In clinical practice, we have been able to improve the survival time and quality of life for patients with rectal cancer through the treatment of “FuZhengXiaoJi” (strengthening the Qi and reducing accumulation).  In this study, we elucidated the core prescriptions for patients with rectal cancer and Qi and blood deficiency syndrome, and we explored the potential mechanisms of the prescriptions using an integrated strategy that coupled data mining with network pharmacology.  A Bron–Kerbosch (BK) algorithm was applied to find the core prescriptions. The active ingredients, targets, activated signaling pathways, and biological functions of core prescriptions were analyzed using network pharmacology and directly associated proteins were docked using molecular docking technology to elucidate the multicomponent, multitarget, and inter-related components associated with TCM systematically.  Data mining identified 3 core prescriptions, and most of the herbs consisted of “FuZhengXiaoJi” Fang. Network pharmacology identified 15 high-degree active ingredients among the 3 core prescriptions and 16 high-degree hub genes linked with both rectal cancer and the 3 core prescriptions. Additional Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of these 16 targets showed that the most significant pathways were MAPK, interleukin-17, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) pathways. From the 16 genes, TGFB1, IL1B, IL10, IL6, PTGS2, and PPARG closely interacted with the tumor microenvironment, and PPARG, MYC, and ERBB2 were closely linked to survival. In molecular docking, quercetin, kaempferol, and lauric acid showed good binding energy to each target.  Data mining, network pharmacology, and molecular docking may help identify core prescriptions, high-degree ingredients, and high-degree hub genes to apply to diseases and treatments. Furthermore, these studies may help discover hub genes that affect the tumor microenvironment and survival. The combination of these tools may help elucidate the relationship between herbs acting on “Zheng” (syndrome) and diseases, thus expanding the understanding of TCM mechanisms. Rectal cancer is a common type of colorectal cancer (CRC). CRC is the second and fourth most common malignant tumor in the world and in China, respectively  ; accordiEffective treatments for CRC include a combination of surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy therapy. However, chemotherapeutics have known limitations, such as their substantial adverse effects, including gastrointestinal reactions, mucositis, bone marrow suppression, neurotoxicity, abnormal liver or kidney function, febrile neutropenia, and fatigue . The seaglycyrrhizae radix et rhizoma), BaiZhu , HuangQi , DangShen (codonopsis radix), and ChenPi (citri reticulatae pericarpium), were identified as associated with significant reductions in chemotherapy-induced gastrointestinal (CIGI) toxicity  [poria), and RenShen (ginseng radix et rhizoma), has alleviated CIGI toxicity, including nausea, vomiting, and diarrhea, better than chemotherapy alone.Traditional Chinese medicine (TCM) has been used to treat CRC for more than 6,000 years with some degree of success . Good ret, etc.) . In addit, etc.) , which cHedyotis diffusa Willd) inhibits VEGF-C-mediated lymphangiogenesis in CRC through multiple signaling pathways [Some herbs increase the effects of radiotherapy and chemotherapy and prevent metastasis. BaiHuaSheSheCao  using data mining to provide novel insights into the management and clinical treatment of this disease. Moreover, this study applied the methods of network pharmacology and molecular docking to explore the mechanisms of action for core prescriptions used to treat rectal cancer. The study procedure is shown in The data used for this study were extracted from the medical records of outpatients and inpatients with rectal cancer in Ruijin Hospital affiliated with Shanghai Jiaotong University School of Medicine from January 2017 to February 2021. This study was a retrospective study. The protocols were approved by the Institutional Human Ethics Committee, Ruijin Hospital Affiliated with Shanghai Jiaotong University School of Medicine.Patients with stage II, III, or IV rectal cancer with a clear pathological diagnosis were included in the study. There were no restrictions with regard to age or sex.The following patients were excluded from the analysis: (1) patients who did not meet the inclusion criteria or who had incomplete data with regard to determinations of treatment efficacy; (2) pregnant or lactating women; (3) patients with a known allergy or those that presented with an adverse reaction during treatment; (4) patients diagnosed with psychiatric conditions; and (5) patients with severe comorbid conditions, such as cardiovascular disease, cerebrovascular disease, impaired liver or kidney function, or a hematopoietic system disease.The following data were collected from the patient's original medical records to establish a database: general information , clinical symptoms, laboratory tests, diagnosis, tongue condition, pulse condition, and prescription (drug names and dosage). All data were double-checked by two operators (Shiyu Ma and Lin Zheng) to ensure the accuracy, consistency, and completeness of the data entered. A network analysis toolbox of MATLAB was used to build a drug-dispensing network, and the Bron–Kerbosch (BK) algorithm was used to mine the core prescriptions for a minimum of eight drugs and a support threshold of 0.03.