of eduction . Finally formula .According to the calculated riskscore, the patients with riskscore higher than the median were defined as high-risk group, otherwise, the low-risk group. Kaplan-Meier (KM) survival curve was used to evaluate the prognosis difference between high- and low-risk patients. The receiver operating characteristic (ROC) curve was used to evaluate the prediction efficacy of our model in a specific time node. The independence of our prognosis model was validated by univariate and multivariate Cox regression analysis .Underlying biological differences can lead to different outcomes in patients. Pathway enrichment analysis was performed based on the GSEA algorithm. The reference gene set was set as Hallmark, c2.cp.kegg.v7.5.1.symbols, and c5.go.v7.5.1.symbols gene set. ClueGO plug-in in cytoscape software was used for the gene pathways enrichment . The datImmune infiltration analysis was conducted using the single sample gene set enrichment analysis (ssGSEA) algorithm . EvaluatP < 0.05 were considered statistically significant. Normal distribution was tested by the Student t-test. Nonnormally distributed variables were compared using the Mann–Whitney U test.All the analysis was performed using the R software version 4.0.0. Values of CRHR2, ITIH1, PRSS48, MAS1L, CYP4Z1, LMO1, TCP10L2, KRT78, IGFBP1, and PITX3 . Meanwhile, a higher TIDE score was found in the high-risk patients, indicating a lower percentage of immunotherapy responders in high-risk group . However, no remarkable correlation was observed between the MAS1L and MSI score and EREG-mRNAsi (Figures Furthermore, we tried to compare the expression level of  Figures . The res samples . Moreove Figures . MAS1L aegulated . GO anall Figure . KEGG anl Figure . Next, w Figures . A negat Figures .MAS1L expression might have a higher M2 macrophages infiltration (MAS1L expression might have a lower sensitivity to docetaxel and paclitaxel (Figures Immune microenvironment played an important role in tumor development. Immune infiltration analysis showed that riskscore was positively correlated with the mast cells, eosinophils, iDC, DC, and macrophages, while negatively correlated with Th2 cells Figures . Also, wltration . Moreoveltration . The res Figures .Lung cancer is a serious public health concern worldwide . Lymph nCRHR2, ITIH1, PRSS48, MAS1L, CYP4Z1, LMO1, TCP10L2, KRT78, IGFBP1, and PITX3. In the clinical practice, detecting the relative expression of these genes can indicate the risk group of patients, as well as their response to chemotherapy and immunotherapy.In our study, we firstly identified the characteristic genes of lymph node metastasis in LUAD through two machine learning algorithms, LASSO logistic regression and SVM-RFE algorithms. Ten characteristic genes were finally identified, including MAS1L, TCP10L2, and CRHR2, which had a good prognosis prediction efficiency in both training and validation cohorts. Univariate and multivariate analysis indicated that our model is a risk factor independent of other clinical features. Next, pathway enrichment analysis was performed to explore the underlying biological differences between high- and low-risk patients. Among three model genes, MAS1L aroused our interest and therefore was selected for further analysis. KM survival curves showed that the patients with higher MAS1L might have better disease-free survival and progression-free survival. Further, pathway enrichment, genomic instability, immune infiltration, and drug sensitivity analysis were performed to in-deep explore the role of MAS1L in LUAD.Next, we performed univariate Cox regression, LASSO regression, and multivariate Cox regression sequentially to construct a prognosis model based on KRAS mutant lung cancer to chemotherapy [BACH1 can activate the transcription of hexokinase 2 and GAPDH and increases glucose uptake, glycolysis rate, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of lung cancer cells [ENO1 [Our result showed that the pathway of MYC target, unfolded protein response, interferon alpha response, DNA repair, reactive oxygen species pathway, and glycolysis were significantly enriched in the high-risk patients. A break in the balance of DNA damage and repair would lead to the accumulation of oncogenes in tumor cells, leading to genomic instability and malignant progression . In lungotherapy . Glycolyotherapy . Wiel eter cells . Zhou etls [ENO1 . The difMAS1L was negatively correlated with the TMB and mRNAsi. In brief, TMB is the number of mutations in tumors, which can reflect the instability of the genome to some extent [Underlying genomic burden lead to the diverse performance of patients. Therefore, we found that the e extent . Generale extent . Tumor se extent . In lunge extent . Interese extent .MAS1L was positively correlated with the mast cells, eosinophils, iDC, DC, and M1 macrophages, while negatively correlated with Th2 cells. Eosinophils are rare multifunctional granulocytes and have been reported to play an antitumor role in cancer. Through manipulating eosinophil-related cytokines CCL11 and IL-5, Simson et al. found a negative correlation between tumor growth and eosinophil infiltration [Recently, the microenvironment of tumor cells located in has gained increasing attention from researchers. Immune cells are an important component of tumor microenvironment. Our result showed that ltration . The actltration . Carreteltration .Some limitations should be noticed. Firstly, the patients enrolled in our study were predominantly western populations, which might lead to underlying race bias and reduce the credibility of our conclusions. Secondly, the location of lymph node metastasis is not fully provided. If the relevant data is further improved in the future, this will increase the stability of our conclusions.