Momordica grosvenori (MG) in spinal cord injury (SCI) by network pharmacology analysis.  This study aimed to explore the molecular mechanism of  We searched for potential active MG compounds using the TCMSP database and the BATMAN-TCM platform. The Swiss target prediction database was used to find MG-related targets and the targets of SCI from the CTD, GeneCards, and DrugBank databases. Following that, a protein-protein interaction (PPI) study was carried out. Cytoscape software was used to calculate the hub gene, and R software was used to evaluate the Gene Ontology (GO) and KEGG enrichment pathways. Finally, molecular docking between the hub protein and important compounds was performed. We verified STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, and RXRA potential targets by quantitative PCR.  We obtained 293 MG-anti-SCI targets with potential therapeutic utility by intersecting 346 MG-related targets and 7214 SCI-related targets. The top 10 identified genes, ranking in descending order of value, were SRC, STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, RXRA, AKT1, CREBBP, and JAK2. Through enrichment analysis and literature search, 10 signaling pathways were screened out. The molecular docking of important drugs and hub targets revealed that some had a higher binding affinity. The results of quantitative PCR indicated that MAPK1, RXRA, and STAT3 were expressed differently in in vitro experiments.  In conclusion, the current work indicated that MG might play an anti-SCI role via multicomponent, multitarget, and multichannel interaction, which presents a novel idea for further research into the precise mechanism of MG-anti-SCI interaction. Spinal cord injury (SCI) is defined as structural damage and loss of whole or partial functional impairment of the spinal cord produced by various factors, often resulting in quadriplegia, paraplegia, or defecation dysfunction. The incidence of SCI on a global scale ranges from 10.4 to 83 cases per million people per year. SCI can affect patients' quality of life and consume plenty of healthcare resources . ComplicClinical medical research has traditionally focused on the best approaches to treat spinal cord injuries. Within eight hours of the injury, methylprednisolone (MPS) can be utilized in some contraindication-free SCI cases, and patients should be advised of potential consequences . StudiesTraditional Chinese medicine (TCM) has attracted the attention of more and more researchers in recent years as a potential treatment for SCI . AdditioNevertheless, MG's primary components and pharmacological effects have not been thoroughly identified, limiting future research and application. As a result, a thorough examination of MG's primary pharmacological components and implications is necessary. System analytic methodologies, such as network pharmacology, are promising for developing fresh approaches to elucidating the drug-gene-disease relationship . The netThe study aimed to develop an analytical approach for validating the effects of MG on SCI using network pharmacology, molecular docking, and experiments. https://tcmsp-e.com/) [https://bionet.ncpsb.org.cn/batman-tcm/) [All of the compounds in MG were searched by using databases and scientific studies, including Traditional Chinese Medicine Systems Pharmacology  , a bioinan-tcm/) . In totaAs TCM, it is very important to screen drugs with appropriate ADME characteristics . The perhttps://www.SwissTargetPrediction.ch/) [The predicted targets of active compounds screened from MG were obtained from the SwissTargetPrediction database (ion.ch/) . The finhttps://www.genecards.org/) [https://ctdbase.org/) [https://go.drugbank.com/) [We obtained potential targets of SCI from the GeneCards database (ds.org/) , the Comse.org/) , and thenk.com/) .Using the STRING website, we calculated and constructed the PPI network interaction diagram . A netwohttps://www.rcsb.org/) [The 3D structure of the ligand was imported from Protein Data Bank  , the lig2 at 37°C. AS were activated with 1 ug/ml of lipopolysaccharide (LPS) to form an inflammatory model and treated with 10 uM kaempferol. The effect of kaempferol on specific target genes, including STAT3, MAPK1, HSP90AA1, PIK3R1, PIK3CA, and RXRA, was observed.We performed in vitro experiments with rat astrocytes (AS). AS was acquired from the ATCC cell bank . AS is cultured in DMEM medium . Cells are stored in a clean cell culture incubator containing 5% CORNA from astrocytes in 6-well plates was extracted using the Axygen RNA kit  according to the manufacturer's instructions. The concentration and purity of the RNA, extracted cellular RNA, was measured using a spectrophotometer. Total RNA was reverse transcribed to cDNA using the TARAKA cDNA synthesis kit (Takara Bio) according to the manufacturer's instructions.https://www.ncbi.nlm.nih.gov/) and the primer sequences are arranged in We have designed gene-specific primer sequences based on the target genes  on the NCBI website  . The detpilosula . M10 (S)pilosula .To directly retrieve the name of the active ingredient's target gene, we entered the SMILE number of the active components (UniProt ID) into the SwissTargetPrediction platform  and delehttps://www.genecards.org/) was performed to predict the potential targets of compounds, and 5716 potential targets were obtained [https://ctdbase.org/) for 3234 possible targets [https://go.drugbank.com/) [The GeneCards database  . After cWe discovered the therapeutic targets at the confluence of MG and SCI, which had potential therapeutic benefits for treating SCI with MG. Venny software was used to intersect the MG and SCI targets, as illustrated in The interaction network is then constructed using Cytoscape software to reflect the connection between active compounds from MG and SCI targets . In FiguWe retrieved 293 therapeutic targets of MG for SCI from the STRING database and constructed a PPI network  consistiGO and KEGG pathway enrichment analysis plays a significant role in network pharmacology research. Through the DOSE, enrichplot, path view, KEGGgraph, Rgraphviz, ClusterProfiler, org.Hs.eg.db, and ggplot2 packages in the R Studio software, we operated GO enrichment analysis on the 293 obtained targets of MG-anti-SCI. As a result, we received a total of 2781 GO items, including 2426 BP, 98 CC, and 255 MF. The top ten enrichment results of BP, CC, and MF are displayed using a bar chart or a bubble chart .In the same way, through the ClusterProfiler, enrichplot, pathview, KEGGgraph, org.Hs.eg.db, ggplot2, and Rgraphviz packages in the R Studio software, using KEGG pathway enrichment analysis on 293 therapeutic targets, we identified 148 KEGG pathways. The top ten KEGG enrichment pathways are displayed in descending order, including lipid and atherosclerosis (hsa05417), EGFR tyrosine kinase inhibitor resistance (hsa01521), neuroactive ligand-receptor interaction (hsa04080), prolactin signaling pathway (hsa04917), apoptosis (hsa04210), AGE-RAGE signaling pathway in diabetic complications (hsa04933), insulin resistance (hsa04931), prostate cancer (hsa05215), chemical carcinogenesis-receptor activation (hsa05207), and endocrine resistance (hsa01522). The visualization of the above KEGG result is shown in We performed molecular docking between the top 10 hinge genes and kaempferol . The result of the molecular docking score, which ranged from 82.5427 to 116.96, is shown in The results show that kaempferol has good docking results with AKT1, RXRA, HSP90AA1, CREBBP, and JAK2. For instance, the scoring function LibDockScore of LibDock molecular docking is based on the ligand and receptor's affinity, relative energy, and docking mode. It is commonly assumed that a LibDockScore greater than 100 implies a stronger binding, and the higher the score, the more stable the shape of the complex formed via docking. The greater the affinity, the higher the ligand's binding strength to the protein .Based on the analysis of kaempferol and hub target data through molecular docking software, the diagram in The expression of hub gene targets  was detected in the inner AS cell by real-time PCR . The divAccording to the report, the random aggregated yearly incidence of traumatic SCI in the Middle East and North Africa (MENA) Region was 23.24 cases per million individuals . IndividInitially, compounds and targets derived from MG were obtained by investigating TCMSP and BATMAN-TCM. OB ≥ 30 and DL ≥ 0.18 were the ADME principles. Then, relevant target proteins were acquired from the SwissTargetPrediction databases (probability > 0), and their UniProt IDs were validated on the UniProt platform.The operational process of the TCMSP and BATMAN-TCM is separated into five steps: (1) The compounds of MG are directly obtained . (2) Screening out related compounds according to ADME standards . (3) The SwissTargetPrediction database is used to retrieve the target of the active component. The likelihood that is higher than 0 is the selected standard. (4) The final target is obtained by deleting duplicates . (5) Checking for and recording the UniProt ID number of the obtained compound's target protein. Finally, 346 MG-related targets and 11 MG-related active molecules were identified.In the second step, 7214 SCI-related targets were acquired by exploring 3 disease databases, including CTD (number: 3234), GeneCards (number: 5716), and DrugBank (number: 15). The website's online tool then constructs a Venn diagram of MG and SCI-related targets and extracts 293 targets from MG that may be beneficial in treating SCI.Finally, the top ten hub genes  were determined to have a higher therapeutic value for MG against SCI and will be examined subsequently.AKT1 phosphorylates and activates the downstream pathway during SCI. The PI3K/Akt signaling pathway can help patients recover from acute SCI . Our finκB, and the pyroptosis signaling pathway, which shows that the active compound candidate kaempferol offers potential for the treatment of SCI [To lower oxidative stress and inflammatory response, kaempferol can downregulate ROS-dependent MAPKs, NF-t of SCI . The molMolecular docking methods are generally considered useful techniques for screening drug candidates . MoleculThrough go enrichment analysis, we obtained the top 10 target genes of 293 targets of MG-anti-SCI, related biological processes are as follows: cellular response to chemical stress (GO-BP:0062197), lipid transport (GO-BP:0006869), steroid metabolic process (GO-BP:0008202), regulation of lipid metabolic process (GO-BP:0019216), lipid localization (GO-BP:0010876), response to lipopolysaccharide (GO-BP:0032496), response to molecule of bacterial origin (GO-BP:0002237), response to drug (GO-BP:0042493), response to oxidative stress (GO-BP:0006979), multi-multicellular organism process (GO-BP:0044706), nuclear receptor activity (GO-MF:0004879), ligand-activated transcription factor activity (GO-MF:0098531), steroid binding (GO-MF:0005496), monocarboxylic acid binding (GO-MF:0033293), protein serine/threonine kinase activity (GO-MF:0004674), phosphatase binding (GO-MF:0019902), protein tyrosine kinase activity (GO-MF:0004713), carboxylic acid binding (GO-MF:0031406), protein phosphatase binding (GO-MF: 0019903), bile acid binding (GO-MF:0032052), apical part of cell (GO-CC:0045177), membrane raft (GO-CC:0045121), membrane microdomain (GO-CC:0098857), membrane region (GO-CC:0098589), ficolin-1-rich granule (GO-CC:0101002), ficolin-1-rich granule lumen (GO-CC:1904813), integral component of presynaptic membrane (GO-CC:0099056), apical plasma membrane (GO-CC:0016324), intrinsic component of presynaptic membrane (GO-CC:0098889), and vacuolar lumen (GO-CC:0005775).The top 10 KEGG pathway enrichment analysis results are provided together with 293 MG targets against SCI. The enriched pathways were categorized, and 10 highly associated pathways were selected as follows: lipid and atherosclerosis (hsa05417), EGFR tyrosine kinase inhibitor resistance (hsa01521), neuroactive ligand-receptor interaction(hsa04080), prolactin signaling pathway (hsa04917), apoptosis (hsa04210), AGE-RAGE signaling pathway in diabetic complications (hsa04933), insulin resistance (hsa04931), prostate cancer (hsa05215), chemical carcinogenesis-receptor activation (hsa05207), and endocrine resistance (hsa01522).in vivo and in vitro experiments.Based on our preliminary findings from the above network pharmacology study, we believe that the primary active components of MG may play a role in the treatment of SCI via the pathways mentioned above and hub genes. Taken together, we anticipate that an increasing number of researchers will commit themselves to clarify the mechanism of TCM's action through network pharmacology, thereby increasing global awareness of this Chinese treasure. In the future, based on the above research results, we will explore the effects of MAPK1, RXRA, and STAT3 on SCI through more