α confidence level [Xi to the total number of drugs in the BF). The larger CMBN value of each group is the final core prescription.The confidence of the mined core prescriptions was evaluated using the confidence based on the whole network (CMBN) score and was supported by the ce level . Both vaIn this work, rectal cancer was considered a disease, and associated genes were collected from the GeneCards database.Information about the chemical ingredients of the three core prescriptions  was gathered from the following literature and data sources: STITCH database, Herb Ingredients' Targets (HIT) database, Chinese Academy of Sciences Chemistry database, Traditional Chinese Medicine Systems Pharmacology database, and the Traditional Chinese Medicine Integrated Database (TCMID).https://pubchem.ncbi.nlm.nih.gov/). The quantitative estimate of drug-likeness (QED) was calculated to prescreen pharmaceutically active compounds in the P1, P2, and P3 prescriptions. Those compounds with QEDs >0.3 were chosen.We collected the chemical ingredients and their structures from the PubChem database (http://herb.ac.cn/). HERB [Potential targets of these ingredients were investigated and analyzed using HERB (/). HERB  is a hig/). HERB . We collhttp://cbbio.cis.umac.mo) [PSOVina (umac.mo) , 21 is ahttp://www.wwpdb.org/), including human interleukin (IL)-6 , human MYC (PDB ID: 1nkp), human matrix metalloproteinase 9 , human IL-10 (PDB ID: 1lk3), human ILIB (PDB ID: 4dep), human transforming growth factor (TGF)-B1 (PDB ID: 3kfd), and human PPARG (PDB ID: 3e00).For each docking assay, six human receptors were retrieved from the Protein Data Bank  enrichment analysis for molecular function (MF), cellular component (CC), and biological process (BP) analyses. Pathway enrichment analysis was based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, Disease Ontology (DO) enrichment analysis, and the Reactome database.To investigate potential pathways regulated by P1, P2, and P3, enrichment analysis was carried out to identify the significant biological profiles of P1, P2, and P3. The hypergeometric http://string-db.org; version 11.0) online database. A combined score >0.4 was considered statistically significant. The plug-in app Molecular Complex Detection  of Cytoscape  was applied for clustering a given network on the basis of its topology to identify densely connected regions. The PPI networks were drawn using Cytoscape, and the significant modules in the PPI networks were identified using MCODE. The criteria for selection were as follows: MCODE scores > 5, degree cutoff = 2, node score cutoff = 0.2, max depth = 100, and k-score = 2. Subsequently, KEGG and GO enrichment analyses were performed for genes in these modules. Hub genes were defined as nodes with degrees ≥10 [Analysis of the functional interactions between proteins may provide insights into the mechanisms of P1, P2, and P3 activity, and a protein-protein interaction (PPI) network was predicted using the Search Tool for the Retrieval of Interacting Genes . GEPIA2 (http://gepia2.cancer-pku.cn/#index) was applied for the survival analysis. TCGA-READ (RNAseq data of level 3 HTSeq-FPKM) was used to study the correlation analysis between genes and other genes after log2 transformation. Results were interpreted with ggplot2 in the R package (version 3.6.3). P values < 0.05 (∗), 0.01 (∗∗), and 0.001 (∗∗∗) indicated different levels of significance in the relationship between genes and genes.The relationship between the expression level of the hub genes and the prognosis of rectal cancer and the correlation of this relationship with different core genes in the tumor microenvironment were analyzed using The Cancer Genome Atlas rectal adenocarcinoma (READ) database . Most patients were elderly; the average age was 66.69 (±8.92) years. Twenty-three patients were men, and 14 were women. The 4 diagnostic conditions were distributed as follows: all 37 patients had a light red tongue and white color; 32 patients had a fine pulse at the left and right wrists; 1 patient had a sunken pulse at the left and right wrists; and 4 patients had a fine string pulse at the left and right wrists. Of these 37 patients, 11 had stage II disease; 12 had stage III; and 14 had stage IV disease. Six patients were receiving chemotherapy; 13 patients had disease progression and recurrence; and 18 patients were in a recovery period.coicis semen), EZhu (curcumae rhizoma), LingZhi (Ganoderma), ShuYangQuan (Solanum lyratum Thunb.), GanCao, DangShen, and ShanZha (crataegi fructus). The top 20 herbs (frequency and degree) are listed in A total of 280 herbs were used in the 563 prescription medication preparations. The following were used most often: BaiZhu, HuangQi, BaiHuaSheSheCao, FuLing, ChenPi, YiYiRen  and compared them with P1, P2, and P3. Among the six patients in the chemotherapy period group, four had prescriptions in which more than 80% of the herbs overlapped with those in P2, suggesting that P2 may be the core prescription for patients during chemotherapy. Among the 13 patients in the progression and recurrence period group, 6 had prescriptions in which more than 80% of the ingredients overlapped with P1, and 5 had prescriptions with 60%–80% overlap with P1, indicating that P1 may be the core prescription for patients in the progression and recurrence period. Among the 18 patients in the recovery period group, 6 had prescriptions in which more than 80% of the overlapped with P3, and 11 had prescriptions with 50%–80% overlap that indicates that P3 may be the core prescription for patients in the recovery period.n = 16 in P1, 30 in P2, and 21 in P3; After screening of the databases, removal of duplicate components, and integration