Pathway-based expression profiles allow for high-level interpretation of transcriptomic data and systematic comparison of dysregulated cellular programs. We have previously demonstrated the efficacy of pathway-based approaches with two different applications: the drug set enrichment analysis and the Gene2drug analysis. Here, we present a software tool that allows to easily convert gene-based profiles to pathway-based profiles and analyze them within the popular R framework. We also provide pre-computed profiles derived from the original Connectivity Map and its next generation release, i.e. the LINCS database.gep2pep and can be freely downloaded from https://bioconductor.org/packages/gep2pep.The tool is implemented as the R/Bioconductor package Bioinformatics online. The use of genome-wide expression profiling technologies has transformed the way in which scientists approach the study of molecular mechanisms. Millions of assays have been performed and public databases have been developed to collect such immense amount of data, thus improving both reproducibility of the original studies and reuseability for novel investigations . Many dipathways for simplicity) as their elementary variables, as opposed to single genes. We demonstrated the efficacy of the approach with two different tools: (i) the drug set enrichment analysis (DSEA)  to pathway expression profiles (PEPs). This allows to develop analytic approaches that use dysregulated gene sets   .gep2pep R/Bioconductor package supports the management of large collections of heterogeneous profiles, exploiting the HDF5 format in combination with the Repo  pair, where c is a condition and p is a pathway. Therefore, each  pair is assigned an Enrichment Score (ES) and its corresponding P-value according to the Kolmogorov–Smirnov test (KST), giving rise to an M × N matrix E of ESs and an M × N matrix P of P-values. gep2pep allows to perform further PEP-based analyses using any of E, P or their element-wise product, according to: −E · log(P). gep2pep also implements merging of multiple PEPs into a single PEP, along the lines of E, P), the resulting merge is the row-wise average of E, and the row-wise aggregation of P by the Fisher’s method.PEPs are created from GEPs using GSEA. Given gep2pep package supports two kinds of pathway-based analyses (see CondSEA) and pathway set enrichment analysis (PathSEA). Both are based on a GSEA-like procedure performed on PEPs as opposed to GEPs. CondSEA first ranks the PEPs row-wise. Then, given a set of conditions as input, it computes their KST within each row. Enriched rows correspond to pathways that are consistently dysregulated across the input conditions. When conditions are drug-induced GEPs, we call this approach DSEA , where the full dataset in the gep2pep format can be obtained (see Together with the package, here we release two large collections of pre-computed PEPs: one derived from the Cmap (ined see .gep2pep, an R/Bioconductor package implementing a pathway-based approach to the analysis of GEPs. We also provided a large collection of pre-computed PEPs. We hope that an offline, structured and carefully documented tool for generic pathway-based approaches will allow more researchers to develop original applications under this new paradigm.We introduced This work has been supported by Fondazione Telethon and Fondazione Veronesi.Conflict of Interest: none declared.btz803_Supplementary_DataClick here for additional data file.


Huangqi Guizhi Wuwu Decoction (HGWD) has been applied in the treatment of joint pain for more than 1000 years in China. Currently, most physicians use HGWD to treat rheumatoid arthritis (RA), and it has proved to have high efficacy. Therefore, it is necessary to explore the potential mechanism of action of HGWD in RA treatment based on network pharmacology and molecular docking methods.  The active compounds of HGWD were collected, and their targets were identified from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and DrugBank database, respectively. The RA-related targets were retrieved by analyzing the differentially expressed genes between RA patients and healthy individuals. Subsequently, the compound-target network of HGWD was constructed and visualized through Cytoscape 3.8.0 software. Protein-protein interaction (PPI) network was constructed to explore the potential mechanisms of HGWD on RA using the plugin BisoGenet of Cytoscape 3.8.0 software. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed in R software . Afterward, molecular docking was used to analyze the binding force of the top 10 active compounds with target proteins of VCAM1, CTNNB1, and JUN. p value <0.05 and |log 2(fold change)| > 0.5 were collected. Moreover, 739 GO entries of HGWD on RA were identified, and 79 pathways were screened based on GO and KEGG analysis. The core target gene of HGWD in RA treatment was JUN. Other key target genes included FOS, CCND1, IL6, E2F2, and ICAM1. It was confirmed that the TNF signaling pathway and IL-17 signaling pathway are important pathways of HGWD in the treatment of RA. The molecular docking results revealed that the top 10 active compounds of HGWD had a strong binding to the target proteins of VCAM1, CTNNB1, and JUN.  Cumulatively, 790 active compounds and 1006 targets of HGWD were identified. A total of 4570 differentially expressed genes of RA with a  HGWD has important active compounds such as quercetin, kaempferol, and beta-sitosterol, which exert its therapeutic effect on multiple targets and multiple pathways. Typisability –6. Curresability , 8. Howesability . TherefoJingui Yaolue, has been used over 1000 years in China to treat joint pain. HGWD was formulated by Zhang Zhongjing during the Han dynasty. It is a mixture of five Chinese medicines including Astragalus membranaceus (Huangqi), Cassia Twig (Guizhi), Radix Paeoniae Alba (Baishao), ginger (Shengjiang), and Chinese date (Dazao). HGWD is considered to have the ability to reinforce Qi and nourish blood, warm and smoothen meridian, and dredge arthritic pain traditionally [The traditional Chinese medicine (TCM) is based on the theory of syndrome differentiation and has long been established as an effective treatment of RA. Huangqi Guizhi Wuwu Decoction (HGWD), a classical prescription described in tionally . Previoutionally . Moreovetionally . HoweverNetwork pharmacology is partly bioinformatics and was first proposed by Hopkins . It has In the present study, network pharmacology was used to establish a compound-target-disease network for exploring the potential HGWD mechanism of action in RA treatment. This study provides a reference for future pharmacological studies and clinical applications. The flow diagram of the network is shown in http://tcmspw.com/tcmsp.php) [Information on the HGWD compounds was retrieved from the Traditional Chinese Medicine Systems Pharmacology Database  . TCMSP imsp.php) . The actmsp.php) . The OB msp.php) . DL propmsp.php) . Subsequhttps://www.drugbank.ca/) to identify the corresponding potential targets of HGWD [Target identification is an important aspect of drug exploration . The tar of HGWD .https://www.ncbi.nlm.nih.gov; series: GSE21959; samples: there were 36 samples, 18 for healthy individuals and 18 for those with rheumatoid arthritis). The genes with a p value <0.05 and |log 2(fold change)| > 0.5 were regarded as differentially expressed genes and RA-related targets.The differentially expressed genes in RA were retrieved from the GEO database  analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) are important methods that describe the features of candidate targets. The two were performed in R software  with the standard  of 0.05 , 28. TheThe 3D protein structure of the three core proteins corresponding to the core targets, VCAM1, CTNNB1, and JUN, was downloaded from the UniProt database. Subsequently, the structure of the active ingredient in HGWD (the top 10 places in the number of targets) was downloaded from the PubChem database and saved in the PDB format. Using PyMOL software, the three proteins were virtually dehydrated and hydrogenated, the original ligands were extracted in each protein, and then were stored separately. AutoDockTools 1.5.6 was utilized to convert compounds, ligands, and proteins into the “pdbqt” format and to define if the location of each protein or its ligands was the active pocket of the protein. Finally, Vina 1.5.6 was run to assess molecular docking. At a binding energy value <0, the molecular proteins were considered to spontaneously bind and interact with each other. Accordingly, the lower the energy is, the more stable the molecular conformation is.A total of 790 compounds of the five herb medicines in HGWD were retrieved from the TCMSP database. This included 87 compounds in Huangqi, 220 in Guizhi, 85 in Baishao, 265 in Shengjiang, and 133 in Dazao. Among them, 74 compounds passed OB ≥ 30% and DL ≥ 0.18 filtering. Specifically, the numbers of candidate compounds in Huangqi, Guizhi, Baishao, Shengjiang, and Dazao were 20, 7, 13, 5, and 29, respectively. The candidate compounds in HGWD used for further analysis are shown in The compound-target network of HGWD was established with the collected compounds and their targets as shown in To elucidate the mechanism by which HGWD ameliorates RA, the potential target network was merged with the RA-related target network to form a core PPI network of 2284 nodes and 53,119 edges . The preDuring the second screening, since the number of genes was limited, only BC average value was used. The candidate targets were further identified, and 111 targets with BC ≥ 348.07  were chosen . EventuaTo further confirm the biological responses from RA treatment with HGWD, GO analysis of the 49 RA-related potential therapeutic target genes was performed based on BP, CC, and MF. The analysis results revealed a total of 739 entries. In BP enrichment analysis, 666 entries were obtained including response to antibiotic, response to alcohol, and response to steroid hormone. In CC enrichment analysis, 34 entries involved in membrane raft, membrane microdomain, membrane region, etc. were obtained. The MF enrichment analysis revealed 39 entries, including kinase regulator activity, protein kinase regulator activity, and serine hydrolase activity. The top 20 terms are shown in p value <0.05. The top 20 KEGG pathways' enrichment analysis is shown in To further show the biological processes of these targets, the KEGG pathway analysis was performed. The analysis results showed that these targets were enriched in 79 pathways with an adjusted The gene-pathway network analysis was constructed according to the significantly enriched pathways and genes which regulate these pathways as shown in Normally, high connectivity compounds are associated with more targets. According to the number of related targets, the top 10 active compounds in HGWD with high connectivity in the compound-target network were selected for molecular docking  [κB) receptor activator ligand RANKL. Consequently, this increases the content of matrix metalloproteinases, possibly promoting cartilage degradation, inhibiting chondroprogenitor cell formation, and stimulating osteoclast bone damaging [The targets of HGWD against RA were enriched in BP, CC, and MF through GO analysis. Results suggested that HGWD mainly regulated response to antibiotics, response to steroid hormones, and response to radiation. Furthermore, it was shown to affect some cellular components and molecular functions including membrane raft, membrane microdomain, membrane region, and kinase regulator activity. In the present study, 79 KEGG pathways including TNF signaling pathway and IL-17 signaling pathway were significantly enriched. IL-17 and TNF- (DAS28) . This wa (DAS28)  found th (DAS28) . It has damaging –53.α plays an important role in the development of RA. On the one hand, it can bind to the TNFR1 receptor on fibroblast synovial cells, promoting the release of inflammatory cytokines such as IL-1, IL-6, and IL-8 and aggravating the damage of the articular cartilage and bone [α can stimulate immune cells in blood to enter the joint through VCAM-1, aggravating the joint injury [TNF-and bone . On the t injury . Besidesα, IL-6, and IL-17 play a crucial role in synovial fibroblast inflammation. Studies have demonstrated that TNF-α upregulates the E2F2 expression by activating the nuclear transcription factor NF-κB pathway. This enhances synovial cell proliferation and synovial tissue thickening leading to joint injury [The gene-pathway network was constructed to explore the main target genes of HGWD against RA. The results revealed that JUN, FOS, CCND1, IL6, E2F2, and ICAM1 are important target genes. A study found that the ERK-JNK-P38 signaling pathways in autoimmune disease are activated, leading to high levels of downstream JUN and Fos protein. Subsequently, the two combine to form dimer transcription factor-activating protein 1 (AP-1), which is involved in the occurrence and development of RA. It regulates the transformation of initial T cells into effector T cells, hence regulating the immune response and inflammatory process –58. Inflt injury . The sert injury . ThroughOur study systematically elucidated the mechanisms of action and molecular targets of HGWD against RA via the network pharmacology approach. Quercetin, kaempferol, and beta-sitosterol regulated most of the targets related to RA. The HGWD can regulate gene function through their related pathways including TNF signaling pathway and IL-17 signaling pathway. The key target genes in the gene-pathway network of HGWD against RA were JUN, FOS, CCND1, IL6, E2F2, and ICAM1. Furthermore, according to molecular docking analysis, important compounds such as stepholidine, stigmasterol, beta-carotene, quercetin, and the core protein CTNNB1, VCAM1, and JUN all have good binding ability. The network pharmacology is a promising suitable approach for the study of TCM formulations.