Saposhnikovia divaricata–Angelica dahurica herb pair (SAHP) in migraine treatment.  This work studied the molecular mechanism of the  The active ingredients of drugs were screened, and potential targets were predicted by the Traditional Chinese Medicine Systems Pharmacology (TCMSP), TCMID, ETCM, and other databases. Migraine-related targets were obtained by harnessing the GeneCards, DrugBank, OMIM, TTD, and other databases. The protein-protein interaction (PPI) network was constructed with STRING software by performing a Venn analysis with bioinformatics. Gene Ontology (GO) functional enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed with the Metascape platform. The component-target-pathway (C-T-P) network was constructed with Cytoscape 3.7.2 software, and molecular docking was assessed with AutoDockVina software.  A total of 183 relevant targets and 39 active ingredients in migraine therapy were obtained from SAHP. The active ingredients and targets were screened according to topological parameters: wogonin, anomalin, imperatorin, prangenin, 2-linoleoylglycerol, and methylenetanshinquinone were identified as key active ingredients. PTGS2, PIK3CA, PIK3CB, PIK3CD, F2, and AR were identified as key targets. The molecular docking results demonstrated high binding activity between the key active ingredients and key targets. A total of 20 important signaling pathways, including neural signaling pathways, calcium signaling pathways, pathways in cancer, cAMP signaling pathways, and PI3K-Akt signaling pathways, were obtained through enrichment analysis.  Migraine with SAHP is mainly treated through anti-inflammatory and analgesic effects. The herb pair can be used for migraine using “multicomponent, multitarget, and multipathway” approaches. Saposhnikovia divaricata–Angelica dahurica herb pair (SAHP) is frequently used in treating migraine and is a commonly used herb pair for migraine treatment [Migraine is a universal chronic paroxysmal neurovascular illness, with recurrent or periodic attacks of throbbing headaches on one or both sides, often accompanied by autonomic symptoms such as nausea, vomiting, and photophobia, which can easily diminish a patient's ability to learn and work, as well as lower the quality of life. The current methods of treating migraine are mainly drug therapy, surgical therapy, and pressure therapy. Although these methods are effective, they have side effects . Faced wreatment . TCM clareatment . Wind-exNetwork pharmacology can predict the mechanism of drug functions in disease therapy as a whole, which is similar to the mechanism of multiconstituents and multitargeting function of traditional Chinese medicine compounds and conforms to the principle of a holistic view of traditional Chinese medicine . Moleculhttps://tcmspw.com/tcmsp.php) database [https://47.100.169.139:8000/tcmid/), and the Encyclopedia of Traditional Chinese Medicine [https://www.tcmip.cn/ETCM/) database, and the relevant literature for those components was excluded from the databases. Initial chemical components were obtained by integrating and deduplicating chemical components.The chemical components of SAHP were collected through the Traditional Chinese Medicine Systems Pharmacology  and the TCMSP were used.To screen initial chemical components and to screen the conditions for the chemical components included in the TCMSP, oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18  were usehttps://www.genecards.org/), DrugBank [https://go.drugbank.com/), Online Mendelian Inheritance in Man [https://www.omim.org/), and the Therapeutic Target Database [https://db.idrblab.org/ttd/) were searched using “migraine” as a keyword, and search results were integrated and deduplicated. The obtained targets were normalized on the UniProt database [https://www.uniprot.org/), and the relevant targets of migraine treatment were acquired.GeneCards  , intersecting targets that are underlying targets for herb pairing for migraine therapy were obtained.By drawing a Venn diagram for the migraine-related targets of SAHP with the jvenn module of the bioinformatics platform  (https:/https://cn.string-db.org/). The abovementioned intersecting targets were introduced into STRING, the species was selected as “homo sapiens,” and the interaction score was set to 0.4 to obtain the PPI network relationship. The PPI network chart was drawn with CytoScape3.7.2 software [PPI analysis is essential for the analysis of biological processes and for understanding complex mechanisms in cells. The PPI network was constructed using STRING  (https:/software . The deghttps://metascape.org/) for bioinformatic enrichment analysis, including a GO analysis of biological processes, molecular functions, and cellular components, and a pathway enrichment analysis of the KEGG. The species was set to “Homo sapiens,” and the significance level was P < 0.01. After the analysis, the top 10 GO functions and top 20 KEGG pathways were chosen, and the results were saved and visualized through the bioinformatics platform.The potential targets of SAHP for migraine therapy were imported into the Metascape platform  , as were core proteins from the PDB database (https://www.rcsb.org/). A 3D structure of the main effective ingredients with the Chem 3D component was saved in ChemOffice in the mol2 format, the minimized force field applicable to ligand energy was MMFF94s, and its energy was minimized. Proteins were dewatered and hydrogenated using PyMOL software (https://www.Pymol.org), and compounds and target protein formats were converted to the pdbqt format with AutoDockTools software [https://autodock.scripps.edu/resources/tools). Molecular docking was performed using AutoDockVina [https://vina.scripps.edu/), and the corresponding calculation results were obtained. Active ingredients and targets were screened using better integration activity through the docking value affinity, in which the value <−4.25 kcal·mol−1 can be considered to have binding activity between ligands and targets, the value <−5.0 kcal·mol−1 can be considered to have better binding activity, and the value <−7.0 kcal·mol−1 has yet greater docking activity [Molecular docking of the core targets chosen from SAHP for migraine treatment and their corresponding active ingredients was performed. Compound structures were downloaded from the PubChem database  in the C-T-P network diagram of the SAHP for the treatment of migraine and the top six active ingredients  underwent molecular docking separately Based on the same method, subsequently, 36 groups of receptor-ligand docking consequences were obtained. During the process of molecular docking, intermolecular forces shall be considered. The intermolecular forces in the study are mainly hydrogen bonds. Saposhnikovia divaricata is considered sweet and spicy, warm in nature, attributed to the bladder meridian, liver meridian, and spleen meridian. It relieves rheumatism and exterior syndrome, overcomes dampness, and relieves pain. Modern pharmacological studies have confirmed that Saposhnikovia divaricata has the functions of antipyretic, analgesic, anti-inflammatory, and antioxidant effects and promotes blood circulation [Angelica dahurica is sweet and warm in nature, attributed to the stomach meridian, large intestine meridian, and lung meridian, and it relieves exterior syndrome and rheumatism and dissipates cold and pain. Angelica dahurica can reduce the behavioral manifestations of migraine, reduce the secretion of ENK, increase the content of β-EP, and decrease the expression of C-FOS [Saposhnikovia divaricata and Angelica dahurica is in line with migraine pathogenesis, and it has a significant curative effect on migraine. It can effectively and safely relieve headache symptoms.Migraine is a common clinical primary headache characterized by high morbidity and a high recurrence rate. The disease has a long course and is hard to recover. It seriously impacts the patient's life, work, and study and significantly reduces their quality of life . Regardiculation . Angelicof C-FOS . The comκB signaling transduction, regulate iNOS, COX-2, TNFα, MAPKs, and CREB pathways, and has a therapeutic effect on inflammatory pain [Angelica dahurica, having anti-inflammatory [Angelica dahurica [Angelica dahurica has anti-inflammatory and analgesic effects, significantly inhibiting the serum level of PGE2 and reducing the content of TNFα in blood [κB pathway to inhibit the expression of inflammatory factors in macrophage THP-1 induced by LPS [This study has six key active ingredients  that were preliminarily screened out using the network pharmacology method of SAHP for migraine treatment. Wogonin can effectively prevent and reduce inflammatory responses by regulating the COX-2 pathway and inflammatory mediators, and it exercises a therapeutic effect on chronic neuroinflammation . Anomaliory pain . Imperatammatory , analgesammatory , and vasammatory  effects.ammatory . Prangendahurica . Animal in blood . 2-Linolin blood . Methyled by LPS .It was found that PTGS2, PIK3CA, PIK3CB, PIK3CD, F2, and AR were key targets combined with the PPI network and the C-T-P network. PTGS2 is a core enzyme in the biosynthesis of prostaglandins, also called cyclooxygenase-2 (COX-2). It overexpresses under mechanical, chemical, and physical stimuli, and it plays a vital role in the formation of inflammation . ResearcκB, and other pathways [κB is an important pathway in the process of migraine; it is associated with upregulating the release of downstream inflammatory mediators and mediating neurogenic inflammation [The results of the KEGG study have indicated that the main pathways of SAHP in migraine therapy are related to neuroactive ligand-receptor interaction, calcium signaling pathways, pathways in cancer, cAMP signaling pathways, and PI3K-Akt signaling pathways. The nerve signal transmission pathway is closely associated with nervous functions, and it is a collection of receptors and ligands associated with the plasma membrane and intracellular and extracellular signaling pathways . These ppathways . cAMP (cpathways . Other rpathways . The actpathways . NF-κB iammation .To conclude, SAHP mainly treats migraine through its anti-inflammatory and analgesic effects. Based on the network pharmacology method and molecular docking technology, this research initially revealed the molecular mechanism of SAHP to treat migraine through multicomponent, multitarget, and multibiological pathway regulation.Herein, we believe that the key active ingredients are wogonin, anomalin, imperatorin, prangenin, 2-linoleoylglycerol, and methylenetanshinquinone components of SAHP for migraine treatment. Such active ingredients may influence neuroactive ligand-receptor interaction, calcium signaling pathways, signaling pathways in cancer, cAMP signaling pathways, and PI3K pathways by acting on multiple targets such as PTGS2, PIK3CA, PIK3CB, PIK3CD, F2, and AR to treat migraine. Through network pharmacology and molecular docking technology, this study introduces the potential mechanism of SAHP in treating migraine, which not only provides a reference for the clinical treatment of migraine but also provides some theoretical support and data support for the development of new drugs to treat migraine.Meanwhile, this study had certain limitations. According to multiple databases, the study mainly predicted the pharmacology mechanism of SAHP in migraine therapy. However, to ensure reliability and rationality of forecast results, it is necessary to conduct further in vivo pharmacological research development of SAHP and conduct further animal or cell experiments.