of data, we confirmed 129, 191, and 164 active ingredients and 315, 439, and 405 major targets in P1, P2, and P3, respectively. Cytoscape 3.7.0 was applied to construct active ingredient-target network diagrams for the active ingredients and targets in P1, P2, and P3; Cytoscape identified the numbers of active ingredients with a degree rank value ≥ 200 for each prescription (As shown in Based on the Chinese Pharmacopoeia (2020 edition) and the Shanghai Concoction Specification (2018 edition), we constructed the Chinese herbal medicine network of core prescriptions using Cytoscape 3.7.0 . The sizP value) < 0.01 to identify the top 100 most significant ontology molecular functions (MFs) of the three core prescriptions, and Venn analysis was performed. We identified 72 biological functions of P1, 94 of P2, and 87 of P3 that overlapped with each other; these functions mainly involved proteins, enzymes, cytokine receptors, nuclear receptors, peptides, and cofactors. The main MF included binding to proteins, enzymes, cytokine receptors, nuclear receptors, peptides, and cofactors, which promotes activities involving protein dimerization, protein kinases, transcription factors, and neurotransmitter receptors. The enrichment of MF mainly focused on energy metabolisms, such as glucuronosyltransferase activity (GO:0015020), UDP-glycosyltransferase activity (GO:0008194), NADP binding (GO:0050661), and other MF pathways with effects on tumor immunity. Other MF pathways, such as TNF receptor superfamily binding (GO:0032813), TNF receptor binding (GO:0005164), and p53 binding (GO:0002039), also have a role in tumor immunity. P1 has effects on fatty acid-binding (GO:0005504), and P3 also promotes norepinephrine binding (GO:0051380) and growth factor receptor binding (GO:0070851).Targets of the P1, P2, and P3 prescriptions were subjected to GO enrichment analysis  by screeP) < 0.01 to identify the top 100 most significant biological processes (BPs) attributable to the three core prescriptions and subsequent Venn analysis revealed that 64 BPs of P1, 90 of P2, and 88 of P3 overlapped. The main BPs were as follows: (1) apoptosis-related effects of tumor, such as negative regulation of programmed cell death (GO:0043069), negative regulation of cell death (GO:0060548), regulation of cell death (GO:0010941), regulation of programmed cell death (GO:0043067), positive regulation of cell death (GO:0010942), and positive regulation of programmed cell death (GO:0043068); (2) oxidative stress, such as response to oxygen levels (GO:0070482) and cellular response to ROS (GO:0034614); (3) regulation of inflammatory response, inflammatory response (GO:0006954), chronic inflammatory response to antigenic stimulus (GO:0002439), and chronic inflammatory response (GO:0002544); (4) stimulation of immune function, such as regulation of immune system processes (GO:0002682), immune system processes (GO:0002376), and regulation of immune response (GO:0050776); and (5) angiogenesis-related BPs, such as VEGF-activated neuropilin signaling pathway (GO:0038190), VEGF-activated platelet-derived growth factor receptor signaling pathway (GO:0038086), and positive regulation of cell proliferation by the VEGF-activated platelet-derived growth factor receptor signaling pathway (GO:0038091).Screening using an FDR (adjusted P) < 0.01. The KEGG enrichment analysis results were mainly associated with oncological diseases. The three core prescriptions converged on pathways such as the IL-17 signaling pathway (ko04657), the TNF signaling pathway (ko04668), and the VEGF signaling pathway (ko04370). P2 and P3 prescriptions also affected the apoptosis pathway (ko04210).The targets of the P1, P2, and P3 prescriptions were subjected to KEGG enrichment analysis , settingP) < 0.05, DO enrichment analysis  < 0.05, Reactome enrichment analysis  was performed using the Reactome tool. Five main pathways were identified: cytokine signaling in the immune system (HSA-1280215), apoptosis (HSA-109581), hemostasis (HSA-109582), metabolism of angiotensinogen to angiotensin (HSA-2022377), and IL-6 signaling (HSA-1059683). In the KEGG analysis (FDR < 0.01), most of these pathways were related to tumor immunity and included the TNF, IL-17, and NF-kB signaling pathways, apoptosis, MAPK signaling, T-cell receptor signaling, platinum drug resistance, and resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors.The first group  found that the targets were mainly related to IL-related receptors, IL-10 signaling (R-HSA-6783783), signaling by ILs (R-HSA-449147), IL-4 and IL-13 signaling (R-HSA-6785807), and cytokine signaling in the immune system (R-HSA-1280215).The CTD database was used to verify the relevance of the core prescription targets to rectal cancer, and an inference score was used to determine the degree of relevance. Twenty-six targets (96.3%) had scores >50, indicating that they were extremely relevant to the diseases and could be regarded as intervention targets in the core prescriptions for rectal cancer .Additional analysis of the 27 targets that coacted against the disease and the 40 high-degree targets that coacted with 3 core prescriptions revealed that 16 of them, namely, MMP2, AKT1, PPARG, IL10, TP53, IL6, PTGS2, TNF, VEGFA, TGFB1, IL1B, MYC, CASP3, MMP9, ESR1, and ERBB2, were coacting targets (among the disease and 3 core prescriptions). The rank values of these 16 targets in each prescription and the inference score in the disease are shown in From the 16 genes, 3 genes  were linked with the survival of rectal cancer . TGFB1, The docking results of the main 15 active ingredients in