Bioinformatic network and pathway analyses of identified genes (n = 235) were performed using ReactomeFIViz in Cytoscape based on the highly reliable Reactome functional interaction network algorithm. This approach merges interactions extracted from human curated pathways with predicted interactions from machine learning. Genome-wide pathway analysis showed total of 16 enriched hierarchical clusters. A total of 278 enriched single pathways were also identified . Gene network analyses showed multiple known and new targets in the p75NTR gene network. This study provides a comprehensive analysis and investigation into the current knowledge of p75NTR signaling networks and pathways. These results also identify several genes and their respective protein products as involved in the p75NTR network, which have not previously been clearly studied in this pathway. These results can be used to generate novel hypotheses to gain a greater understanding of p75NTR in acute brain injuries, neurodegenerative diseases and general response to cellular damage.P75 neurotrophic receptor (p75NTR) is an important receptor for the role of neurotrophins in modulating brain plasticity and apoptosis. The current understanding of the role of p75NTR in cellular adaptation following pathological insults remains blurred, which makes p75NTR’s related signaling networks an interesting and challenging initial point of investigation. We identified  These neurotrophins and their receptors function in an extensively well-regulated mechanism resulting in a delicate equilibrium that is highly responsive to nervous system injury and change3. Neurotrophins bind to two distinct types of receptors including p75 neurotrophin receptor (p75NTR) with a lower affinity, as well as to the tropomyosin receptor kinases (Trk) receptors, which function as tyrosine kinase receptors with high affinity and selectivity4. Precursor forms of neurotrophins acts as potent ligands for p75NTR and can induce apoptosis, while lacking affinity to Trk receptors5. The Trk receptors activate numerous downstream pathways including the MEK/MAPK pathway, extracellular signal-regulated kinase (Erk), the PI3K-Akt pathway, and the phospholipase C gamma pathway6. In contrast the p75NTR does not have intrinsic tyrosine kinase activity, but rather associates with other transmembrane proteins including the Trks, Nogo and myelin associated glycoprotein (MAG) for its downstream effects5.Neurotrophins are secreted dimeric proteins, including BDNF, the most well-known, as well as nerve growth factor (NGF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4)7. Similarly, a down-regulation of both Nogo and p75NTR promoted improved stroke recovery in another pre-clinical model of middle cerebral artery occlusion8. Nogo associates with p75NTR by MAG, oligodendrocyte myelin glycoprotein or Nogo that activates RhoA leading to inhibition of neurite outgrowth5. In-line with downregulation exhibiting possible neuroprotective and stimulatory effects, activation of p75NTR by proneurotrophins in culture induces a pro-apoptotic effect on neurons9. This is particularly interesting since cleaved neurotrophins promoted recovery through Trks9. In another model of central nervous system (CNS) injury, a small-molecule modulator of the p75NTR receptor demonstrated that modulation of this receptor and its associated pathways results in improved outcomes following traumatic brain injuries10. All of these studies point to the strong role of neurotrophins and p75NTR in the response to CNS injury.This modulating role of p75NTR have been shown in several animal model studies. Inhibition of p75NTR and its associated Nogo co-receptor exerts a neuroprotective effect in models of middle cerebral artery occlusion11. This mechanism of p75NTR mediated apoptosis without Trk receptor activation likely plays a significant role in AD pathogenesis11. Interestingly, further findings by Yao et al. (2015) demonstrated physiological neuroprotective effects of p75NTR ectodomain against amyloid β peptide toxicity in the brain of AD patients12. In addition, AD patients have increased levels of p75NTR and proBDNF in hippocampal tissue samples and a greater proBDNF/BDNF ratio in cerebrospinal fluid that may result in imbalance of death and survival counter-regulation mechanisms13. Of note, in AD an increased level of proNGF leads to p75NTR activation and apoptosis in the absence of TrkA14. P75NTR has been shown to have similarities in the biology of Amyloid precursor protein (APP) which plays a significant role in AD patophysiology15. Also, patients with amyotrophic lateral sclerosis (ALS), another neurodegenerative disease, revealed highly increased p75NTR ectodomain concentrations in urine when compared to healthy controls. In addition, high concentrations of p75NTR ectodomain correlated to progression of ALS disease16.Viewing the role of neurotrophins in exhibiting neuroprotective effects from a slightly different viewpoint, it has previously been suggested that Alzheimer’s disease (AD) amyloid β peptide also acts as ligand to p75NTR, but not Trk receptors while inducing neuronal apoptosis18. With p75NTR being studied in various different diseases and fields suggests that it may play an important central role in cellular response to injury. The body of evidence surrounding p75NTR is diffusely spread out over numerous fields suggesting that a review of these separate bodies of work in one study may identify new connections, pathways, and mechanisms that were previously identified as isolated characteristics.Understanding the role that p75NTR and associated molecules may play in AD also supports the notion that these molecular mechanisms should not be underestimated in the pathophysiological response to acute brain injury. Especially when considering the significant role that plasticity and associated adaptive mechanisms may play in these insults, including chronic traumatic encephalopathyp75NTR interaction networks and pathways to understand how these genes form networks, connections, and signaling pathways across the broad literature that involves p75NTR. By using machine learning educated linkage gene analysis, we aimed to identify new gene and protein candidates that may be involved in a network with p75NTR, but have not been widely identified or established as possible targets in the literature regarding acute and chronic response to cellular injury.Herein, we investigate the published literature through machine-learning approaches to study the role of p75NTR and its related gene networks in an attempt to elucidate new factors that may be important to future development of therapeutic targets. We hypothesized that by using already published results of p75NTR signaling, we would be able to investigate p75NTR and its related genes, we performed an extensive literature search using PubMed on published articles from the past 20 years (1998/11/13–2018/11/13) related to p75NTR. The final query term was: (p75[All Fields] AND ("neurons"[MeSH Terms] OR "neurons"[All Fields] OR "neuron"[All Fields])) OR (p75[All Fields] AND ("brain"[MeSH Terms] OR "brain"[All Fields])) OR  AND ("1998/11/13"[PDAT] : "2018/11/13"[PDAT]). The query resulted in a total of 2041 publications. We used R statistical software with “PubMed.mineR” package to mine all gene names appearing in the 2041 publication abstracts19 (Core-team R 2015). Appropriate gene names were used in accordance with HUGO Gene Nomenclature Committee (HGNC) approved symbols (genenames.org). The results of data mining were adjudicated by the authors JK and AS.In order to identify p75NTR gene and its role in gene networks and pathways. Network analyses were performed with ReactomeFIViz in Cytoscape (https://www.cytoscape.org/) based on a highly reliable Reactome functional interaction (FI) network. The entire FI network was constructed by merging interactions extracted from human curated pathways with interactions predicted using a machine learning approach21. Network analyses were followed with Reactome pathway analyses, and false discovery rate (FDR) corrected p-value < 0.05 was considered to be significant in order to avoid false positive results22. Visualization of genome-wide pathway enrichment analysis and generation of hierarchical cluster figure was performed with Reactome analysis tool provided in reactome.org20.A flow of the study is presented in Fig. Institutional ethics board evaluation was not applicable due to the nature of this study. Good scientific practice was used throughout this study.p75NTR and its role in neuronal signaling . The five most enriched pathways  were pathways in cancer, signaling by interleukins, signaling by NGF, melanoma, and proteoglycans in cancer. A list of the top 20 pathways are presented in Table We further identified enriched pathways related to 235 p75NTR and related genes connect to each other with related functions  genes and linkage genes in relation to the p75NTR’s gene networks and pathways. We identified p75NTR related genes, gene