The Lands Pathway is a fundamental biochemical process named for its discovery by William EM Lands and revealed in a series of seminal papers published in the Journal of Biological Chemistry between 1958–65. It describes the selective placement in phospholipids of acyl chains, by phospholipid acyltransferases. This pathway has formed a core component of our knowledge of phospholipid and also diglyceride metabolism in mammalian tissues for over 60 years now. Our understanding of how the Lands pathways are enzymatically mediated via large families of related gene products that display both substrate and tissue specificity has grown exponentially since. Recent studies building on this are starting to reveal key roles for the Lands pathway in specific scenarios, in particular inflammation, immunity and inflammation. This review will cover the Lands cycle from historical perspectives first, then present new information on how this important cycle forms a central regulatory node connecting fatty acyl and phospholipid metabolism and how its altered regulation may present new opportunities for therapeutic intervention in human disease. However, this turned out not to be the case, with a far higher level of incorporation of 14C in the FA compartment of the PL pool than expected. This led him to propose that the formation of lysoPL from PL was not simply a degradative end reaction for PL metabolism, but that a cycle existed where by exchange of FA can take place, via a lysoPL intermediate [William (Bill) EM Lands was a Biological Chemist born in 1930 in Chillilcothe, Missouri. Following a BS in Chemistry in Michigan, a PhD in Biological Chemistry from Illinois, and one year as Post Doc Fellow in California Institute of Technology, he spent 25 years on faculty at University of Michigan, starting around 1956. It was during this time that his seminal work on lipid metabolism included his discovery of the Lands Pathway, ultimately named after his own work. This pathway describes how selective fatty acyl (FA) placement is accomplished by lipid acyltransferases and phospholipases during recycling of glycerophospholipids (GPL), or more simply phospholipids (PL). It was uncovered in a series of seminal articles, reproduced in part during the centenary of JBC in 2005 . At the These seminal studies set the groundwork for our understanding of PL metabolism, and were then followed by several decades research charactering the large families of acyl transferases that mediate these reactions using FA-CoA ester and lysoPL substrates. Notably, the Lands cycle doesn't exist in isolation, but alongside other biosynthetic pathways such as Kennedy Pathway or CDP-choline pathway, identified in 1956 by Eugene Kennedy as the primary pathway for PC biosynthesis . For an While the reaction of the Lands Pathway may appear relatively straightforward, comprising the esterification of two substrates to form a single product, in reality this is far more complex with the different gene products showing a high level of specificity for particular FAs and PLs, leading to a hugely complex pattern of tissue specific PL composition. Furthermore, there are several mechanisms by which this can be accomplished, as outlined below. A summary figure of the Pathway is shown in in vitro and in vivo for PC [By the late 1990's, methods that allowed the separation and analysis of molecular species of PL had provided details on tissue specific composition, primarily in macrophages and liver . Studieo for PC . Complimo for PC . Aside fo for PC . The thio for PC . Last, Co for PC .While great strides had been made in elucidation of biochemical processes regulating PL recycling, at that time, the identities of the enzymes themselves was limited. Protein purification by detergent solubilisation, chromatography and SDS–polyacrylamide gel electrophoresis had been applied to the analysis of PC and PI acyltransferases in bovine heart , 32, 33,in vitro please see [During the 2000's, following the sequencing of genomes and once candidate genes were identified, many products of cDNAs were tested for enzymatic function relating to phospholipid remodelling. Enzymes that were called lysophospholipid acyltransferases (LPLATs) were suggested to be organised into two main families of proteins, then termed lysophosphatidic acid acyltransferases (LPAATs) and membrane bound O-acyltransferases (MBOATs) . For reaease see . Howeverease see . Notablyease see .AGPAT designated LPLATx members are named LPLAT1-10 and 9b, based on the new nomenclature. They have four conserved motifs which based on AlphaFold predictions are believed to define their enzyme active site and recognition of substrate. LPLAT8 and 9 are considered monotopic membrane proteins, with LPLAT8–10 predicted to be anchored via N-terminal helices. These LPLATs are widely expressed in mammalian tissues, and are mainly localised to the ER membrane, although some are also found on Golgi, mitochondrial, nuclear membrane or lipid droplets . As examMBOATs with LPLATx activity comprise LPLATs11–14, and they share four conserved motifs, that are distinct to those found in AGPAT family members. These LPLATs have several transmembrane domains, and a conserved histidine suggested to be the active site residue . There aBeyond lysoPL acyltransferases, when PL recycling involves a fatty acyl-CoA, other enzymes are involved. These include phospholipases , and also Co-A ligases, which themselves also comprise large families of structurally related proteins showing a high level of tissue specificity and differential regulation of gene expression. First, by hydrolysing FA from a PL, phospholipases provide the substrate for acyl-CoA formation. Human PLA2 enzymes which cleave FA at Sn2 are termed Groups I-VII, X and XII, with several having numerous members. These groups comprise many structurally related proteins expressed by diverse cell types, with some showing calcium sensitivity, while others are secretory forms. Many are actively involved in providing FA substrate for eicosanoid and prostaglandin biosynthesis, as well as PL recycling. PLA1 on the other hand, cleaves FA at Sn1. The best known, PLA1A removes FA from PS, generating lysoPS and is secreted from human platelets . A full The generation of fatty acyl-CoAs is catalysed by a large family of enzymes, called acyl-CoA synthetases (ACS). There are ∼26 mammalian ACS isoforms, which differ in their preference for FA substrates based on chain length , 59. TheAlthough the Lands pathway was discovered decades ago, for a long time it was mainly studied from the point of view of its underpinning biochemical mechanisms. More recent research is shedding light on key roles of this fundamental cycle in human health and disease, particularly in development and innate immunity. Indeed, disruption of metabolism of lysoPL via Lands and closely related pathways is associated with several diseases, such as atherosclerosis, vascular dementia and Alzheimer's disease .http://www.informatics.jax.org/). In relation to liver disease, several studies have seen that LPLAT11 (MBOAT7) mutations are associated with development and severity of non-alcoholic fatty liver disease (NAFLD) [http://www.informatics.jax.org/diseasePortal/genoCluster/view/57 246). Along with this, mice globally lacking MBOAT5 die soon after birth and also show significant alterations in liver glyceride levels [Studies using knockout mice have revealed underpinning roles of MBOAT family members in biology. The lack of LPLAT11 (MBOAT7) in mice, which is specific for lysoPI, and prefers 20:4 as FA substrate, leads to disordered cortical lamination and delayed neuron migration, and novel loss of function mutations were associated with intellectual disability , 69. Mic (NAFLD) , while m (NAFLD)  (http://e levels , 50. To e levels , 74.in vitro [A relatively recent finding has been the involvement of Lands Pathway enzymes in the generation of oxygenated PL species, following agonist activation of circulating blood cells such as platelets and neutrophils . As one in vitro .in vitro and in vivo, where the calcium dependent association of factors with PS is significantly enhanced by the presence of HETE-PLs in the membrane itself [in vitro, using recombinant enzymes [eoxPL formation appears to be a central part of innate immunity, since the cells that generate them are all essential to our immediate response to acute injury and trauma. Platelets, neutrophils, monocytes and eosinophils all form eoxPL acutely following agonist activation, and recent studies have shown that these lipids may play key roles in the innate immune response itself, reviewed in . EoxPL, e itself , 83. Here itself . The mose itself . This ise itself . A major enzymes .Aside from their role as substrates for PL recycling, lysoPL are bioactive lipids that signal through interacting with G protein coupled receptors . They alSn1 and an acetate bound at Sn2. Several studies have shown an essential role for two LPLAT isoforms in regulating its formation, namely LPLAT8 and 9, formerly known as LPCAT1 and 2 respectively. LPLAT8 was proposed to be involved in formation of noninflammatory PAF [PAF is an unusual phosphatidylcholine species which is characterised by a plasmalogen bond at tory PAF , while Ltory PAF , 99, invtory PAF  and a prtory PAF . Both LPtory PAF . For mortory PAF .The Lands Pathway has been known about since the end of the 1950's, when it was first noted by Bill Lands that a distinct metabolic pathway for PL recycling existed. This is one of the key processes by which membrane lipid composition is controlled across tissues and cells. Since then, our knowledge of the function and importance of the enzymes and products of this pathway has grown significantly and study of the role of lysoPL themselves in health and disease, and novel oxygenated PLs generated by this cycle is a major focus of ongoing research. With the advent of new generation LC/MS/MS methods, revisiting Lands pathway enzyme specificities in complex tissues in health and disease would be timely, since older studies mainly characterised their biochemistry using cloned enzymes. Currently in most studies, only existing levels of lipids are measured, with no analysis of their rapid dynamics of formation and degradation. This is a major disadvantage of current methods, and one example of an approach that addresses this gap is shown here in the case of a time resolved analysis of hepatocyte glyceride metabolism . One topThe Lands Pathway is a fundamental underpinning biochemical process, first elucidated in the 1950's.It is required for membrane biogenesis, and also formation of bioactive lipid mediators, and controlled in a cell and tissue dependent manner by numerous related gene products.New appreciation of the Lands Pathway in cell biology and immunology is being realised through lipidomics studies, and future directions will reveal mechanistic insights into the detailed roles of this cycle in human health and disease.