the core prescription with genes are shown in In this study, we identified the 3 core prescriptions  used to treat rectal cancer. We also found that P1, P2, and P3 may be potential core prescriptions for patients during the tumor progression and recurrence periods, the chemotherapy period, and the recovery period, respectively. The results revealed that the core prescriptions are involved in three major biological processes, tumor apoptosis, angiogenesis, and immune anti-inflammatory effects, through 15 main active ingredients and 16 potential targets and that the prescriptions activated the following pathways: signaling by ILs , MAPK siA deficiency of cellular immune function in patients with malignant tumors is closely associated with the onset, development, and prognosis of tumors . In TCM,albiziae cortex) can be used for these patients to improve sleep and anxiety symptoms. In addition, QuanXie, a component of P1 can be used to prevent cancer metastasis and improve the poor prognosis [granati pericarpium) stops diarrhea and bleeding, so it may improve the adverse effect of oxaliplatin. P3 may be the core prescription for patients during the recovery period. The use of Qi-tonifying and spleen-invigorating herbs, such as DangShen, ShanZha, and ChenPi, in P3, helps improve the gastrointestinal function of convalescent patients and promotes recovery.We analyzed herbal prescriptions for patients in different subgroups , and compared them with P1, P2, and P3. P1 may be the core prescription for patients during the progression and recurrence period. Patients in the progression and recurrence period may be anxious, be irritable, and have insomnia because of the disease progression and recurrence, and HeHuanPi  genes were common to the prescriptions. IL is an important immune and anti-inflammatory cytokine controlling cell growth, differentiation, and migration. IL-6 is a key proinflammatory cytokine within the gut , and it κB activity in intestinal epithelial cells through the JAK-STAT signaling pathway. It is well accepted that NFκB plays a central role in inflammatory and innate immune responses, and the inhibition of NFκB activity may interfere with carcinogenesis and slows tumor development.IL-10 functions as an essential immunoregulator in inflammation and inflammation-associated cancer. IL-10 mainly blocks NFα, and VEGF in colorectal carcinoma cells. Our data suggest a significant role for Akt1 in tumor cell migration and invasion [We identified AKT1, MMP2, and MMP9 as potential targets. Some studies have found that Akt1 signaling significantly suppresses the expression of MMP2, MMP9, HIF1invasion . Akt1 paκB-signaling pathways, which play crucial roles in CRC [The molecular docking results found that quercetin, apigenin, and other flavonoids exhibit good binding energy for each target, indicating that they have potential as drug treatments, though pharmacodynamic studies are needed. Flavonoids could affect cancer risk through anti-inflammatory and anti-tumor activities, mainly by inhibiting cycloxygenase-2 (COX2) in colon cancer cells, which is associated with a reduced risk of CRC . Flavonos in CRC . Quercets in CRC , 38. In s in CRC . Lauric s in CRC . Lauric s in CRC .β1 had a significant impact on multiple genes in the tumor microenvironment. In patients with CRC, a stroma-expressed gene program enriched for TGF-β has been linked to poor prognosis and metastasis formation. Thus, TGF-β signaling plays a key role in instructing the tumor microenvironment in late-stage CRC. TGF-β, TNFα, and NFκB are capable of cooperatively mediating the development of EMT. The integrated pathway involving TGF-β/Snail with TNFα/NFκB may be the principal axis that associates cancer cells to their microenvironment during the EMT process and exerts a critical role in CRC development and prognosis, which results in a poor prognosis for patients [β1 and IL-6 are crucial cytokines for Th17 differentiation and are upregulated in the tumor tissue of patients with CRC. CRC-conditioned macrophages regulate the EMT program to enhance CRC cell migration and invasion by secreting IL-6 [From the 16 core genes identified in this study, we found 3 genes linked with the survival of rectal cancer and 5 genes related to the tumor microenvironment . Some stpatients . TGF-β1 ing IL-6 .β families of proteins are especially relevant. The AKT/HIF1α signaling pathway, which regulates the expression of VEGF to promote angiogenesis, also provides potential therapeutic opportunities for the treatment of CRC [γ agonists can regulate glucose and lipid metabolism. Current evidence indicates that obesity and overweight status are risks that contribute to CRC. PPARγ can participate in primordial cell activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. PPARG gene activity might be one of the targets of miRNA-34a and a conceivable therapeutic target for CRC [We also evaluated the impact of ATK1 and VEGFA activities on the tumor microenvironment. Angiogenesis is highly regulated by various factors involved in different signaling pathways. Among these pathways, the VEGF and the TGF-t of CRC . PPARγ a for CRC .Our results support and enhance the current understanding of the therapeutic effects of TCM “Zheng” (syndrome) on rectal cancer. We have demonstrated an effective strategy combining network pharmacology and data mining to understand the mechanisms of action of core prescriptions for patients with rectal cancer and Qi and blood deficiency syndrome. Our findings may facilitate the generation of new hypotheses to reveal the mechanisms of TCM treatment for rectal cancer.