networks, and pathways while identifying new associated p75NTR network genes. While a portion of these results validate previously published mechanistic links, the advantage of reviewing vast amounts of previously unconnected literature through this approach also identified new genes and proteins for future studies. The pathway analyses provide a general overview of the functions of p75NTR in a network with its closely related genes. The data presented here will also serve as a useful resource to the research community in querying potential biomarkers, therapeutic targets, and potential areas of future studies.The complex role of p75NTR in brain plasticity and apoptosis has proven a challenging subject of investigation leaving researchers without a full understanding of the role it plays in various pathophysiological aspects. Herein we investigated previously published literature using data mining and machine learning approaches to gain a better understanding of 27. The most enriched pathways identified validated the roles of previous studies, such as the finding that p75NTR has been shown to be highly expressed in human cancers including melanoma29. The link of p75NTR to cancer pathology is particularly interesting, given its role in immune modulation, matrix remodeling, and cellular adaptation. However, there are currently no studies demonstrating p75NTR’s effects on modulating the immune response in cancer, nor in acute or chronic brain disease suggesting this area may warrant further investigation, noting also the fact that p75NTR belongs to tumor necrosis factor receptor superfamily. The pathway analyses that resulted provide a general overview of the functions of p75NTR in a network with its closely related genes. Overall, 278 enriched pathways and 16 hierarchical pathway clusters were identified generating vast amounts of data that may be useful for validation and future research by other investigators.Our results obtained from genome-wide cluster and pathway analyses validated the current understanding of p75NTR in that it plays a role in various pathways including programmed cell death, immune system modulation, signal transduction, developmental biology, gene expression regulation, and extracellular matrix organizationp75NTR identified genes. GRB2 has been shown to be directly downstream of TrkA acting as a signaling adaptor protein30. Associations or links of p75NTR and GRB2 are not well established. Interestingly, in the brain tissue of scrapie infected rodents, BDNF, TrkB, phospho-TrkB, GRB2 and p75NTR were all significantly down-regulated supporting the direct association identified in our results between GRB2 and p75NTR32. Viral infection etiology of AD has been a widely known controversial topic. This makes the finding about scrapie viral infection particularly interesting. Results of three independent AD cohorts showed disruption of molecular, genetic, and clinical networks by human herpesvirus33. However, the role of p75NTR in possible viral pathophysiology of AD remain unknown, but these results in conjunction with previous works may shed some light generating new hypotheses and studies.The gene network analyses in combination with the focused subnetwork analysis using linkage genes identified functionally related genes that were not part of our original datamined UBC, the gene for polyubiquitin precursor protein, which is known to have a role in proteasome degradation34. Conjugation of ubiquitins has been well established as highly important for protein degradation and the associated role that plays in larger cellular process such as DNA repair, cell cycle regulation, kinase modification and endocytosis system35. Loss of UBC is associated with the pathophysiological molecular factors of AD via decreased proteasome degradation system, which may be thought of as cells inefficiently removing malfunctioning, damaged, or old proteins36. These connections with p75NTR underline the important role in AD’s pathophysiology17. However, it is important to understand that the role of p75NTR in AD pathophysiology is only a model for which p75NTR likely acts as a central protein signaler in response to various elements of cellular damage.Another linkage gene identified was 38. As shown in several studies, MAPKs are downstream targets of Trks and p75NTR39. Our results suggested that NGF and p75NTR link with MAPK1, and NGF activates MAPK3, which are in line with previously published studies40. An interpretation for these interactions in the broader context of a disease can be seen with inhibition of ERK1/2 following the acute phase of stroke promoting long-term functional outcome and enhanced later-stage recovery processes in rats41. Other experiments using preclinical rat models have shown how MAPK activity also plays a fundamental role during postnatal neurogenesis when hippocampal apoptosis and synaptogenesis are occurring42. The significant connections between MAPK and p75NTR highlight that p75NTR should also be further investigated in relation to these disease processes, as well as finding possible targets for future medical therapy.MAPK1/ERK2 and MAPK3/ERK1 pathways were also identified and their roles have been reported in apoptosis, neuronal repair, and axonal growthSRC, CTNNB1, and EP300 were identified in the same linkage gene network with p75NTR. Interestingly, inhibition of SRC family kinases improved cognitive function in rats after intraventricular hemorrhage43. Indeed, these observations further increase the interest of p75NTR and its role following hemorrhagic insult as another form of pathological damage cells suffer acutely. Similarly, CTNNB1 encodes a β-catenin protein that increases proNGF leading to p75NTR activation ultimately promoting neuronal growth44. Further investigation showed that modulation of β-catenin pathway was neuroprotective after intracerebral hemorrhage in rats45. The role of p75NTR, however, was not previously studied in these aspects. Our results suggest an association between CTNNB1 and p75NTR possibly demonstrating a substantial role for p75NTR in the stroke response.In addition, SORT1), lingo (LINGO1) and NRAGE (MAGED1) have been linked to p75NTR functions regulating apoptosis, axonal outgrowth and transporting pro-neurotrophins39. The genes did not result in the analyses directly. Interestingly however, MAGEL2 was identified in our analysis that belong to the same melanoma-associated antigen (MAGE) family than NRAGE, and shares strong homology to NRAGE and other proteins in MAGE-family. As NRAGE is involved in the p75NTR mediated programmed cell death, MAGEL2 is linked to neurodevelopmental disorders such as Prader-Willi syndrome and Schaaf-Yang syndrome. This suggest significant importance of MAGEL2 in neuronal development in humans46. Previous animal studies have shown that mTOR and autophagy pathways are dysregulated in Magel2 null mice models47. In our analysis, MAGEL2 was directly linked to transcription factor E2F1 that was directly linked to p75NTR. This suggests a role of necdin-related MAGE proteins in p75NTR functions, supported by preclinical observations48.Previously, sortilin (UBC, and EP300 have not been extensively studied in the context of brain plasticity. These two linkage genes and their encoded proteins could be interesting targets for future studies to explore in patients with acute brain injury or neurodegenerative diseases. This also highlights the benefit of taking large scale approaches through data mining, which allows for an overview of the interactions that may exist between different studies, but have not previously been discovered to exist.Two of seven subnetwork linkage genes identified here, namely Our study was limited to in silico analyses, and confirmatory in vitro or in vivo analyses were not performed. However, the research approach used herein has the advantage of interrogating a large dataset through a systematical approach which incorporates all currently available knowledge. Analyzing such a search with powerful bioinformatic tools educated with machine learning algorithm from human curated pathways allows for broad investigation of connections that may have previously been missed, or not looked for in the first place. Confirmation of discussed associations are warranted.p75NTR. This study incorporates current knowledge using a large dataset approach that increases the overall understanding of complex p75NTR networks. These results suggest both new possible target genes for further investigation in p75NTR research, while also validating previously conducted research in identifying pathways, genes, and clusters that highlight p75NTR’s biological function. These results can be used to generate novel hypotheses to gain a greater understanding of p75NTR in acute or chronic brain injuries, other neurodegenerative diseases, and the general response to cellular injury.We provide the single largest comprehensive gene and functional network library of Supplementary Information 1.Supplementary Information 2.


Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp.) and the biosynthesis pathway for pyridoxal 5′-phosphate and its vitamers (vitamin B6), found in S. aureus. Using current genomic data, we discuss the possibilities of inhibition of enzymes in the pathway and review the current state of the art in the scientific literature.Antibiotic resistance is a worldwide concern that requires a concerted action from physicians, patients, governmental agencies, and academia to prevent infections and the spread of resistance, track resistant bacteria, improve the use of current antibiotics, and develop new antibiotics. Despite the efforts spent so far, the current antibiotics in the market are restricted to only five general targets/pathways highlighting the need for basic research focusing on the discovery and evaluation of new potential targets. Here we interrogate two biosynthetic pathways as potentially druggable pathways in bacteria. The biosynthesis pathway for thiamine (vitamin B1), absent in humans, but found in many bacteria, including organisms in the group of the ESKAPE pathogens ( Antibiotic resistance is an urgent threat to human health and requires urgent actions from physicians, patients, industries, governmental agencies, and the academic community worldwide. According to the last document from the Centers for Disease Control and Prevention (CDC) regarding antibiotic resistance in the United States, from 2013, the number of people with serious infections caused by resistant bacteria reaches 2 million every year, with at least 23,000 deaths per year directly caused by these infections  theAntibiotic resistance, as a threat to human health, should be addressed in multiple and simultaneous ways. For the academia, an interesting way to address antibiotic resistance is the discovery and validation of new targets/pathways that could be specifically targeted by new antibiotic candidates. It is worthy of note that, according to Kelly and Davies , no new Escherichia coli genome, 303 genes, were shown to be composed of essential genes , the activity of several carbohydrate metabolism enzymes is impaired, including pyruvate dehydrogenase, which connects glycolysis and the citric acid cycle . Other tsubtilis .Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp.)? (ii) What enzymes are present, and what do we know about them?The pathways involved in the synthesis of vitamins, in particular, vitamin B1 (thiamin) and B6 , seem to be of great relevance, since they are involved in central processes in the metabolism of carbohydrates and amino acids, and the corresponding enzymes are found in most bacteria, fungi, and plants but not in humans , favorinPlasmodium falciparum , a KEGG search for the maps of thiamin and pyridoxal metabolism was done, looking for the existing genes in commercial strains for each of the ESKAPE organisms. For the sake of comparison, some additional resistant strains were listed, with no differences in the observed genes for the pathways under study. A similar search was carried out for vitamin B6 metabolism (map 00750) in KEGG, interrogating the existing enzymes for the ESKAPE pathogens in comparison with humans. The analysis was focused in the enzymes in the biosynthesis of B1 and B6 vitamins in an attempt to identify the most promising targets for future medicinal chemistry studies. Finally, a table listing the existing genes for each pathogen was compiled and is presented in the following sections.The analyses provided here are the result of the interrogation of the enzymes in the biosynthesis pathways for thiamin and pyridoxine phosphate or pyridoxal phosphate using the KEGG database , 15. Fordruggability of the identified promising targets.Using the existing literature, we sought to identify whenever possible the cases of enzymes with functional redundancy with another enzyme for the same organism. The identified cases were discussed as less promising targets for medicinal chemistry campaigns. Additionally, the existing data for enzyme inhibition using natural or synthetic compounds was compiled to provide initial proof-of-concept clues about the When necessary, the sequence of an enzyme of the pathway was used to search for homologs using BLAST  searchesThe biosynthesis pathway for thiamin involves two branches, as shown in Q9H3S4. Other than that, the remaining enzymes in the biosynthetic pathway for thiamin are missing in humans, making them very attractive for the design of chemical probes that could be used as proof-of-concept compounds.According to the KEGG pathway database, humans lack many of the enzymes of this pathway, but not all of them. In humans, a TPK (TPK1) enzyme is found, with UNIPROT ID E. faecium (ATCC 8459) in the KEGG database did not identify genes for ThiE, ThiM, and TenA. For this species, only ThiD and TPK were positively identified. However, a BLAST search for TenA homologous within the non-redundant database restricted to E. faecium (TAXID 1352) identified a single result for thiaminase II (GenBank: SAM74984.1), four results for ThiE , and two results for ThiM . The small number of hits in the BLAST search may suggest some issues in the annotation.The analysis of the thiamin pathway for Enterococcus faecalis thiamin pathway is compared with the pathway observed for E. faecium, many differences are found. The KEGG pathway for E. faecalis (ATCC 29212) shows that the entire pathway, as described in E. faecium. Additionally, there is no change in the repertoire of enzymes in the pathway for the Vancomycin-resistant strain V583 compared to the ATCC 29121 strain.Interestingly, when the S. aureus (NCTC 8325) and K. pneumoniae (subsp. pneumoniae ATCC 43816 KPPR1), the KEGG pathway indicates that all the enzymes in the pathway are observed, with no changes to an S. aureus-resistant strain such as COL (MRSA) compared to the NCTC 8325. For A. baumannii (ATCC 14978), all enzymes are observed but TPK. Instead, thiamin phosphate may be converted directly to thiamin diphosphate by a thiamin-monophosphate kinase, and then converted to thiamin triphosphate by an adenylate kinase. In the case of P. aeruginosa (NCGM 1900), some enzymes are missing: ThiD, TenA, TPK. Finally, Enterobacter sp. (638) has ThiD, ThiE, ThiM and misses TenA and TPK.For The overall panel of enzymes for the thiamin pathway for the ESKAPE pathogens is summarized in Bacillus subtilis, the deaminase activity is somewhat redundant with the ThiC (ThiA) activity, since both activities provide HMP or HMP phosphate, which can be further phosphorylated by ThiD , and ThiM function can be dispensable since thiazole phosphate can be synthesized P. aeruginosa, which lacks ThiD, and E. faecium, which lacks ThiE, according to the KEGG data. For ThiE, in particular, it was reported that 4-amino-2-trifluoromethyl-5-hydromethylpyrimidine (CF3-HMP), an HMP analog, can be converted by ThiD to CF3-HMP pyrophosphate, which in turn inhibits ThiE , B. subtilis [PDB ID 2I5B (Thermus thermophilus (PDB ID 1UB0), A. baumannii (PDB ID 4YL5), Bacteroides thetaiotaomicron (PDB ID 3MBH), and the bifunctional enzyme from Saccharomyces cerevisiae (PDB ID 3RM5). Typically, ThiDs are folded as typical ribokinases, with a central eight-stranded β-sheet surrounded by eight α-helices, and structural studies suggest that some surface loops may have a structural change based on the presence of the nucleotide  has a dual role, phosphorylating pyridoxal and pyridoxine in the pyridoxal de novo biosynthesis pathway as well as HMP in the thiamin biosynthesis pathway, with a KM almost 20 times greater for HMP than for pyridoxal and kcat values three times faster for pyridoxal (S. aureus ThiD (SaThiD), with less efficiency, though.Interestingly, the yridoxal . So, SaPSaThiD was shown to be potentially inhibited by Rugulactone (Ru0), a natural product, as well as by its derivatives Ru1 and Ru2, with IC50 values ranging from 14 to 32 μM , B. subtilis [PDB IDs 1G4T, 3O15, 3O16, 1G69, 1G4E, 1G67, 1G4P, 1YAD (Mycobacterium tuberculosis (3O63), and for the bifunctional enzyme from Candida glabrata [PDB IDs 3NL2 and 3NM1  is a close homolog of Bacillus subtilis Pdx1 [81% identity, PDB ID 2NV1 . S. aureus Pdx2 has Geobacillus kaustophilus [PDB ID 4WXY (G.s stearothermophilus (PDB ID 1Q7R), and B. subtillis [PDB IDs 2NV0 and 2NV2  interact with 12 Pdx2 enzymes. However, Pdx2 molecules do not interact with each other in this complex -helices . The mostopology . Curious complex .in vitro inhibitory effects on the enzyme Pdx1 from P. falciparum  and B6  may be interesting molecular targets for the development of new chemical probes aiming to inhibit the synthesis of these essential cofactors in pathogenic organisms. Model compounds, mainly based on substrate analogs showed that inhibition of the vitamin B1 biosynthetic enzymes, ThiD and ThiE, as well as Pdx1, Pdx2, and PdxK in the pyridoxal 5′-phosphate biosynthesis pathway have shown an initial proof of concept for the model, and it is up to the scientific community and/or researcher in the industry to explore these targets further.The datasets generated for this study are available on request to the corresponding authors.CW, IP, and AN conceived the project. LM and RG analyzed the thiamine pathway, while AB and LD analyzed the pyridoxal pathway. All authors wrote and approved the manuscript.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.