Intrauterine adhesion (IUA) represents an endometrial repair disorder that is associated with menstrual disorders, recurrent pregnancy loss, and infertility. This study aimed to explore the underlying biological mechanisms of Guishen Huoxue decoction for the treatment of IUA based on network pharmacology.  The selection of active compounds for Guishen Huoxue decoction and prediction of relevant targets were performed by the TCMSP and Swiss Target Prediction databases, respectively. The targets of IUA were obtained by three databases, including Online Mendelian Inheritance in Man (OMIM), DisGeNET, and GeneCards. The drug-disease regulatory network was constructed via Cytoscape software, following the acquisition of common genes of active compounds of drug Guishen Huoxue decoction and disease IUA, which was carried out through Venny software. Protein-protein interaction (PPI) network and function enrichment analyses were performed.  According to the data obtained from TCMSP, a total of 200 potential active compounds of Guishen Huoxue decoction and their related targets (1068) were screened by the Swiss Target Prediction database. 1303 disease targets and 134 common targets were identified. The drug-disease regulatory network showed that 165 active compounds were found to be involved in the treatment of IUA. Among 134 common targets, AKT1, SRC, TP53, VEGFA, and IL-6 were predicted as core genes against IUA. PI3K-Akt, Rap1, Ras, and AGE-RAGE were the main signaling pathways that participated in the treatment of Guishen Huoxue decoction for IUA.  The active compounds of Guishen Huoxue decoction confer therapeutic effects against IUA by regulating fibrosis, inflammation, and oxidative stress through major signaling pathways such as PI3K-Akt and AGE-RAGE. Intrauterine adhesion (IUA), also known as endometrial fibrosis or Asherman's syndrome, was first proposed by Joseph Asherman in 1948 . With thNo descriptions of IUA were found in ancient traditional Chinese medical books, but the symptoms of IUA belong to the categories of “hypomenorrhea,” “amenorrhea,” “abdominal pain,” and “infertility” in traditional Chinese drugs. Most studies believed that this disease was caused by operations in the intrauterine, and the operations damaged the uterus and uterine vessels, resulting in kidney deficiency, insufficiency of vital energy and blood, and blockage of blood stasis , 13. ModNetwork pharmacology is a new discipline used to analyze the network of biological systems and select specific signal nodes to design multitarget drug molecules . This sthttps://tcmspw.com/tcmsp.php) was performed to obtain the active compounds of Guishen Huoxue decoction, consisting of Paeonia lactiflora, Ligusticum wallichii, root of red-rooted salvia, Angelica sinensis, fruit of Chinese wolfberry, Astragalus membranaceus, Spatholobus stem, fruit of glossy privet, Chinese yam, fruit of medicinal cornel, prepared rehmannia root, seed of Chinese dodder. Oral bioavailability (OB) ≥ 30% and drug-like (DL) ≥ 0.18 were considered the screening conditions of the main active compounds, and the potential targets of the screened compounds were identified using the Swiss Target Prediction (https://swisstargetprediction.ch/) database, with a prediction score greater than zero. The UniProt database (https://www.uniprot.org/) was used for converting target information into gene symbols.The traditional Chinese drug systems pharmacology database and analysis platform (TCMSP) (https://omim.org/), DisGeNET (https://www.disgenet.org/), and GeneCards (https://www.genecards.org/) were carried out to obtain the disease-related targets (only “Homo sapiens”) using “Asherman's Syndrome” and “intrauterine adhesion” as the keywords. A gene symbol was obtained with the help of the UniProt database (https://www.uniprot.org/).Databases including Online Mendelian Inheritance in Man (OMIM) , with the parameter of “Homo sapiens” as protein type and “moderate confidence” (>0.400) as the minimum interaction critical value. The PPI network was visualized by importing the TSV-based file to Cytoscape 3.7.2 software, and the visual analysis included a topology analysis, gene cluster analysis, and core gene screening. On the basis of the degree value obtained, the core genes were sorted out.The PPI network was constructed based on common targets of Guishen Huoxue Decoction and IUA mapping into the STRING database  was applied to perform the GO and KEGG analysis of common targets of Guishen Huoxue decoction and IUA. Go enrichment analysis clarified gene function from three aspects: biological process (BP), cellular component (CC), and molecular function (MF). KEGG reflects a particular pathway of diseases with significant gene concentrations.The primary biological process and related signal pathways of Guishen Huoxue decoction in the treatment of IUA were explored by functional enrichment analysis. On the basis of R software, Bioconductor software  were found following duplicate removal. A total of 1169 targets related to potential active compounds were screened using the Swiss Target Prediction database.As listed in Using “Asherman syndrome” and “intrauterine adhesions” as the searching keywords, the results of three databases, including OMIM, DisGeNET, and GeneCards, indicated that 1303 disease-related records were obtained after duplicate removal.The Wayne diagram was mapped using 1169 targets associated with potential active compounds of Guishen Huoxue decoction and 934 disease-related IUA targets via Venny 2.1 software. A total of 134 common target genes were shown in the Wayne diagram . The druThe PPI network , containThe GO enrichment analysis was conducted following the performance of the R language on 134 common genes. Go analysis indicated that the common genes were enriched in 2291 BP terms, mainly including peptidyl-tyrosine phosphorylation, peptidyl-tyrosine modification, and positive regulation of cell adhesion . In termIUA is mainly characterized by hypomenorrhea or amenorrhea . The purβ1/Smad Pathway [Paeonia lactiflora, Ligusticum wallichii, the root of red-rooted salvia, Angelica sinensis, fruit of Chinese wolfberry, Astragalus membranaceus, Spatholobus stem, fruit of glossy privet, Chinese yam, fruit of medicinal cornel, prepared rehmannia root, seed of Chinese seed of Chinese dodder, improves renal function and promotes blood circulation [In recent years, traditional Chinese drugs have enriched the development of therapy for uterus-related diseases. Ding et al. revealed that as a classic traditional prescription, Bushen Huoxue Huatan decoction improved polycystic ovary syndrome through regulating hormones, reversing insulin resistance, and alleviating inflammation reactions, resulting in fertility improvement . Feng et Pathway . Similarculation , 24.In this study, we performed drug-disease regulatory network construction and the PPI network construction to obtain the core genes of Guishen Huoxue decoction for the treatment of IUA, and the core genes were analyzed by GO function enrichment and KEGG pathway enrichment to explore their potential molecular mechanisms. A total of 134 common targets were obtained from Guishen Huoxue decoction and IUA, and five core genes, including AKT1, SRC, TP53, VEGFA, and IL-6, were mainly enriched in PI3K/Akt, Rap1, Ras, and AGE-RAGE signaling pathways. IUA is a fibrotic disease, that is, the serious damage of the endometrial basal layer promotes a large number of fibrocyte proliferations and excessive deposition of extracellular matrix, leading to the partial or complete replacement of endometrial tissue by fibrous tissue . VEGF isAKT1 plays a key role in cell growth and survival in various diseases. Nie et al. manifested that  AKT1 medIn summary, our study based on network pharmacology shows that AKT1, SRC, TP53, VEGFA, and IL-6 in Guishen Huoxue decoction might be potential biomarkers for the treatment of IUA, and Guishen Huoxue decoction regulates fibrosis, inflammatory response, and oxidative stress response through several main signaling pathways including PI3K-Akt, Rap1, Ras, and AGE-RAGE. This study has conducted a preliminary theoretical discussion on the use of Guishen Huoxue decoction in the treatment of IUA. In the future, we need to verify our results through molecular docking analysis of drug-disease targets and active chemical compounds as well as experimental validation in cell and animal models.