Deep RNA sequencing experiment was employed to detect putative single nucleotide polymorphisms (SNP) in mammary epithelial cells between two diverse cattle breeds (Jersey and Kashmiri) to understand the variations in the coding regions that reflect differences in milk production traits. The low milk-producing Kashmiri cattle are being replaced by crossbreeding practices with Jersey cattle with the aim of improving milk production. However, crossbred animals are prone to infections and various other diseases resulting in unsustainable milk production. In this study, we tend to identify high-impact SNPs from Jersey and Kashmiri cows (utilizing RNA-Seq data) to delineate key pathways mediating milk production traits in both breeds. A total of 607 (442 SNPs and 169 INDELs) and 684 (464 SNPs and 220 INDELs) high-impact variants were found specific to Jersey and Kashmir cattle, respectively. Based on our results, we conclude that in Jersey cattle, genes with high-impact SNPs were enriched in nucleotide excision repair pathway, ABC transporter, and metabolic pathways like glycerolipid metabolism, pyrimidine metabolism, and amino acid synthesis . Whereas, in Kashmiri cattle, the most enriched pathways include endocytosis pathway, innate immunity pathway, antigen processing pathway, insulin resistance pathway, and signaling pathways like TGF beta and AMPK which could be a possible defense mechanism against mammary gland infections. A varied set of SNPs in both breeds, suggests a clear differentiation at the genomic level; further analysis of high-impact SNPs are required to delineate their effect on these pathways. Cow milk is an essential natural product which provides a medium for nutrients including growth and immune factors to offspring and a valued raw material for human food . It playSingle nucleotide polymorphisms markers are being rapidly used in selective breeding programmers for improving phenotypic selections of the animals through genomic selection (GS), gene-assisted selection (GAS), and marker-assisted selection (MAS) methods . It is rHigh-throughput RNA sequencing technologies have provided unprecedented prospects in functional and comparative genomic research, including gene expression, genome annotation and pathway analysis, non-coding RNA discovery, and SNP detection and profiling . It has Kashmiri and Jersey cattle are two important milk animals of the Indian northern state, Kashmir which contributes significantly to the total milk production in the state. Kashmiri indigenous cattle are small, hardy, and well adapted to the hilly areas of this region and differ greatly from Jersey cattle in dairy production traits. Whereas Jersey is a well-established exotic dairy breed which has been utilized in crossbreeding programs to enhance the milk production capability of Kashmiri cattle .In the present study, we used the transcriptome data of Jersey and Kashmiri cattle with the objective to identify the single nucleotide polymorphism in coding regions that were associated with milk production traits. Additionally, the study is aimed to benefit the marker-assisted selection programs by cataloging the differential SNP profile for the improvement of milk production in Kashmiri indigenous cattle.SRR6324372. The samples were obtained at three different lactation stages from three Jersey and three Kashmiri cattle’s breed in similar conditions at Sher-e-Kashmir University of Agricultural Sciences and Technology dairy farm, Mountain Livestock Research Institute (MLRI), Kashmir, India. The sample collection from the healthy animals including sample preparation and RNA sequencing were explained in the study by Transcriptome data of the Jersey and Kashmiri cattle were downloaded from NCBI SRA database with accession number Bovine genetic variants from dbSNP 2.0 build 153 dated: Aug 8 2019 were incorporated in SNP calling to populate the RS_ID column of the known SNPs. Filtering  of generated variants and annotation were performed using VCFtools version 0.1.8 and SnpEff program v4.1. To further evaluate the biological significance of the genes with high-impact variations, the KEGG pathway enrichment analysis was performed using nKOBAS server version 3 .In our previous study after analyzing the inter- and intrastage gene expression profiling between Jersey and Kashmiri cattle, we observed a vast diversity in terms of gene expression while huge similarity between the breeds was also witnessed. Differentially expressed genes in both Kashmiri and Jersey were enriched for multicellular organismal process, receptor activity, catalytic activity, signal transducer activity, macromolecular complex, and developmental processes. Most of the identified pathways responsible for milk production in Jersey include JAK-STAT, p38 MAPK, and PI3 kinase whereas antioxidant genes like RPLPO and RPS28 are highly expressed in Kashmiri cattle . The preThe trimming process removed 0.21% of reads and only 4.2% of duplicated reads were rejected. At the mapping step, around 99.7% of the reads were mapped against the reference genome (Bostaurus assembly ARS-UCD1.2.99) . A totalp-value < 0.05) of signaling pathways (like AMP activated protein kinase (AMPK) and tumor growth factor (TGF) beta), insulin resistance, and high antigen processing in Kashmiri cows. Jersey cattle enrichment analysis shows genes with high-impact SNPs were involved mainly in nucleic acid  and nucleotide  metabolism pathways. Interestingly, the activity of primary immunodeficiency pathway, nucleotide excision repair pathway, glycerolipid metabolism, and phosphatidylinositol signaling pathway were significantly enriched in Jersey cattle  genes with SNPs in Kashmiri cows were mainly involved in the binding process (enzyme and ribonucleotide binding) and antigen processing  and release (exocytosis) are