We aimed to evaluate microvascular changes on optical coherence tomography angiography (OCTA) in patients with rhegmatogenous retinal detachment (RRD) who underwent silicone oil (SO) tamponade and compare changes according to macular involvement.This retrospective study included 48 patients with unilateral RRD who underwent vitrectomy and SO tamponade and were stable after SO removal. Control data were obtained from the fellow healthy eye. Ophthalmic examinations, including best corrective visual acuity (BCVA) and OCTA, were conducted. Differences in vascular density (VD) in different sections of the macula and differences in the foveal avascular zone (FAZ) were analyzed between the affected eyes and control eyes. Subgroup analyses according to macular involvement were performed.Baseline BCVA and duration of SO tamponade were associated with postoperative BCVA . The average VD in the deep capillary plexus (DCP) and the VD of the nasal parafoveal area in both the superficial capillary plexus (SCP) and the DCP decreased relative to those in the control eyes . The FAZ area in the DCP and in the SCP also increased when compared with that in the controls . In addition, the macular-off RRD group had lower VD in the nasal parafoveal area of the DCP than the macular-on RRD group.SO tamponade could cause microvascular changes, especially in the nasal parafoveal area. The macular-off RRD group were affected more than the macular-on RRD group. Rhegmatogenous retinal detachment (RRD) is an important cause of blindness worldwide and occurs in 10,000 people per year , 3.Although SO is an effective intraocular tamponade due to its high viscosity and high surface tension, it has been associated with cataract progression, increased intraocular pressure, emulsified oil droplets, and band keratopathy . UnexplaRecently, several studies have reported changes related to SO tamponade using optical coherence tomography (OCT) and microperimetry. These changes are widely assumed to be linked to SO-induced toxicity and ischemia, which result in damage to ganglion cells and the inter-nuclear synapsis , 9.OCT angiography (OCTA) is a noninvasive imaging modality used to display retinal and choroidal blood vessels. It can assess the vascular density, structures of the retinal bloodstream, and figures of the foveal avascular zone (FAZ). Microvascular changes related to retinal detachment have been described in current OCTA researches. Some investigators have reported microvascular changes in the superficial capillary plexus (SCP) and in the deep capillary plexus (DCP) in patients with RRD after vitrectomy using SO or gas tamponade –12. In cTherefore, this study aimed to evaluate retinal microvascular changes in the FAZ and parafoveal segments after SO tamponade and compare these changes, according to macular involvement, between macular-on and macular-off RRD using OCTA.This study was a retrospective review of consecutive cases. Patients who presented to the Department of Ophthalmology of Seoul St. Mary’s Hospital in Seoul, Korea, between January 2018 and January 2020 with a confirmed diagnosis of RRD and underwent pars plana vitrectomy using SO tamponade were included. The healthy control data were extracted from the patients’ unaffected fellow eye. The study followed by tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Seoul St. Mary’s Hospital and the Catholic University of Korea. The need to obtain informed patient consent was waived due to the retrospective study design.The inclusion criteria were the successful repair of primary RRD following surgery with SO tamponade and a minimum of 3 months of follow-up after SO removal. The exclusion criteria were traumatic or tractional RD, the co-occurrence of any other ocular disease related to ocular circulation , history of previous intraocular surgery, and severe media opacity.All patients underwent baseline ophthalmic examinations, which included best-corrected visual acuity (BCVA), slit-lamp examination, color fundus photography, swept-source OCT, and OCTA imaging  on the day or the day before the operation. We assessed the postoperative scans, which were obtained 3 months after silicone oil removal, and the findings were compared. BCVA was converted into the logarithm of the minimum angle of resolution (logMAR) for statistical analysis.OCTA scans were obtained with DRI OCT Triton (Topcon) for a 4.5 × 4.5-mm square centred upon the fovea. Patients having low-quality images with a signal strength index of under 50 were excluded. OCTA images were evaluated using automatic segmentation. We analyzed the vascular density (VD) of the superficial and deep retinal vascular zones  within the 1- and 3-mm inner and outer circles, respectively, using the built-in software program. The FAZ area was measured by manual delineation and was calculated.® Vision system, Alcon Laboratories Inc., Fort Worth, TX, USA) was performed by a single surgeon (YGP). Briefly, after vitreous removal, subretinal fluid drainage and endolaser were performed, and SO  was injected into the vitreous cavity. After at least 3 months of SO tamponade, the SO was removed with confirmation of retinal reattachment  scale  and undeThe mean BCVA changed from 1.46 ± 0.07 logMAR at baseline to 0.65 ± 0.42 logMAR 3 months after SO removal (p <0.05). In the multivariate analysis, baseline BCVA and duration of SO tamponade were significantly associated with postoperative BCVA among all preoperative factors  .2 vs 0.26±0.13 mm2, p = 0.043; 0.56 ± 0.15 mm2 vs 0.27 ± 0.12 mm2, p = 0.002, respectively; The average parafoveal VD of the DCP in the case data was significantly lower than that in the control data . A decrease in the average VD of the DCP was identified in patients with SO tamponade. Particularly, the mean VD of the nasal area was significantly lower in the SCP and DCP layers.The macular-off RRD group had a lower VD in the nasal parafoveal area of the DCP compared to that in the macular-on RRD group (p = 0.048). The FAZ area of the DCP was also larger in the macular-off than that in the macular-on RRD group . This may be secondary to ischemic damage to the retina. Studies on other ischemic retinal diseases have already determined that the DCP might be more susceptible to ischemia than the SCP owing to anatomical differences . The DCPAs we determined the parafoveal VD in four different areas , the changes in each area can be divided and compared. We proved that significant nasal parafoveal changes occur in the SCP and the DCP in SO tamponade eyes when compared with those in fellow control eyes. The average VD in the DCP was also significantly different between the groups. Several hypotheses could explain these changes. First, the change in OCTA parameters may reflect the microvascular changes in the papillomacular bundle (PMB), which is particularly more vulnerable to ischemic changes compared to other areas. Pellegrin et al.  reportedAn enlarged FAZ area can represent microvascular alterations in the fovea. This has been previously reported in various ischemic retinal diseases, and it is related to poor visual outcomes , 26. McKThe safety issues and pathogenesis related to SO tamponade are still not completely understood. Retinal toxicity may be caused by the mechanical pressure of the SO, which blocks the exchange of oxygen between the vitreous and the retina, resulting in metabolic derangement of the retina . MacularIn general, OCTA is used to detect subtle vascular alterations in patients with RRD treated with SO tamponade. According to our results, distinct changes can be observed in the nasal parafoveal area in patients with RRD treated with SO tamponade.There are several limitations to our study. First, this study was a retrospective observational study. A longitudinal study should be required to explain the microvascular changes after a successful surgery with SO tamponade. Second, the sample size was relatively small to draw clear conclusions. Third, we acquired the main outcome measures at least 3 months after primary vitrectomy and 3 months after SO removal. This is a sufficient period to detect the improvement of post-surgical edema and inflammation. However, long-term follow-up studies after SO removal may reveal a recovery of vascular alterations. Finally, OCTA image artifacts can interfere with precise evaluation of retinal microvasculature because projection artifacts might disturb imagining of the deep layer. Further prospective studies would provide data to better understand changes in the vascular parameters and their relationship with functional and anatomical results.BCVA improved significantly following a successful retinal reattachment surgery. Baseline BCVA and the duration of SO tamponade were associated with postoperative BCVA. The average VD in the DCP and the VD of the nasal parafoveal area in both the DCP and SCP decreased relative to those in the control eye. The FAZ area in the DCP and SCP significantly increased when compared with that in the control eye. Our results suggest that SO tamponade could lead to negative microvascular changes, especially in the nasal parafoveal area of both the SCP and DCP. Moreover, the macular-off RRD group had a lower VD in the nasal parafoveal area of the DCP than the macular-on RRD group, potentially suggesting that macular involvement could influence the microvascular changes of the vessels. Further prospective studies would provide data to better understand these changes.S1 Fig Fundus photography of before and after silicone oil removal.  Measurements of foveal avascular zone area and  results of vessel density calculations using optical coherence tomography angiography .(TIF)Click here for additional data file.


Penetration of a flexible and steerable needle into a soft target material is a complex problem to be modelled, involving several mechanical challenges. In the present paper, an adaptive finite element algorithm is developed to simulate the penetration of a steerable needle in brain-like gelatine material, where the penetration path is not predetermined. The geometry of the needle tip induces asymmetric tractions along the tool–substrate frictional interfaces, generating a bending action on the needle in addition to combined normal and shear loading in the region where fracture takes place during penetration. The fracture process is described by a cohesive zone model, and the direction of crack propagation is determined by the distribution of strain energy density in the tissue surrounding the tip. Simulation results of deep needle penetration for a programmable bevel-tip needle design, where steering can be controlled by changing the offset between interlocked needle segments, are mainly discussed in terms of penetration force versus displacement along with a detailed description of the needle tip trajectories. It is shown that such results are strongly dependent on the relative stiffness of needle and tissue and on the tip offset. The simulated relationship between programmable bevel offset and needle curvature is found to be approximately linear, confirming empirical results derived experimentally in a previous work. The proposed model enables a detailed analysis of the tool–tissue interactions during needle penetration, providing a reliable means to optimise the design of surgical catheters and aid pre-operative planning.The online version of this article (10.1007/s10237-020-01310-x) contains supplementary material, which is available to authorised users. PBNs possess a structure with a number of interlocked segments that slide relative to one another, with motion inspired by the egg-laying channel of certain wasps. PBNs are capable of 3D steering, thanks to a bevel tip and a programmable offset between the segments . The stage ends once the strain energy accumulates to a critical value, at which a crack is initiated.Cut propagation until full penetration:Once a crack is initiated, the external work is also consumed by frictional dissipation and work of fracture. Following initial rupture, there is a relaxation of the elastic strain energy (region 2a) and beginning of the cut propagation under increasing force (region 2b).Sliding after full penetration:When the indenter has penetrated the material for the whole depth and breaks through the other end, there is another stage of relaxation, and the frictional resistance is the only remaining contribution (region 3).The mechanics of needle penetration is about the complex interaction between needle and target tissue. The detailed insertion behaviour depends on a great number of variables, e.g. the characteristics of the indenter, the properties of the target material and the penetration rate  designed at Imperial College, London  has the capability to account for the nonlinear behaviour of soft materials and allows the inclusion of the contact constraints imposed by the penetration of the needle. In two-dimensional implementations of CZM, the fracture process zone is lumped into a line ahead of the crack tip, and the crack surfaces interaction is defined through a stress–displacement relationship describing the damaging mechanisms taking place close to the crack tip. The length of the cohesive process zone ace Fig. a:3\documDepending on the shape of the stress–displacement relationship, different types of failure can be described: for quasi-brittle materials, a possible choice is given by the bilinear curve  the FE analysis and (ii) a post-processing step. The complete formulation is summarised in Fig. Part (i) is carried out with the commercial software SIMULIA Abaqus 2017 (Dassault Systèmes), using the implicit static solver and accounting for nonlinear geometry. Each iteration describes the penetration of the needle from the initial configuration . The needle is prevented from buckling by moving between two rigid vertical surfaces, which are frictionless, fixed and represent delivery through a trocar, which is common for flexible catheters. Contact constraints are enforced using a Lagrange multipliers method, with a frictional interaction between the needle and the substrate described by the Coulomb frictional law. Since the material of the substrate is highly deformable, it was necessary to introduce an initial notch to ease the convergence at the stage of indentation, as shown in Fig. The refined inner region of the substrate is remeshed at each iteration of the penetration algorithm, in order to accommodate the advancement of the needle. The remeshing stage is carried out in MATLAB, using the mesh generator DistMesh , measured at a specified distance At this point, a criterion to determine the crack propagation direction needs to be defined. Several alternatives have been proposed in the literature Supplementary material 2 (mp4 24585 KB)Below is the link to the electronic supplementary material.