Plant specialized metabolites (PSMs) play essential roles in the adaptation to harsh environments and function in plant defense responses. PSMs act as key components of defense-related signaling pathways and trigger the extensive expression of defense-related genes. In addition, PSMs serve as antioxidants, participating in the scavenging of rapidly rising reactive oxygen species, and as chelators, participating in the chelation of toxins under stress conditions. PSMs include nitrogen-containing chemical compounds, terpenoids/isoprenoids, and phenolics. Each category of secondary metabolites has a specific biosynthetic pathway, including precursors, intermediates, and end products. The basic biosynthetic pathways of representative PSMs are summarized, providing potential target enzymes of stress-mediated regulation and responses. Multiple metabolic pathways share the same origin, and the common enzymes are frequently to be the targets of metabolic regulation. Most biosynthetic pathways are controlled by different environmental and genetic factors. Here, we summarized the effects of environmental factors, including abiotic and biotic stresses, on PSM biosynthesis in various plants. We also discuss the positive and negative transcription factors involved in various PSM biosynthetic pathways. The potential target genes of the stress-related transcription factors were also summarized. We further found that the downstream targets of these Transcription factors (TFs) are frequently enriched in the synthesis pathway of precursors, suggesting an effective role of precursors in enhancing of terminal products. The present review provides valuable insights regarding screening targets and regulators involved in PSM-mediated plant protection in non-model plants. The ability of plants to synthesize an extremely wide arsenal of diverse metabolites makes them preeminent chemists . TraditiOn the basis of their core structures, PSMs form three major categories: nitrogen-containing chemical compounds, terpenoids/isoprenoids, and phenolics . NitrogeTaxus media, quinine from Cinchona officinalis, withanolide from Physalis angulate, and artemisinin from Artemisia annua, are widely applied in the treatment of a variety of serious diseases -norcoclaurine by norcoclaurine synthase -3′-hydroxy-N-methylcoclaurine (S)-3′-hydroxy-N-methylcoclaurine to (S)-reticuline, a branch-point product, in the production of morphine, tetrahydropalmatine, sanguinarine, and noscapine, is conducted by 3′-hydroxy-N-methylcoclaurine 4′-hydroxylase  and three-units isopentenyl diphosphates (IPP) . Both DMArabidopsis as an example. Isopentenyl phosphate kinase, common to most plants, catalyzes the conversion of isopentenyl monophosphate and dimethylallyl monophosphate to IPP and DMAPP , and 4-coumarate: CoA ligase , strong light, high-temperature, and drought increase the accumulation of glucosinolates , and sesquiterpene abscisic acid (ABA) . In lettAvena spp.) to combat various diseases . Many key enzyme-encoding genes involved in PSM biosynthesis are the downstream targets of different TFs .C. roseus, an ORCA3 TF regulates the expression of TIA biosynthetic pathway-related genes, such as GEISSOSCHIZINE SYNTHASE, STRICTOSIDINE SYNTHASE, and DEACETYLVINDOLINE ACETYLTRANSFERASE , WRKY40a and WRKY40b participate in the BIA biosynthetic pathway by regulating the TYDC, NCS, CYP80G, and 7OMT genes  REDUCTASE, which are key genes in the artemisinin biosynthetic pathway (A number of TFs are involved in the terpenoid biosynthetic pathway . ArtemisSE genes . AaWRKY1 pathway .Gossypium arboretum), GaWRKY1 regulates the conversion of sesquiterpenes to gossypol, which plays a role in responses to fungal infection (Hevea brasiliensis). HbWRKY1 and HbEREBP1 are positive and negative regulators, respectively, of latex biosynthesis induced by wounding , PpMYB17 positively controls the flavonoid biosynthetic pathway by activating the promoters of PpCHS, PpCHI, PpF3H, PpFLS, and PpUFGT (Pyrus × bretschneideri) TF, PbWRKY75 affects flavonoid biosynthesis by regulating the expression of PbDFR, PbUFGT, and PbMYB10b , StWRKY13 promotes anthocyanin biosynthesis in tubers by activating the promoters of StCHS, StF3H, StDFR, and StANS (MdHY5 (In d MtCHS2 . In appl11 genes . In buckOX genes . In Fago of F3’H . In peard PpUFGT . AnotherPbMYB10b . In Poputh KNAT7 . In potand StANS . The oveS (MdHY5 .ADT6, which catalyzes the key step that supplies phenylalanine (MYB15L in red-fleshed apple calli represses anthocyanin accumulation and cold tolerance (Arabidopsis MYB60 in lettuce plants significantly reduces the production and accumulation of anthocyanin pigments by inhibiting the expression of DIHYDROFLAVONOL-4-REDUCTASE gene (MYB2-1 expression causes the purple color in cabbage leaves, suggesting that it encodes a potential negative regulator of the flavonoid biosynthetic pathway (Ginkgo biloba, a negative regulator, GbMYBF2, affects flavonoid biosynthesis by down-regulating several key genes, such as GbPAL, GbANS, GbFLS, and GbCHS2 (Brassica napus WRKY TF, BnWRKY41-1, acts as a repressor of anthocyanin biosynthesis (S. miltiorrhiza, SmbHLH60 was identified as a negative regulator in anthocyanin biosynthesis mainly via SmDRF gene (Furthermore, many negative regulators of the flavonoid biosynthetic pathway have been identified in different plant species. AtMYB4 and its close homologs AtMYB7 and AtMYB32 inhibit flavonoid accumulation by down-regulating the expression of lalanine . The oveolerance . Homodimolerance . The oveASE gene . The los pathway . In Ginkd GbCHS2 . A Brassynthesis . In S. mDRF gene .Environmental signals pass through cell membrane through a large number of TFs to activate downstream functional genes. To date, a number of TF involved in metabolic pathway have been identified in different plants. Our review summarizes a network that is involved in the transcriptional regulation of PSM biosynthesis under environmental stresses . To dateArabidopsis, medicinal plant C. roseus, and woody plant poplar, these results also provide good guides for the regulation in other plants. This review summarizes the key enzymes and TFs involved in PSM biosynthetic pathways, providing valuable insights for screening targets and regulators in non-model plants.Plants produce a large number of PSMs having diversified structures, and they play important physiological and ecological roles in stress tolerance. The biosynthesis of stress-related PSMs is controlled by environmental and genetic factors. Artificial regulation of PSM biosynthesis is helpful to enhance plant resistance to environmental stresses. We summarized potential genetic and environmental factors and their targets, particularly in MYB, bHLH, and WRKY families. For plant genetic improvement, overexpression of activating TFs or inhibition of expression of inhibitory TFs can increase the yield of PSM and enhance the resistance of plants to environmental stress. We further found that the downstream targets of these TFs are frequently enriched in the synthesis pathway of precursors, suggesting an effective role of precursors in enhancing of terminal products. Although most of PSM-related TFs have been identified in different plant species, including the model plant RC was involved in the review writing. RC and ZC were involved in manuscript refinement. XZ and CS initiated the idea of the review and were involved in the manuscript writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Eucommia ulmoides Oliver is one of the commonly used traditional Chinese medicines for the treatment of osteoporosis, and iridoid glycosides are considered to be its active ingredients against osteoporosis. This study aims to clarify the chemical components and molecular mechanism of iridoid glycosides of Eucommia ulmoides Oliver in the treatment of osteoporosis by integrating network pharmacology and molecular simulations. The active iridoid glycosides and their potential targets were retrieved from text mining as well as Swiss Target Prediction, TargetNet database, and STITCH databases. At the same time, DisGeNET, GeneCards, and Therapeutic Target Database were used to search for the targets associated with osteoporosis. A protein–protein interaction network was built to analyze the interactions between targets. Then, DAVID bioinformatics resources and R 3.6.3 project were used to carry out Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Moreover, interactions between active compounds and potential targets were investigated through molecular docking, molecular dynamic simulation, and binding free energy analysis. The results showed that a total of 12 iridoid glycosides were identified as the active iridoid glycosides of Eucommia ulmoides Oliver in the treatment of osteoporosis. Among them, aucubin, reptoside, geniposide and ajugoside were the core compounds. The enrichment analysis suggested iridoid glycosides of Eucommia ulmoides Oliver prevented osteoporosis mainly through PI3K-Akt signaling pathway, MAPK signaling pathway and Estrogen signaling pathway. Molecular docking results indicated that the 12 iridoid glycosides had good binding ability with 25 hub target proteins, which played a critical role in the treatment of osteoporosis. Molecular dynamic and molecular mechanics Poisson–Boltzmann surface area results revealed these compounds showed stable binding to the active sites of the target proteins during the simulations. In conclusion, our research demonstrated that iridoid glycosides of Eucommia ulmoides Oliver in the treatment of osteoporosis involved a multi-component, multi-target and multi-pathway mechanism, which provided new suggestions and theoretical support for treating osteoporosis. The etiology of OP is very complex, including the interaction of endocrine, nutritional status, genetic, physiological and immune factors3. The imbalance between bone formation of osteoblasts and bone resorption of osteoclasts is the underlying cause of OP4. The treatment of OP depends on drug therapy, including bisphosphonate, selective estrogen receptor modulator, mixed steroid receptor agonist, monoclonal antibody against RANKL, parathyroid hormone analogue and so on5. These drugs can alleviate bone loss and improve clinical symptoms to a certain extent, but their long-term clinical application is limited by low tolerance, severe side effects and high cost6. Therefore, it is of great significance to develop more safe, effective and economical drugs for the treatment of OP.Osteoporosis (OP) is a chronic epidemic characterized by low bone mass and deterioration of bone microarchitecture, which leads to increased bone fragility and fracture risk, and caused a heavy economic burden to society7. In the theoretical system of TCM, OP is recognized as bone atrophy or arthralgia syndrome caused by kidney deciency8. Eucommia ulmoides Oliver (EU) is one of the most important nourishing medicinal materials in TCM. It has been found that EU can effectively prevent bone loss, improve bone biomechanical strength, prevent the deterioration of trabecular bone microarchitecture and cure OP11. Modern researches have considered that iridoid glycosides are the main pharmacological ingredients of EU14. There are many studies showing that iridoid glycosides