governed by the physiological phase of the mammary gland . Furthervia glycerolipid pathway converts glycerol into glucose thus providing sufficient energy for maintaining high milk production  and other important pathways like phosphatidylinositol signaling, Antifolate resistance, ABC transporters pathway, and nucleotide excision repair. Amino acid (AA) metabolism plays a critical role in milk production by regulating maternal endocrine status, blood flow through the lactating mammary gland, and activation of mechanistic  target rapamycin (mTOR) signaling . The higoduction .In Kashmiri cattle, we found variations in the major histocompatibility complex (MHC) region. In cattle, polymorphism in the MHC class II genes influences both the magnitude and specificity of antigen-specific T cells to various diseases like mastitis . In our We also found variations in genes regulating TGF-β  and mTOR signaling in Kashmiri cattle , 2 Bhat. SMAD4 pIt is widely accepted that mTOR is a key regulator of milk protein synthesis, and most reports were concerned with the role of AAs in the regulation of P-mTOR on Ser2448 in milk protein synthesis . mTORC1 The SNP analysis based on RNA sequencing demonstrated a clear distinction between Jersey and Kashmiri cattle in milk production traits. In Kashmiri cattle, the high-impact SNP variants were involved in adaptive immunity and resistance against mammary gland infectious diseases. Whereas, in Jersey cattle, enriched pathways were mainly involved in maintenance of lactation and production. These findings provide insights in genetic variation of the breeds that can be used for genomic selection of the animals.https://www.ncbi.nlm.nih.gov/sra/SRR6324372.Publicly available datasets were analyzed in this study. This data can be found here: SA designed the study and wrote the manuscript. BB and MY performed the data analysis. SB and SM helped in the writing of the manuscript. MR and MI helped in data interpretation and analysis. RS and NG did proofreading of the manuscript. All authors revised and approved the final version of 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.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.


Helicobacter pylori (HP)-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma has increased over the last two decades, whereas that of HP-positive gastric MALT lymphoma has decreased. Although the role of first-line antibiotics in the treatment of HP-negative gastric MALT lymphomas remains ambiguous, several case series have reported that a first-line HP eradication therapy (HPE)-like regimen could result in complete remission in a proportion of patients with localized HP-negative gastric MALT lymphoma. Previous sporadic reports have indicated that certain patients with extragastric MALT lymphoma can respond to first-line antibiotic treatment as well. These findings suggest that, in contrast to antibiotic-unresponsive tumors, antibiotic-responsive tumors may be recognized within the spectrum of HP-negative MALT lymphoma. In addition to conventional chemotherapy and immunochemotherapy, macrolide antibiotics and immunomodulatory drugs have been previously used and demonstrated to be efficacious. This article provides the spectrum and therapeutics for HP-negative MALT lymphoma.The prevalence of Helicobacter pylori (HP)-unrelated mucosa-associated lymphoid tissue (MALT) lymphoma includes the majority of extragastric MALT lymphomas and a small proportion of gastric MALT lymphomas. Although the role of first-line antibiotics in treating HP-negative gastric MALT lymphomas remains controversial, HP eradication therapy (HPE)-like regimens may result in approximately 20–30% complete remission (CR) for patients with localized HP-negative gastric MALT lymphoma. In these patients, H. heilmannii, H. bizzozeronii, and H. suis were detected in sporadic gastric biopsy specimens. Extragastric MALT lymphoma is conventionally treated with radiotherapy for localized disease and systemic chemotherapy for advanced and metastatic diseases. However, a proportion of extragastric MALT lymphomas, such as ocular adnexal lesions and small intestinal lesions, were reported to be controlled by antibiotics for Chlamydophila psittaci and Campylobacter jejuni, respectively. Some extragastric MALT lymphomas may even respond to first-line HPE. These findings suggest that some antibiotic-responsive tumors may exist in the family of HP-negative MALT lymphomas. Two mechanisms underlying the antibiotic responsiveness of HP-negative MALT lymphoma have been proposed. First, an HPE-like regimen may eradicate the antigens of unknown bacteria. Second, clarithromycin (the main component of HPE) may have direct or indirect antineoplastic effects, thus contributing to the CR of these tumors. For antibiotic-unresponsive HP-negative MALT lymphoma, high-dose macrolides and immunomodulatory drugs, such as thalidomide and lenalidomide, have reported sporadic success. Further investigation of new treatment regimens is warranted.  