MEIOB, was previously found to play key roles in DNA double-strand break (DSB) repair. In this study, we aimed to investigate the effects and mechanisms of MEIOB in the carcinogenesis of triple-negative breast cancers (TNBCs).The newly defined cancer-testis (CT) gene, MEIOB. Cox regression analysis was used to evaluate the association between MEIOB expression and the prognosis of human TNBC. The effects of MEIOB on cell proliferation and migration in TNBCs were also assessed in vitro. Patient-derived xenograft (PDX) models were used to assess the sensitivity of breast cancers with active MEIOB to PARP1 inhibitors.The Cancer Genome Atlas database was used to quantify the expression of MEIOB as a CT gene whose expression was restricted to the testes and breast tumors, especially TNBCs. Its activation was significantly associated with poor survival in breast cancer patients . In addition, we found that MEIOB was oncogenic and significantly promoted the proliferation of TNBC cells. Further analysis showed that MEIOB participated in DSB repair in TNBCs. However, in contrast to its function in meiosis, it mediated homologous recombination deficiency (HRD) through the activation of polyADP-ribose polymerase (PARP)1 by interacting with YBX1. Furthermore, activated MEIOB was shown to confer sensitivity to PARP inhibitors, which was confirmed in PDX models.We confirmed MEIOB played an oncogenic role in TNBC through its involvement in HRD. In addition, dysregulation of MEIOB sensitized TNBC cells to PARP inhibitors, so MEIOB may be a therapeutic target of PARP1 inhibitors in TNBC. Due to the absence of these three receptors in TNBC, chemotherapy remains the standard-of-care for TNBC patients both in early and advanced stages3. Although chemotherapy can improve the conditions of patients with early TNBC, the recurrence and metastasis rate of TNBC patients is high for patients with advanced disease6. Thus, novel targeted approaches are urgently needed for the treatment of TNBC. Recently, olaparib, a polyADP-ribose polymerase (PARP) inhibitor, was approved by the US Food and Drug Administration for the treatment of breast cancers with BRCA1/2 mutations7. BRCA1/2 mutations in cancers are prototypic molecular alterations that confer homologous recombination deficiency (HRD) and sensitivity to DNA damaging therapy9. In addition, some studies show that cancers with genetic deficiencies involved in homologous recombination repair other than BRCA mutations, such as deficiencies in ATM, ATR, PALB2, and FANC, are also highly susceptible to PARP inhibitor treatment11. A group of cancer-testis (CT) genes is essential for homologous recombination13. These genes include the meiotic topoisomerase that catalyzes DNA double-strand breaks14, components of the synaptonemal complex (SYCP1)16, and multiple proteins that mediate homologue alignment or recombination (MEIOB)18. Luo et al.17 revealed that MEIOB is involved in highly ordered DNA doubled-strand break (DSB) repair during meiotic homologous recombination, by forming a complex with its cofactor, SPATA22. In our previous study, we identified MEIOB as a new CT gene involved in the carcinogenesis of lung cancer19. Nevertheless, its role and precise mechanism in TNBC remain unknown. We therefore characterized the involvement of MEIOB in the DNA repair process in TNBC patients, and further examined whether dysregulated MEIOB in TNBC conferred sensitivity to PARP inhibitors.Triple-negative breast cancer (TNBC) accounts for approximately 10%–15% of all breast cancers and is defined as breast cancer that is negative for the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2)MEIOB in breast cancer tissues by reanalyzing the raw RNA sequencing data of 1,058 patients from The Cancer Genome Atlas (TCGA) datasets. A standard STAR-HTSeq-DESeq2 pipeline was used to quantify gene expression20. We used normalized read counts > 5 as the cutoff to define the expression of MEIOB. The clinical information was obtained from Firehose Broad GDAC . To define signature 3, we converted all mutation data from WGS datasets into a matrix (M) composed of 96 features comprising mutation counts for each mutation type  using each possible 5′ and 3′ context for all samples, and applied the R package deconstructSigs12 to determine the proportion of each known mutational signature. The tissue array used in immunohistochemistry (IHC) analysis was obtained from Shanghai Outdo Biotech, Shanghai, China. This study was approved by the ethics committee of Nanjing Medical University , and all patients completed the Clinical Sample Informed Consent Form.We determined the expression of 21. The cells were cultured in Dulbecco’s Minimal Eagle’s Medium (DMEM)  containing 10% fetal bovine serum  and incubated at 37 °C in 5% CO2 and saturated humidity. The transient overexpression or knockdown of MEIOB was performed using siRNA or MEIOB plasmids. The 3 cell lines were cultured in low glucose DMEM. All cell lines were cultured at 37 °C in a humidified chamber with 5% CO2, tested negative for mycoplasma , and were authenticated using short tandem repeat profiling within the last 3 years .MDA-MB-231 and MDA-MB-468 cells were obtained from the American Type Culture Collection , and the SUM1315MO2 cell line was provided by Stephen Ethier (University of Michigan), as previously describedSupplementary Table S1.Total RNA was extracted using TRIzol reagent  and reverse transcribed using the PrimeScript RT Reagent Kit . Expression of cDNA was quantified using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) with an ABI 7900HT System . Primer sequences and their respective amplicon sizes for RT-PCR are summarized in g for 15 min at 4 °C, the cell debris was removed, and the supernatant (cell lysate) was used for Western blot. Protein concentrations were measured using a BCA assay . Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes . The membranes were blocked in blocking buffer  at room temperature for 1.5 h. Then, the membranes were incubated at 4 °C overnight with primary antibody diluted in blocking buffer, followed by incubation with the corresponding secondary anti-IgG horseradish peroxidase conjugate  for 1.5 h. The antibody binding was visualized with ECL solution . The expression of proteins was assessed by immunoblotting and was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The antibodies were as follows: anti-GAPDH , anti-MEIOB , anti- green fluorescent protein , anti-RAD51 , anti-YBX1 , anti-PARP1 , and anti-PAR .The cells were washed 3 times with phosphate-buffered saline (PBS), and the total protein was isolated using protein lysis buffer. After centrifugation at 12,000 × CCK-8 was purchased from Dojindo Molecular Technologies . Briefly, 2 h before each indicated time point, 10 μL of the CCK-8 solution was added to each well in a plate containing 100 μL of DMEM. Then, the absorbance at 450 nm was recorded using a microplate absorbance reader. Each count was determined as an average of 3 repeats, and each data point was the average of at least 3 experiments. All data were normalized to the control group.MEIOB knockdown or MEIOB-overexpressing MDA-MB-231 cells and MEIOB-overexpressing SUM1315MO2 cells was performed using 6-well dishes (500 cells/well), with 6 wells per condition. The number of colonies containing more than 50 cells was counted. Cells were fixed and then stained with Crystal Violet. Images were captured under a microscope, and 3 wells were counted by 2 independent investigators.The colony formation assay of 4 cells/well. A volume of 100 μL of the cells was added to the upper chamber, while 600 μL of DMEM containing 10% fetal bovine serum was added to the lower chamber and incubated at 37 °C in 5% CO2. The cells attaching to the upper surface of the membrane were removed with a cotton swab, and the cells on the underside were fixed and stained with Giemsa  for 3–5 min and counted (9 random fields) by 2 independent investigators. The results were normalized to those of the controls.Migration and invasion assays were performed using Transwell migration chambers . The cells were seeded at a density of 4 × 10The cell cycle was analyzed by flow cytometry. For cell cycle analysis, transfected lung cancer cells were suspended in 75% ethanol overnight and centrifuged at 1,000 rpm. The cell pellets were washed twice with PBS and re-suspended in PBS containing 50 mg/mL propidium iodide and 100 g/mL DNase-free RNase A. The cell suspension was incubated for 30 min at 37 °C and analyzed by flow cytometry. Cell cycle distribution was further analyzed with Cell Quest software  and Mod Fit LT .Xenografts were formalin-fixed for at least 24 h and paraffin-embedded. Sections were evaluated by hematoxylin & eosin staining. IHC was performed on additional sections to detect MEIOB , PARP1 , and Ki67  following the manufacturers’ instructions.22. We are grateful to Prof. Mansour for providing vectors used in the DSB repair reporter assay. The GFP-based repair substrates were cloned using pEGFP-N1  and pBluescriptII-KS  as backbones, with pGC for homology-directed GC. For the GC substrate, pGC, the 18-bp I-SceI recognition site was inserted into the unique BcgI site of pEGFP-N1, thereby inactivating the GFP-coding sequence. This intermediate, named pGC-intermedI, and linked to a modified fragment of pBluescriptII-KS, was generated as previously described22. To induce DSBs, SUM1315MO2 cells containing the stably integrated reporter construct for gene conversion of pGC were transfected with the I-SceI expression vector, pCMV3xnls-I-SceI (1 μg), using Fugene HD  as a transfection reagent. Forty-eight hours after transfection, the cells were assessed for green fluorescence using Western blot and flow cytometry .DSB repair efficiency was measured using a DSB reporter assay as previously describedFor immunofluorescence staining, tissues were first washed in PBS 3 times to remove excess Optimal Cutting Temperature (OCT) buffer, and then they were blocked with goat serum for 30 min at room temperature. Then, they were incubated with primary antibodies at 4 °C overnight. Before incubation with secondary antibodies, the tissues were washed 3 times in PBS. The 4′,6-diamidino-2-phenylindole (DAPI) stain was simultaneously added with 2 antibodies  from different species for 2 h at room temperature. After washing the sections 3 times with PBS and mounting them with mounting medium, the images were acquired by confocal microscopy. The reagents were as follows: DAPI stain , anti-MEIOB , and anti-γ-H2AX .23. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee and the Ethics Committee of Nanjing Medical University . We selected 6 of the established breast tumor samples, including 3 MEIOB-negative and 3 MEIOB-positive tumors, for further use in xenograft models.Patient-derived xenografts were generated from primary or metastatic breast tumors using previously described proceduresMEIOB gene expression data from TCGA data were analyzed for differential expression in tumor tissues vs. adjacent normal tissues. The association between MEIOB expression in all tumor tissue samples and survival was analyzed by Cox regression. Kaplan-Meier analysis was used to fit the survival curves, and significance was evaluated using the log-rank test. For experimental data, continuous values are described as the mean ± SE and tested by analysis of variance. A value of P < 0.05 was accepted as statistically significant. All statistical analyses of in vitro and in vivo assays were performed using Prism 6.01 .The data that support the findings of this study are available from the corresponding author upon reasonable request.MEIOB, we conducted RT-PCR using cDNA of 16 normal human adult tissues, including the testis. The results showed that the expression of MEIOB in the testis was higher than that in any other normal tissue , which was consistent with the results from the GTEx database. Furthermore, we found that MEIOB was aberrantly expressed in breast cancer tumor tissues based on TCGA data set analyses , and the ratio of expression was the highest in TNBCs when compared with other subtypes of breast cancer . The results of IHC analysis of breast cancer tissues confirmed the upregulation of MEIOB protein levels in TNBCs when compared with both luminal cancers and adjacent tissues . In addition, Kaplan-Meier analysis showed that high MEIOB expression was positively correlated with poor overall survival in breast cancer patients, especially in TNBC patients from TCGA database . These findings suggested the tumor-promoting roles of MEIOB in the tumorigenesis and prognosis of breast cancer, especially TNBC.To thoroughly evaluate the expression pattern of Supplementary Figure S1A and S1B), indicating that MEIOB might play an important role in a subset of TNBCs. Furthermore, immunofluorescence staining showed that MEIOB was localized in the nuclei of MDA-MB-231 cells . To better understand the role of MEIOB in breast cancer, we then knocked down MEIOB by using siRNA in MDA-MB-231 cells  and found that depletion of MEIOB in MDA-MB-231 cells led to a significant decrease in both cell proliferation and migration (2C). We also overexpressed MEIOB in MDA-MB-231 cells , which significantly increased cell proliferation and migration (2F). These results were further confirmed in SUM1315MO2 cells . Thus, we concluded that MEIOB may promote tumor proliferation in TNBCs.To validate the hypothesis that MEIOB affected the viability of breast cancer cells, we first conducted RT-PCR and Western blot to examine the mRNA and protein expressions of MEIOB in various types of breast cancer cell lines. The results showed that both mRNA and protein levels of MEIOB were dramatically upregulated in two TNBC cell lines, MDA-MB-231 and MDA-MB-468, while they were significantly lower in either normal breast epithelium or luminal breast cancer cells (27. We therefore treated SUM1315MO2 cells with cisplatin (20 μmol/L) to induce the DNA damage response and then measured the degree of DNA damage by monitoring anti-γ-H2AX nuclear staining signals28. The accumulation of γ-H2AX was significantly decreased in MEIOB-overexpressing SUM1315MO2 cells treated with cisplatin (3B), indicating that MEIOB promoted the DSB repair response in TNBC cells.Cisplatin, a common chemotherapy agent that creates DNA interstrand and intrastrand crosslinks, can lead to DNA DSBs after its removal in both replicating yeast and mammalian somatic cells, and has been used to treat many breast cancer types, including TNBC29. Our results further showed that the overexpression of MEIOB in SUM1315MO2 cells resulted in cell cycle arrest at the G1 phase, which indicated that MEIOB might be absent from homologous recombination while being involved in the non-homologous end joining (NHEJ) repair process . As reported in past studies, CDK4 and CDK2 are necessary for cells to enter the G1 phase from the G0 phase30. In MEIOB-overexpressing SUM1315MO2 cells, we observed increased expressions of CDK2 and CDK4, which further confirmed that MEIOB induced SUM1315MO2 cells to maintain G1/G0 phase arrest . DSBs that occur during G1 phase are mainly repaired through NHEJ, whereas those formed during S and G2 phases are predominantly repaired by homologous recombination mechanisms31. Furthermore, the pHR-GFP reporter vector was used to assess the effect of MEIOB expression on DNA damage repair by homologous recombination in SUM1315MO2 breast cancer cells32. Homologous recombination activity was assessed by quantifying GFP protein expression and GFP-positive cells 72 h after co-transfection of pHR-GFP and pSce. As a result, we found that GFP intensity was significantly downregulated in MEIOB-overexpressing SUM1315MO2 cells when compared with control cells . Similar results were observed in GFP-positive cells, as detected by flow cytometry (3G). Thus, we concluded that MEIOB overexpression in SUM1315MO2 cells resulted in HRD.Homologous recombination is inhibited during the G1 phase of the cell cycle, but is active in both the S and G2 phasesin vitro to inhibit PARG24 degradation of its polyADP-ribose, interacted with MEIOB . This interaction was further verified by co-immunoprecipitation analysis of FLAG-tagged MEIOB . We also found that the score of signature 3 in YBX1 high-expressing tumors was significantly higher than that in YBX1 low-expressing tumors in TCGA breast cancer datasets . Next, we found that MEIOB caused an increase in PARP1 polyADP-ribose levels and a decrease in RAD51 expression, which is a key marker of the homologous recombination repair process 33. Finally, the activated PAR-related DNA repair process in MEIOB-overexpressing cells was again silenced after YBX1 was knocked down . Taken together, our results suggested that MEIOB was involved in PARP1-related HRD, probably by interacting with YBX1.To determine the mechanism by which MEIOB affected the DNA damage repair process, we performed liquid chromatography-tandem mass spectrometry to identify the proteins interacting with MEIOB. The results showed that the YBX1 protein, which can physically interact with PARP1 34. According to the results described above, we hypothesized that MEIOB might result in genomic alterations similar to the loss of BRCA1/2. Notably, we found that the score of signature 3 was correlated with MEIOB and was significantly higher in patients with high MEIOB expression in TCGA breast cancer datasets (5B). To exclude the influence of BRCA1/2, we found that MEIOB expression was mutually exclusive with mutations in BRCA1/2 in both breast cancers and TNBCs . These results suggested that MEIOB might be a new biomarker of HRD in TNBC patients, except in patients with BRCA1/2 mutations.Biallelic inactivation of BRCA1/2 is associated with a pattern of HRD known as signature 3Figure 6A shows that MEIOB-expressing MDA-MB-231 and MDA-MB-468 cells were more sensitive to AG-14361 than MEIOB-null SUM1315MO2 cells. Similar results were observed in cells treated with olaparib.We hypothesized that TNBC cells overexpressing MEIOB would be more sensitive to PARP inhibitors than MEIOB-null cells. We evaluated the sensitivity of TNBC cells with different MEIOB expression patterns to PARP1 inhibitors, by treating cells with multiple concentrations of the PARP1 inhibitors, AG-14361 and olaparib.  Supplementary Figure S4A. Fresh TNBC tissues were obtained from patients and divided into two groups according to MEIOB protein expression levels (MEIOB-null and MEIOB-expressing) . All tumors were implanted subcutaneously in NOD/SCID mice. Both groups were treated with olaparib when the patient-derived tumor volume had reached 200 mm3. Tumor volumes were measured once a week after treatment with olaparib . Six weeks after administration, the tumors were harvested and weighed . The results showed that the volumes of tumors expressing MEIOB significantly decreased to less than 30% of MEIOB-null tumors after treatment with olaparib , suggesting that MEIOB-expressing tumors, in vivo, were more sensitive to olaparib than MEIOB-null tumors.We then established a patient-derived xenograft (PDX) mouse model of TNBC that had characteristics similar to those of human primary tumors. A schematic of the PDX model is shown inFigure 6E). In addition, PARP1 was strongly inhibited by olaparib in MEIOB-expressing tumors . These results indicated that tumors that expressed elevated levels of MEIOB were more sensitive to olaparib.In olaparib-treated PDX tumors, IHC labeling was conducted using antibodies to cell proliferation markers, Ki67 and PARP1. After treatment with olaparib, the number of Ki67-positive proliferating cells was dramatically decreased in the MEIOB-expressing tumors when compared with the MEIOB-null tumors 36. Representative gene expression levels involved in the DNA repair process were then verified by RT-PCR. Supplementary Figure S4C shows that expressions of the PARPBP37, POLQ38, and RTEL139 genes, which participate in NHEJ, were inhibited by olaparib. A previous study showed that olaparib monotherapy provided a significant benefit over standard therapy in breast cancers with BRCA mutations7. Taken together, these results suggested that PARP inhibitors may have additional applications in the treatment of TNBC.To validate the downstream mechanisms of olaparib treatment, RNA-seq was performed with the olaparib-treated and untreated MEIOB-expressing tumors. We then selected differentially expressed genes for further Gene Ontology (GO) analysis. The results confirmed that olaparib therapy led to an accumulation of DNA DSBs and cell cycle arrest  and in vivo (PDX models) showed that the overexpression of MEIOB increased the sensitivity of TNBC to PARP1 inhibitors in patients without BRCA1/2 mutations. Thus, it is possible that MEIOB dysregulation might be considered a biomarker for the selection of TNBC patients that could be responsive to PARP inhibition.TNBC is the most aggressive subtype of breast cancer46. In contrast, it was reported that MEIOB binds with SPATA2217 and plays key roles in the early repair of the meiotic homologous recombination repair process18. This suggested that MEIOB plays different roles during two biological processes; MEIOB strongly activates the homologous recombination repair pathway in meiosis18; and in a different manner, it induces another major DSB repair pathway, error-prone NHEJ, and results in HRD in TNBC. In TNBCs, MEIOB binds to YBX1, a protein that physically interacts with PARP1 to inhibit its polyADP-ribose degradation47. YBX1 was previously found to interfere with PARP1 activation on the damaged DNA by multimerization or interaction with its DNA-binding domain48. Furthermore, we observed an increase in PAR caused by MEIOB, which was attenuated after YBX1 was knocked down. These results led us to hypothesize that YBX1 may function as a cofactor of MEIOB in the synthesis of PAR.In this study, MEIOB was involved in the error-prone NHEJ repair process in TNBCs, which was characterized by an increase in PAR and a reduction in RAD51Supplementary Figure S4D).Taken together, our study showed that the CT gene, MEIOB, promoted breast cancer cell viability and mediated HRD by activating the PARP1-dependent DNA damage response. Additionally, MEIOB sensitized TNBC to PARP1 inhibitors, which might expand the use of PARP1 inhibitors for the treatment of MEIOB-positive TNBCs (Click here for additional data file.