play a primary role in bone resorption and bone remodeling. Iridoid glycosides can increase the differentiation and activity of osteoblasts, promote bone formation, inhibit the generation of osteoclasts, reduce osteoclast activity and limit bone resorption15. Such as aucubin could improve osteoblast differentiation in MG63 cells16. Geniposide could induce the proliferation and differentiation of the MC3T3-E1 cells17. Catalpol could suppress osteoclastogenesis and attenuate osteoclast-derived bone resorption18. Asperuloside could inhibit osteoclasts differentiation and reduce the number of osteoclasts19. Monotropein could promote the formation of osteoblastic and decreases the production of pro-inflammatory cytokines in osteoblasts20. Swertiamarin could significantly increase the expression level of OPG and against the activity of osteoclast21. These results indicated that iridoid glycosides exhibited potential preventive and therapeutic effects on OP. However, there are few studies on the chemical components and molecular mechanisms of iridoid glycosides of Eucommia ulmoides Oliver (IGEUs) in the treatment of OP.Traditional Chinese medicine (TCM) has a long history in China. It is more and more popular with the advantages of good curative effect, few side effects and affordable price23. Molecular docking is an important method to verify the reliability of network pharmacology in predicting drugs and targets24. Molecular dynamic (MD) simulation is frequently used to observe the dynamic process of complex conformations and provide more realistic trajectories over time25. Therefore, the purpose of this study is to clarify the hypothesis of a multi-component, multi-target and multi-pathway mechanism of IGEUs in the treatment of OP through network pharmacology, molecular docking and MD simulation, so as to provide a theoretical basis for future research. The specific research process is shown in Fig. In view of the complex chemical composition of TCM, network pharmacology has become a powerful tool to explore TCM from the system and molecular levelhttps://www.cnki.net/), PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Web of Science (https://www.webofscience.com/) databases27, which summarized the contents, biological functions and pharmacological effects of main active components from different parts of EU36. In addition, the PubChem ID, CAS, canonical smiles and 2D structure of the IGEUs were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)37. The targets of the IGEUs were predicted by using the Swiss Target Prediction webserver (http://www.swisstargetprediction.ch/), the TargetNet database (http://targetnet.scbdd.com/) and the STITCH webserver (http://stitch.embl.de/)40. The IGEU-predicted target network is based on the active components of IGEUs and their potential targets. The targets related to OP were obtained from the DisGeNET database (https://www.disgenet.org/), GeneCards database (https://www.genecards.org/) and Therapeutic Target Database (http://db.idrblab.net/ttd/)43. “Osteoporosis” was the keyword and “Homo sapiens” was the organism used when searching for the targets, which were verified using UniProt ID, and the target names were standardized into official gene symbols with the UniProt database (https://www.uniprot.org/)44. The common targets between the compounds and the disease were obtained by Venny2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) with a Venn diagram45.In this study, the IGEUs were obtained from China National Knowledge Infrastructure (https://string-db.org/) to construct a protein–protein interaction (PPI) network46. The organism was chosen as “Homo sapiens”, the minimum interaction threshold was selected as “medium confidence > 0.4”, disconnected nodes in the network were hidden, and other parameters remained the default settings. Cytoscape 3.8.0 was used to construct and visualize the PPI network47. At the same time, we analyzed the PPI network using the plug-in of “Analyze Network” tool. Network topology analysis, which contains many topological parameters, was applied to network pharmacology, among which edgecount is the most important parameter48. The edgecount of a node refers to the number of other nodes that interact with it49. We selected the nodes with more than twice the median edgecount of all nodes to construct the hub PPI. The correlated targets in hub PPI were identified as the hub targets.The common targets between IGEUs and OP were imported to the STRING11.0 database (https://david.ncifcrf.gov/home.jsp) to analyze the Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (www.kegg.jp/kegg/kegg1.html)51. The adjusted p-value was used to save the enrichment analysis results of GO and KEGG pathway, and the threshold of statistical significance was set to p < 0.05. Use R 3.6.3 (https://www.r-project.org/) software to visualize the results. The compound—hub target—pathway network was constructed by Cytoscape 3.8.0 (https://cytoscape.org/). We defined the compounds with edgecount greater than twice the median edgecount as the core compounds in this network for further analysis.In this study, the hub targets of IGEUs in the treatment of OP were introduced into DAVID bioinformatics resources (https://www.rcsb.org/), and the MOL2 structures of the compounds were downloaded from the TCMSP database (https://tcmspw.com/tcmsp.ph)53. SYBYL-X 2.1.1 was used to carry out the molecular docking of core compounds (ligands) with hub targets (receptors) and determine their binding activity54. The Total score is a complex score obtained by docking the receptor and ligand with corresponding parameters using Surflex-Dock procedure. It is generally believed that when the conformation of the ligand and receptor complex is stable, the higher the Total score, the higher the affinity of the receptor and ligand55. Total Score > 4.0 indicates certain binding activity, Total Score > 5.0 indicates good binding activity, while Total Score > 7.0 indicates strong binding activity56. PyMOL 2.4 (https://pymol.org) software was used to visualize the docking results57.Molecular docking was used to confirm the interactions between the compounds and the hub targets of IGEUs in treating OP and to verify the accuracy of the network pharmacology prediction. The 3D structures of the target proteins were downloaded from the RCSB database (http://atb.uq.edu.au/) prior to MD simulation to generate an initial topology for the ligands58. MD simulation of the complex was performed using GROMACS 2019.659. GROMOS96 54a7 force field was applied to the system, and dodecahedron water box consisting of a TIP3P water model was used to solvate the system. Na+ and Cl− ions were also solvated in the box to neutralize the system charge. Next, energy optimization process was performed using the steepest descent method. For equilibration simulation, 100 ps NVT equilibration was performed by using the velocity rescaling thermostat coupling method to keep the temperature constant at 300 K. Then, 100 ps NPT equilibration was performed. To treat the long-range coulombic interactions, the PME method was used. Production run of MD simulation was performed till 50 ns for each protein–ligand complex. Consequently, root mean square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg), solvent-accessible surface area (SASA) and hydrogen bonds were calculated according to the trajectory for further analysis. The binding free energies (ΔGbind) including electrostatic interactions (ΔEelec), Vander Waals interactions (ΔEvdW), non-polar solvation energy (ΔGSASA) and polar solvation energy (ΔGpolar) were calculated using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method implemented in GROMACS compatible tool “g_mmpbsa”61. In addition, the energy of each residue was decomposed, and the energy decomposition could be analyzed to determine the contribution of key residues to binding.The selected compounds were prepared with ATB webserver  terms, 35 molecular function (MF) terms and 25 cellular component (CC) terms, with lue Fig. . The res64. Therefore, the above three signaling pathways and related targets were considered as the candidate pathways for further validation. The compound—hub target—pathway network, including 46 nodes and 173 edges  and ERK2 (MAPK1) promoted osteoblast differentiation via phosphorylation of Runx284. Estrogen signaling pathway is essential for the development of OP. Estrogen is an important regulatory hormone in the human body, and its physiological role is mainly to regulate the transcription and translation of target genes by acting on the estrogen receptors of tissue cells85. As the major estrogen receptor subtype in bone tissue, ESR1 also plays an important role in regulating bone metabolism. Estrogen deficiency is one of the main causes of postmenopausal OP, which indicates the role of ESR1 in human bone homeostasis86. Studies have shown that the ratio of RANKL/OPG increased after estrogen cessation, which further leading to the increase of bone resorption87.In this study, we identified effective compounds, target proteins and important pathways for IGEUs in the treatment of OP. Network analysis showed that aucubin, geniposide, reptoside and ajugoside were the core compounds of IGEUs against OP. Aucubin had strong anti-OP activity, which can not only increased the differentiation of osteoblasts, promote the increase of cortical bone thickness and bone density, but also prevented the apoptosis of osteoclasts88. The RMSD showed that all complexes reached equilibrium till 50 ns, and the RMSF analysis proved the flexibility of the active amino acid residues, which facilitated the ligand binding. Similarly, the Rg and SASA analysis showed that the complexes were less affected by the solvent. Furthermore, the hydrogen bond assessment results were consistent with the results of molecular docking. The binding free energy analysis revealed that all core IGEUs showed stable binding at the binding pocket of the target proteins during the simulation time.Then, molecular docking technology was used to verify the binding of hub targets with core compounds. Results showed that most hub targets had a certain binding activity, and AKT1, ESR1, MAPK1 and MAPK3 had a strong binding activity, indicating they could tightly integrate with corresponding compounds. We further study the interactions between proteins and ligands using MD simulation, which can present the fluctuation and movement of residues at any specific time in motionThrough network pharmacology, molecular docking and MD simulation analysis in this study, we believed that the active compounds of IGEUs could affect the differentiation and survival of osteoclasts and osteoblast, regulate the balance of bone resorption and osteogenesis, and achieve the purpose of treating OP.The present study combined network pharmacology, molecular docking and molecular dynamic methods for the first time to reveal the pharmacological mechanism of IGEUs in the treatment of OP. Our data demonstrated that IGEUs could treat OP through a multi-component, multi-target and multi-pathway mechanism. In conclusion, this valuable finding may provide theoretical support for the clinical application of IGEUs and molecular design of therapeutic targets for OP.