Helicobacter pylori (HP) infection . Ou. OuBurkhClarithromycin, a macrolide antibiotic, is commonly used as the main component of the HPE regimen ,7,8. In In addition to the direct anti-neoplastic effects of clarithromycin, several studies have demonstrated its immunomodulatory effects, including the activation of NK cells and CD8+ cells and costimulatory molecules, CD80, and alteration of cytokine production ,91. In vFurther exploration of the precise mechanisms of lymphomagenesis for this spectrum of antibiotic-responsive HP-negative MALT lymphoma is warranted because a 2-week antibiotic regimen has fewer adverse effects than conventional radiotherapy or chemotherapy.n = 11) provided similar local control, freedom from treatment failure, and OS when compared with those receiving ISRT with 30 Gy (n = 21) for treating HP-independent gastric MALT lymphoma ..n = 2; Pn = 16; lenalidomide plus rituximab, n = 34), Kiesewetter et al. showed that lenalidomide-based regimens provided an ORR of 74% (54% CR), and estimated the median PFS and the 5-year OS rate to be 72 months and 92%, respectively  in treating 18 patients with MALT lymphoma (including five with HP-negative gastric and 13 with extragastric lesions), Kiesewetter et al. showed that lenalidomide resulted in an ORR of 61.1% (six with CR and five with PR) . In theiectively  128]. I. In = 16®] 15 mg daily, day 1–21; cyclophosphamide 50 mg daily, day 1–21, each cycle every 28 days) for antibiotic-unresponsive and relapsed or refractory MALT lymphoma. Hopefully, these two ongoing trials will elucidate whether relapsed/refractory MALT lymphomas, including HP-negative MALT lymphoma, are responsive to lenalidomide-based combined immunomodulatory agent treatment.Based on the optimal response of single agents, clarithromycin or lenalidomide, the ongoing phase II study designed by the IELSG aims to explore the treatment efficacy and toxicity of combined clarithromycin and lenalidomide  for relapsed or refractory MALT lymphoma . Previous studies have reported that low-dose cyclophosphamide has immunomodulatory effects and overcomes the resistance of lenalidomide in patients with multiple myeloma (MM) ,131,132.C. jejuni, C. psittaci, and B. burgdorferi. Interestingly, sporadic reports indicate that certain patients with extragastric MALT lymphoma respond to first-line HPE. In contrast to antibiotic-unresponsive tumors, antibiotic-responsive tumors may be recognized within the spectrum of HP-negative MALT lymphomas. Further identification of unknown bacteria that participate in the lymphomagenesis, and exploration of the precise mechanisms and immune reactions of this spectrum of antibiotic-responsive HP-negative MALT lymphoma, are warranted. Based on the promising results and low toxicities of clarithromycin and lenalidomide for relapsed or refractory MALT lymphoma, further investigations of new chemotherapy-free treatment regimens with high efficacy and minimal adverse effects for antibiotic-unresponsive HP-negative MALT lymphoma are needed.In addition to HP-gastric MALT lymphoma, specific HP-negative gastric MALT lymphomas respond to first-line HPE. Considering that most patients with gastric MALT lymphoma present with localized disease and an indolent clinical course, the administration of HPE as first-line treatment for newly diagnosed gastric MALT lymphoma without life-threatening emergencies is recommended, because HPE not only eradicates HP and NHPH, but also diminishes antigen stimuli or triggers an immune response against lymphoma cells. Although conventional therapy remains the standard treatment for HP-negative extragastric MALT lymphoma, subtypes of extragastric MALT lymphoma are responsive to specific antibiotics against


The outbreak of Coronavirus disease 2019 (COVID-19) has become an international public health crisis, and the number of cases with dengue co-infection has raised concerns. Unfortunately, treatment options are currently limited or even unavailable. Thus, the aim of our study was to explore the underlying mechanisms and identify potential therapeutic targets for co-infection.To further understand the mechanisms underlying co-infection, we used a series of bioinformatics analyses to build host factor interaction networks and elucidate biological process and molecular function categories, pathway activity, tissue-specific enrichment, and potential therapeutic agents.We explored the pathologic mechanisms of COVID-19 and dengue co-infection, including predisposing genes, significant pathways, biological functions, and possible drugs for intervention. In total, 460 shared host factors were collected; among them, CCL4 and AhR targets were important. To further analyze biological functions, we created a protein-protein interaction (PPI) network and performed Molecular Complex Detection (MCODE) analysis. In addition, common signaling pathways were acquired, and the toll-like receptor and NOD-like receptor signaling pathways exerted a significant effect on the interaction. Upregulated genes were identified based on the activity score of dysregulated genes, such as IL-1, Hippo, and TNF-α. We also conducted tissue-specific enrichment analysis and found ICAM-1 and CCL2 to be highly expressed in the lung. Finally, candidate drugs were screened, including resveratrol, genistein, and dexamethasone.This study probes host factor interaction networks for COVID-19 and dengue and provides potential drugs for clinical practice. Although the findings need to be verified, they contribute to the treatment of co-infection and the management of respiratory disease. There are an estimated 100–400 million infections each year, and approximately half of the world’s population is now at risk of contracting the disease.Dengue, a mosquito-borne viral infection caused by four dengue virus serotypes (DENV 1-4), is found in tropical and subtropical climate areas, mainly in urban and semiurban regions . AccordiThere is some cross-reactivity and common pathological processes, such as capillary leakage, thrombocytopenia, and coagulopathy, between SARS-CoV-2 and DENV , 4, makiSerological overlap makes it difficult to identify symptoms, and co-infection complicates each disease. The COVID-19 pandemic and dengue fever recurrence epidemic in tropical countries have become global health issues. One case of co-infection has been reported in Mayotte, a French island in the Indian Ocean . Due to It is necessary to prepare for dengue outbreaks immediately under the premise of controlling the COVID-19 pandemic. The commissioners of Lancet Commission on Dengue and Arboviral Diseases announced that an approach should be