Objective: The traditional Chinese medicine Caulis Sargentodoxae is widely used in the treatment of ulcerative colitis (UC), but the mechanism remains unknown. The present study aims to reveal its effective components, targets and pathways through network pharmacology and bioinformatics approaches.Materials and methods: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to identify effective components. The ligand-based targets prediction was achieved through SwissTargetPrediction and TargetNet. UC-related targets were identified using Gene Expression Omnibus (GEO) data and DisGeNET. The common targets of disease and components were constructed and analyzed by PPI network. Lastly, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses are used to explain the functions of these common targets. Components-Targets-Pathways network was visualized and analyzed to further reveal the connection between the components and targets.Results: Eight active components and 102 key targets were identified to play an important role in UC. These targets were related to regulation of protein serine/threonine kinase activity, positive regulation of cell motility, response to molecule of bacterial origin, response to toxic substance, ERK1 and ERK2 cascade, peptidyl-tyrosine modification, inositol lipid-mediated signaling, cellular response to drug, regulation of inflammatory response and leukocyte migration. Moreover, HIF-1 signaling pathway and PI3K-Akt signaling pathway were the key targets involved in UC-related signaling pathways.Conclusion: The eight active components of Caulis Sargentodoxae mainly play a therapeutic role for UC through synergistic regulation of HIF-1 signaling pathway and PI3K-Akt signaling pathway. Ulcerative colitis (UC) is a disease caused by a genetically predisposed host’s immune disorder to antigens in the intestine, and its incidence and prevalence are increasing globally ,2. The dThe use of herbs to treat various diseases has a long history. In addition, traditional Chinese medicine shows obvious advantages in the treatment of UC, such as low recurrence rate and fewer side effects . Some naNetwork pharmacology methods are rapidly evolving and applied to find new treatment opportunities and reposition of approved drugs. They can also effectively map the unexplored targets of natural products, thereby providing drug space for proteins related to various complex diseases . The purhttps://tcmspw.com/tcmsp.php) [Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) is a manually curated database containing 12144 herbal compounds and related pharmacokinetic properties (msp.php) . Oral bihttp://www.swisstargetprediction.ch/). The principle is to train the prediction model by multivariate logistic regression fitting of various size related subsets of known active molecules to weighted 2D and 3D similarity parameters [http://targetnet.scbdd.com/calcnet/index/). Its server will predict the activity of user-submitted molecules in 623 human proteins by building a high-quality QSAR model for each human protein [https://pubchem.ncbi.nlm.nih.gov/). Subsequently, the SMILES of each component were uploaded to two databases for targets prediction.Drug-targets prediction technology, which can predict component targets with high throughput and quickly, has been widely used in the preliminary research of drug development. The current prediction technologies mainly include ligands-based prediction methods , targets-based prediction methods (such as reverse molecular docking) and machine learning methods. SwissTargetPrediction is a ligand-based targets prediction tool for bioactive small molecules (rameters . TargetN protein . The strP<0.05 and log2 (fold change) > 2 or log2 (fold change) < −2 were considered as differential genes, they have played an important role in the development of UC.Gene Expression Omnibus (GEO) is an ever-increasing platform for storing high-throughput sequencing data. Integrating sequencing information from different centers and performing gene differential expression analysis will facilitate the identification of disease treatment targets. We identified three series from GEO that all came from GPL6244, among which GSE73661 contains 23 active UC tissues and 12 normal tissues, GSE75214 contains 74 active UC tissues and 11 normal tissues, GSE48958 contains 7 active UC tissues and 8 normal tissues. Genes with adjusted https://www.disgenet.org/). It currently covers more than 24000 diseases and traits, 17000 genes and 117000 genomic variants [DisGeNET integrated data from public databases, GWASs, animal models and scientific literature, and collected a large number of variants and genes associated with human diseases (variants . UC-relahttps://string-db.org/) can carry out direct  and indirect  interactions of proteins to achieve a comprehensive and objective network [Homo sapiens’, and the minimum required interaction score threshold ‘highest confidence (≥0.9)’ was set to obtain protein interaction information. Cytoscape v3.7.2 was used for visualization and in-depth analysis of the network. The hub nodes were identified in each network by calculating the size of the degree.STRING , a network enrichment analysis tool, for GO and KEGG enrichment analyses. The reference set was selected as human genome protein encoding. And the enrichment method was selected as over-representaion analysis.Gene ontology (GO) is a gene function classification system that comprehensively describes the attributes of genes and gene products of various species through dynamically updated GO terms. It is used to describe the biological process (BP), the cellular component (CC) and molecular function (MF) of gene sets of interest. Kyoto Encyclopedia of Genes and Genomes (KEGG) is a knowledge base for systematic analysis of gene function, which can describe pathway data involved in genes of interest, including connection of metabolism, membrane transport, signal transduction and cell cycle, etc. We used WebGestalt ’ in STRING. A network with 102 nodes and 618 edges was obtained . Based oUsing WebGestalt to conduct bioinformatics analysis of the above key targets. We identified the top ten GO terms and pathways that were significantly enriched, respectively  and 6. IIn UC, the pathways mainly regulated by Caulis Sargentodoxae were Pathways in cancer, Proteoglycans in cancer, Endocrine resistance, Prostate cancer, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, MicroRNAs in cancer, Fluid shear stress and atherosclerosis, PI3K-Akt signaling pathway and EGFR tyrosine kinase inhibitor resistance . Among tThe key targets of Caulis Sargentodoxae enriched to HIF-1 signaling pathway were BCL2, EGFR, ERBB2, FLT1, IGF1R, INSR, MAPK1, MKNK2, NOS2, NOS3, PIK3CA, PIK3CD, PRKCB, RELA, RPS6KB1 and SERPINE1 . The key targets of Caulis Sargentodoxae enriched to PI3K-Akt signaling pathway were BCL2, CASP, CCND1, CCNE1, EGFR, ERBB2, FGFR1, FLT1, HSP90AA1, IGF1R, INSR, KDR, MAPK1, MCL1, MDM2, MET, NOS3, NTRK1, PIK3CA, PIK3CD, PIK3CG, RAC1, RELA, RPS6KB1 . In the PPI network, EGFR, PRKCB, MAPK1, HSP90AA1, RELA, RAC1, PIK3CA were also identified as hub genes.Moreover, we constructed components-targets-pathways network with 8 components, 102 hub targets and HIF-1 signaling pathway and PI3K-Akt signaling pathway . The netAlthough drugs such as aminosalicylate, glucocorticoids, antibiotics and anti-TNFα play a good role in the treatment of UC, they still fail to achieve healing results and often cause side effects . In ChinCombined with the bioinformatics research on diseases, we can more clearly define the targets and specific mechanisms of Chinese medicine on the diseases, and identify key active molecules. This is of great significance to the development of natural medicines and the treatment of diseases. In our study, we successfully identified eight active molecules and two important pathways related to UC through network pharmacology and bioinformatics. They played a synergistic role in regulating HIF-1 signaling pathway and PI3K-Akt signaling pathway. Current evidences showed that Treg/Th17 imbalance is closely related to UC. The mTOR-HIF-1α-Th17 and STAT3-HIF-1α-Th17 pathways play an important role in the development of Th17 and the activation of IL-17. In addition, HIF-1α can bind to Foxp3, thereby affecting the development and function of Treg . PI3K caAccording to Components-Targets-Pathways network, the degrees of physcion, emodin, 3-(4-Hydroxyphenyl)acrylic acid 4-hydroxyphenethyl ester and meso-dihydroguaiaretic acid were the highest. Physcion is an anthraquinone compound commonly found in various plants, and shows anti-cancer properties in a variety of tumors. It can significantly inhibit the production of LPS/IFN-γ-induced NO in various cells via attenuating iNOS . It can Overall, our results suggested that Caulis Sargentodoxae can treat UC through regulating HIF-1 signaling pathway and PI3K-Akt signaling pathway, but this effect is a synergistic effect of eight active components. It is necessary to further compare the therapeutic effects of Caulis Sargentodoxae and its different components in animal models, and further evaluate these molecules in terms of safety and higher efficacy. Analyzing and verifying the effective dose and toxicity of different molecules by using pharmacology and toxicology experiments will also have important significance for clinical applications.Click here for additional data file.