Clostridium autoethanogenum. We not only developed a computational workflow to design and analyzehundreds of novel biochemical pathways for EG production but alsodemonstrated a successful pathway construction in the chosen host.The EG production was achieved using a two-plasmid system to bypassunfeasible expression levels and potential toxic enzymatic interactions.Although only a yield of 0.029 g EG/g fructose was achieved and thereforerequiring further strain engineering efforts to optimize the designedstrain, this work demonstrates an important proof-of-concept approachto computationally design and experimentally implement fully syntheticmetabolic pathways in a metabolically highly specific, non-model hostorganism.The platform chemicalethylene glycol (EG) is used to manufacturevarious commodity chemicals of industrial importance, but largelyremains synthesized from fossil fuels. Although several novel metabolicpathways have been reported for its bioproduction in model organisms,none has been reported for gas-fermenting, non-model acetogenic chassisorganisms. Here, we describe a novel, synthetic biochemical pathwayto convert acetate into EG in the industrially important gas-fermentingacetogen, Syntheticbiology, through the application of genetic and metabolicengineering technologies, contributes to the development of sustainablebioprocesses, enabling the bioproduction of value-added commoditychemicals from renewable resources. This discipline is essential toreduce our dependence on fossil fuel-based petrochemical industries,which negatively impact the environment and significantly contributeto the current climate emergency through greenhouse gas emissions.3 Considering its high market value and demand with an estimated globalproduction of 65 million tons in 2024,4 sustainable EG bioproduction is highly beneficial as compared totraditional fossil fuel-based chemical processes from both industrialand environmental points of view. In fact, several EG-producing biochemicalpathways have previously been implemented in model organisms. EG production,for instance, has been reported from xylose in Escherichiacoli is an important industrialsolvent that is widely used as an antifreeze agent and a precursorfor several polyesters such as the plastic, polyethylene terephthalate.chiacoli and Sacc E. coli, and from2 and CO) and synthesis gas , are superior to sugars, as they are widely availablefrom a diverse range of waste feedstocks,15 and their availability does not compete with limited arable landor crop production. For example, acetogens, the Gram-positive anaerobicbacteria, show great potential for industrial applications due totheir ability to grow autotrophically via the Wood–Ljungdahl(WL) pathway.17 This metabolic property allowsthem to use CO2 or CO as their sole carbon source to synthesizeacetyl-CoA, further converted into acetate and other species-specificproducts such as ethanol via gas fermentation. Considering their diversemetabolic abilities and potential for industrial applications, researchefforts in the past decade have been focused on developing genetictools for acetogens21 and applying them for metabolic engineering purposes,24 allowing sustainable production of several value-added chemicalswhile fixing CO2. The mesophilic acetogen, Clostridium autoethanogenum,.39 A preselectionprocess was applied based on the following pathway-pruning criteriato choose the best candidate pathway: pathway length; requirementfor external metabolites; gene availability; and gene origin, furtherdiscussed in the C. autoethanogenum genome-scale metabolic model40 and the Flux Balance Analysis (FBA) tool41 in the COBRA toolbox.42 The group contribution method (GCM)43 and the online tool, eQuilibrator44 wereexplored for a thermodynamics-based feasibility analysis of the designedsynthetic pathway for EG manufacture.Synthetic metabolic pathwaysfrom acetate to EG were designed with the cheminformatics tools, FromMetabolite to Metabolite (FRM)E. coli TOP10 and sExpress45 were grown in Luria–Bertani(LB) medium at 37 °C. LB was supplemented with 15 g/L of agarfor plates. The medium was also supplemented with 500 μg/mLof erythromycin, 25 μg/mL of chloramphenicol, and 50 μg/mLof kanamycin where appropriate. C. autoethanogenum DSM 10061 C24 (henceforth C. autoethanogenum C24)46 and its derivatives were grownin YTF , solidified with 15 g/L of agar and supplemented with 6 μg/mLof clarithromycin and 7.5 μg/mL of thiamphenicol when needed.5 mM of β-lactose and 5 mM of theophylline were added to inducegene expression when required. C. autoethanogenum C24 and its derivatives were grown at 37 °C in a Don Whitleyanaerobic chamber .The bacterialstrains and plasmids used in this study are listed in aceA, ghrA, aldA, and fucO were amplified from E. coli genomic DNA with Q5 High-Fidelity DNA polymerase. The primers, synthesized by Sigma-Aldrich, are listed in TableS1 in the Supporting Information. aceA, ghrA, and aldA werecloned into pMTL83251 with SacI-SpeI, SpeI-HpaI, and HpaI-XbaI, respectively,downstream of the PtdcB promoter.46fucO was fused to the riboswitch-Pfdx promoter47 inan NEBuilder HiFi DNA Assembly  reactionand cloned into pMTL84151 with NotI and NheI. Cloning steps were performedin E. coli TOP10. The plasmids werethen transformed into E. coli sExpressfor conjugation into C. autoethanogenum C24 as previously described.45 pMTL83251-EGwas first introduced into C. autoethanogenum C24, and pMTL84151-fucO was conjugated in a secondconjugation step. Plasmids and transformants were confirmed by Sangersequencing by Eurofins Genomics (Eurofins Genomics Germany GmbH). C. autoethanogenum C24 strains were stored in cryotubesat −80 °C in 15% dimethyl sulfoxide.The four genes: C.autoethanogenum C24 EG was grown in YTF, supplementedwith 6 μg/mL of clarithromycin and 7.5 μg/mL of thiamphenicolat 37 °C in an anaerobic chamber .5 mM of β-lactose and/or 5 mM of theophylline was added to thecultures when required for induction of promoters. At each timepoint,OD600 was measured for growth curves, and 1 mL of culturewas collected and centrifuged at 13,000g for 5 min.Supernatants were stored at −20 °C in cryotubes untilHPLC analysis was performed. Supernatant samples were diluted 1:1with 50 mM valerate in 0.005 M sulfuric acid; after vortexing, eachsample was filtered into a HPLC vial. The analysis of metaboliteswas performed using a Thermo Scientific Ultimate 3000 HPLC systemequipped with UV/vis and RI detectors and an Aminex column  (Bio-Rad laboratories) kept at 35°C. Slightly acidified water was used (0.005 M H2SO4) as the mobile phase with a flow rate of 0.5 mL/min.E. coli, S. cerevisiae, and C. glutamicum; thus, progressing toward its sustainable production. The studyreported here explored the possibility of producing this importanttarget chemical in a non-model organism such as the acetogen, C. autoethanogenum, which provides a great opportunityfor simultaneous CO2 fixation and EG production due tothe organism’s gas fermentation capability. The described steps,illustrated in C. autoethanogenum for the design,analysis, and expression of a de novo pathway forEG bioproduction in this host organism.Synthetic biology, mediated through genetic andmetabolic engineering,has been considered as the future for sustainable bioproduction ofvarious target chemicals. Although historically synthetic biologyapproaches relied on introducing existing pathways in a specific hostmicroorganism, recent engineering progress, as well as the developmentof state-of-the-art computational tools and methods have allowed thedesign and implementation of novel synthetic pathways, dramaticallyexpanding the catalogue of products synthesized by chassis organisms.For example, the industrial platform chemical EG has previously beenreported to be produced by 38 and MRE39—were predominantly employed here dueto their user-friendly interface with superior pathway predictioncapabilities. These tools create pathways in a retrosynthetic manner,50 linking a target product, that is, EG, to a starting metabolite,that is, acetate. Acetate was chosen as the starting point due toits high production rates during gas fermentation by acetogens. Theconversion of acetate into other target products would also unlikelybe detrimental to C. autoethanogenum growth because acetate production through the conversion of acetyl-CoAvia the WL pathway generates one mole of ATP under autotrophic conditions.51 In addition, both tools create pathways using only biologicallyknown reactions listed in the Kyoto Encyclopedia of Genes and Genomes(KEGG) database.52 Other tools such asATLAS of Biochemistry53 design pathwayswith novel reactions, but were not explored in this study to avoidthe extensive protein engineering efforts required for experimentalimplementation of novel reactions. To further select the best candidatesamong the extensive number of pathways generated by FMM and MRE, fourmain pruning criteria  were first applied in a more rationalpreselection to exclude inadequate pathways , required for correct protein folding and maycomplicate protein expression in a phylogenetically distant organismwith different intracellular conditions. As such, it is advisableto select genes from closely related organisms to the chosen host,when possible, to avoid additional protein engineering efforts, whichinevitably further limits the number of candidate genes. Using thesedifferent selection criteria, several pathways (example pathways shownin the Supporting Information) were designedto convert acetate into EG in C. autoethanogenum. However, only the selected pathway that was further analyzed andsuccessfully constructed in this acetogen is shown in Although myriad computational cheminformaticstools are now available to design synthetic biochemical pathways,the two tools—FMMpathways 1. As sucC. autoethanogenum.40 Using the COBRA toolbox,41 FBA was performed to predict pathway yield from three substrates and compared to thetheoretical yields estimated from the degree of reductance of substratesand the product, as previously described54  from CO usingtheC. autoethanogenum GEM was observedas compared to the theoretical EG yield from CO (0.44 g EG/g CO);however, this issue was not further addressed as it seemed negligibleand did not influence later analyses.To predict the feasibility and yieldof the selected pathway 2, it wascribed54 3. Interedictions 3; this r43 and theonline tool, eQuilibrator44 were used tocalculate the pathway’s overall thermodynamic feasibility.Although both methods agreed that the pathway had an overall negativestandard Gibbs free energy change (ΔrG′°) value, the values themselves were very differentdepending on the metabolite concentrations or the calculation methodsused  stand out as major obstacleswhen starting pathway implementation but are often missed during computationalanalysis. As such, computational analyses can undeniably act as afirst compatibility assay to predict pathway feasibility or yield,but biases and gaps remain in computational methods, preventing predictionof some notable difficulties important for pathway implementationin a host organism.While the computational analysis described in this study remainsextremely useful to test the feasibility of a pathway in the contextof a specific host to allow the identification of pathway bottlenecksor other obstacles before its time-consuming implementation, thereare still some aspects that cannot be predicted accurately using the C. autoethanogenum. Therefore, heterologousexpression of the pathway’s genes in this host was exploredand implemented. First, the genes required for expressing all thepathway enzymes were placed as a synthetic operon, under the controlof the PtdcB promoter.46 This lactose-inducible system allows fine-tuned targetexpression as previously described.46 Forthis step, the 4-step (reactions 3–6) and 6-step (reactions1–6) versions of the pathway .In fact, while attempting to quantify protein expression to confirmthat the chosen expression system was not preventing target gene expressionto synthesize EG with these operons, the fucO gene. In thisapproach, expression at the PtcdB promoterwas controlled by theClostridiodes difficile sigma R factor TcdR, inserted in C. autoethanogenum genome and controlled by the lactose-inducible promoter PbgaL, itself activated by the transcriptional regulatorBgaR to allow a two-level expression control, as described by Woodset al.46 Therefore, induction of genesregulated by PtcdB was achieved by additionof lactose in the medium. Moreover, the riboswitch used for fucO expression prevented gene expression in the absenceof theophylline by forming a stem loop structure sequestering theribosome-binding sequence (RBS); thereby, preventing ribosome bindingand gene expression. When theophylline was added, the RBS was releasedfrom the stem loop structure to allow for ribosome binding and geneexpression. This theophylline-inducible riboswitch was fused to thestrong constitutive Pfdx promoter, derivedfromC. sporogenesferredoxin gene, aspreviously described by Cañadas et al.47 In addition, the two plasmids used for operon and fucO expression carried different Gram-positive replicons and selectionmarkers to limit unwanted recombination between these two vectors.The computational analysesdescribed above led to the conclusionthat the designed synthetic pathway for EG production would be feasiblein pathway 2 were coucO gene2 was sysectively 2, were e pathway 2, was plC. autoethanogenum . Simultaneousmaintenance of both plasmids led to the production of EG inC. autoethanogenum, even when no inducer was added and 3.06%(transformant 2) of the maximum yield predicted by FBA with fructoseas the main substrate 2. Althouttleneck 5 and to C. autoethanogenum. Importantly, this study describesa workflow to design, prune, and analyze synthetic pathways with computationaltools for a specific value-added chemical and successful implementationof the pathway expression in a microbial chassis, leading to bioproductionof the target chemical. Although the workflow described here is promisingand opens the door for the bioproduction of other target chemicalsinC. autoethanogenum, much more workis still needed to optimize the designed pathway and the engineeredstrain to improve yield and productivity in order to render it viablefor industrial applications. Improving expression levels of differenttarget genes seems particularly important for successful EG productionas shown by the suspected unviable expression of original fucO. As such, other expression systems could be exploredto increase gene expression levels while maintaining viability. Inaddition, other candidate genes from other organisms homologous tothe ones implemented here might also increase pathway efficiency dueto having different enzyme kinetics and substrate specificities. Similarly,rational or randomized protein engineering might be useful to furtherimprove enzyme parameters at later stages of pathway optimization.Additional metabolic engineering strategies could also be explored,for example, to increase cofactor pools or downregulate competingpathways to achieve maximal EG production inC. autoethanogenum. The results described here clearly highlight the gap remainingbetween computational predictions and experimental implementationof synthetic metabolic pathways in a non-model chassis, which promotesadditional efforts for developing more reliable data-informed computationalmodels to truly access the full potential of synthetic biology andmetabolic engineering for sustainable production of various targetproducts, especially in non-model organisms with constrained metabolism.EGis an important industrial platform chemical for the productionof various value-added target commodities. Due to its high demandand numerous applications, its sustainable bioproduction is crucialto reduce the demands on fossil fuels for its availability. In fact,such production has been reported for EG in three model microorganismsusing several engineered metabolic pathways. Here, we described thedesign and implementation of a novel synthetic pathway, allowing theconversion of acetate to EG in the industrially important acetogen,


On the other hand, there is insufficient evidence to link this pathway to axial elongation by controlling its main cellular processes, such as convergent extension and cell proliferation. However, conclusions are premature until more studies incorporating diverse arthropods are carried out.Wnt signaling pathways are recognized for having major roles in tissue patterning and cell proliferation. In the last years, remarkable progress has been made in elucidating the molecular and cellular mechanisms that underlie sequential segmentation and axial elongation in various arthropods, and the canonical Wnt pathway has emerged as an essential factor in these processes. Here we review, with a comparative perspective, the current evidence concerning the participation of this pathway during posterior growth, its degree of conservation among the different subphyla within Arthropoda and its relationship with the rest of the gene regulatory network involved. Furthermore, we discuss how this signaling pathway could regulate segmentation to establish this repetitive pattern and, at the same time, probably modulate different cellular processes precisely coupled to axial elongation. Based on the information collected, we suggest that this pathway plays an organizing role in the formation of the body segments through the regulation of the dynamic expression of segmentation genes,  The embinsects) . This deinsects) . The estinsects) .The segmented body plan of panarthropods is orchestrated by a wide variety of cellular mechanisms and genetic regulators, which can vary depending on the organism in question.β-catenin cytoplasm accumulation and subsequent nuclear translocation to the coactivation of specific gene transcription, and the noncanonical Wnt/planar cell polarity (Wnt/PCP) and Wnt/Ca2+ pathways, which mediate cytoskeleton dynamics and cell movements through directional information or intracellular Ca2+ release, respectively  that has been proposed to have taken place in the common ancestor of spiders and scorpions (Arachnopulmonata) . Furthericerates . This leications . Intereson e.g., .Drosophila wnt9 (Dwnt4) activates cell motility during ovarian morphogenesis through the noncanonical pathway and salivary gland development through the canonical pathway —of practically all the panarthropods analyzed except for the tardigrades, which do not have a proper SAZ .There is also an imbalance in the analysis of the different Drosophila, all segments are patterned almost simultaneously during syncytial blastoderm in what is called long germ segmentation , co-receptor Arrow in Tribolium, Oncopeltus, Gryllus and Parasteatoda , or final effectors (the transcription factors Gb-armadillo/β-catenin in Gryllus and Of-pangolin in Oncopeltus) of the canonical Wnt pathway, lead to the formation of truncated embryos with no posterior segments  in the beetle and a normal-sized abdomen in the case of Oncopeltus embryos  and triple  RNAi injections of the wnt genes that are most clearly expressed in the SAZ , which are sequentially expressed–both temporally and spatially–during segmentation in Tribolium, but also in the long-germ Drosophila embryo. Furthermore, one of the same authors proposed a transition from the posterior expression of caudal/dichaete to dichaete/opa expression at the anterior region of the SAZ, resembling the wavefront limit operating in vertebrates  or inhibiting (at the anterior SAZ) this ligand. However, it was previously found that Wnt8 would be necessary for the dynamic expression of Pt-delta in the same spider  Studies in different arthropods are lacking to determine a possible ancestral regulatory route of posterior growth.i.e., odd, eve, run, hairy, paired, delta), and the most widely described regulatory network involved is Wnt > Caudal > Pair-Rule/Delta - with some variations possible due to evolutionary divergences or even experimental limitations -, where Wnt signaling has a regulatory effect on the timing of segment border definition.2) Virtually all arthropods studied to date have shown some level of dynamic/oscillatory expression in the SAZ (3) Notch signaling, as in vertebrates, could be involved in single-cell oscillations coupling in some arthropods and not others, where we have not yet discovered the gene(s) involved in the oscillatory synchronization.As it has been suggested that the Notch pathway does not present a functional role in the segmentation process in the onychophoran astaneum , the regbyx mori . Thus, fbyx mori :1) StudiSequential segment addition in most arthropods is accompanied by simultaneous germband extension along the anterior-posterior axis. This complex reorganization of the developing body must include, in a species-specific manner, a variable, mutually dependent, and tightly regulated arrangement of cellular behaviors, including oriented (or not) mitotic divisions, cell rearrangements, and changes in cellular density, cell shape, and cell size, among others.wnt (wnt3a and wnt8 in zebrafish) and brachyury, so that posterior elongation depends on this molecular feedback . The first studies on CE were carried out in involved . Furtherreported . In addiosophila  and is csteatoda , leads uongation .However, we will not have an answer to our question until a more significant number of studies have been carried out with a particular focus on the cellular behaviors that take place during posterior elongation in a diverse group of arthropods.After reviewing the Wnt pathway’s role in axial elongation and segmentation processes in arthropods, more questions than answers arise.e.g., maxillopod crustaceans, non-insect hexapods, non-spider chelicerates). Furthermore, given the apparent overlapping between some ligands, it would be precious to have a detailed cellular-level analysis of the expression patterns, hopefully including double in situ hybridizations.We found the complete Wnt ligand repertoire from 42 species of panarthropods. However, the expression pattern of all the ligands has been published only in twelve. Thus, a more diverse palette of species is lacking in the analysis, especially non/underrepresented lineages , will provide the basis for well-supported comparative analyses. In the long term, this will allow us to identify conserved and diverged aspects of the regulatory role of the Wnt pathway on posterior body elongation and segmentation. Furthermore, adding imaging tools to this analysis, like tissue-specific transgenic lines carrying fluorescent reporters for time-lapse imaging, will be essential to understanding the contribution of Wnt signaling in the highly dynamic cellular and molecular network that organizes posterior growth in most panarthropods.The incorporation of functional studies in more organisms, including non-model panarthropods with interesting phylogenetic positions as early-branching clades (