adopted for the prevention and detection of dengue and other arboviral diseases during the COVID-19 pandemic in tropical and subtropical regions . Co-infeIn our study, we compared SARS-CoV-2 and DENV by bioinformatics analysis and obtained 460 common core targets and 30 common critical pathways through Gene Ontology and KEGG enrichment to identify host factor interaction networks between the two viruses. More in-depth and more comprehensive analyses of enormous amounts of data on RNA viruses such as SARS-CoV-2 and DENV can reveal the molecular mechanisms by which viruses infect hosts and can also help in designing therapies.https://www.ncbi.nlm.nih.gov/geo). GEO is an international public repository that archives and freely distributes microarrays, next-generation sequencing, and other forms of high-throughput functional genomics data submitted by the research community. The GSE147507 dataset involves the transcriptional response to SARS-CoV-2 infection by expression profiling with high-throughput sequencing and detailed transcriptomic analysis of blood, lung, and bronchus samples from COVID-19 cases  database (19 cases . The GSE19 cases . We obtahttps://pubchem.ncbi.nlm.nih.gov/#query=covid-19), Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/), and DisGeNET (https://www.disgenet.org/covid/diseases/summary/). PubChem is a public repository for archiving biological data of small molecules obtained through high-throughput RNA interference screening, aimed at identifying critical genes responsible for a biological process or disease condition , DisGeNET (https://www.disgenet.org/), and GeneCards (https://www.genecards.org/) at the same time and screened the top 500 hits in the three databases. Among them, the GSE38246  and input into the STRING website  (https://cytoscape.org/) (http://apps.cytoscape.org/apps/cytohubba) (https://metascape.org/) (Common host factors between COVID-19- and dengue-related targets were identified using the web tools provided by the Van de Peer Lab (db.org/)  to generpe.org/) , 27. Cyttohubba)  was applpe.org/)  and expohttp://bioinformatics.sdstate.edu/go/) was used for WIKI pathway enrichment analysis.We used R software to perform Gene Ontology and KEGG pathway enrichment analyses of genes in common host factor interaction networks. We chose a two-sided hypergeometric test and Bonferroni step down for P-value correction (P < 0.05). The ShinyGO v0.61 platform (https://speed2.sys-bio.net/) (SPEED2 (io.net/)  is a sighttps://www.gtexportal.org/home/), which will help researchers understand inherited susceptibility to disease by identifying regions of the genome that influence whether and how much a gene is expressed (Genotype-Tissue Expression (GTEx) was performed by using the GTEx Project . We used STITCH to predict related drugs that might provide intervention or treatment for these diseases.STITCH  integrates information about drug-target relationship information for over 68,000 different chemicals, including 2,200 drugs, and connects them to 1.5 million genes , c-JUN seems to protect coronavirus-infected cells from apoptosis . InteresIn total, 11 different plates were obtained with the MCODE module in analysis of 460 common host factors. The main function reflected by plate B was interferon (IFN) signaling.Both SARS-CoV-2 and DENV can promote activation of immune cells, leading to the release of proinflammatory cytokines, such as IFN, and causing capillary leakage, thrombocytopenia, and coagulopathy, pathophysiological similarities between COVID-19 and dengue . In a stTo investigate interaction networks at the molecular level, functional enrichment analysis, including functional annotations of significant factors and pathway enrichment analysis, was carried out. GO was utilized to classify common factors. In this study, the classification was executed according to biological process and molecular function categories. Some targets were identified as being associated with the response to stress, response to an organic substance, response to cytokine in biological processes, and response to signaling receptor binding, identical protein binding, enzyme binding, and cytokine receptor binding in molecular functions. The cytokine response was significant.SARS-CoV-2 infection gradually induces an excessive inflammatory response and dysregulated immune defense, which results in tissue damage and causes acute lung injury (ALI) and even ARDS. The mechanism may be due to the massive release of cytokines and chemokines, which might be a characteristic of COVID-19 . The cytThe pathogenesis of dengue includes cytokine storms, vascular leakage, complement activation, autoimmunity, and antibody-dependent enhancement. Through research on patients with dengue hemorrhagic fever or dengue shock syndrome, it was observed that cytokines are produced and change rapidly during the course of the illness. T cells, macrophages, monocytes, and mast cells can produce several soluble factors, such as TNFα, IL-6, IL-8, and IL-10, to increase the vascular permeability of primary endothelial cells .Through KEGG and WIKI, we analyzed the biological function of pathways in host factor interaction networks, such as influenza A, measles, and hepatitis B in KEGG and the TLR signaling pathway, regulation of Toll-like receptor signaling pathway, and spinal cord injury in WIKI.via ACE2, SARS-CoV-2 RNA is detected by TLRs .WZ conceived and designed the study and plotted the figures based on network bioinformatics, using online databases. HW and CL conducted data analysis and performed literature searches. QY, TW, and PW carried out literature searches and studies of the background of the disease. XL, YJ, and SZ reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by grants from the “Double First-Class” and High-level University Discipline Collaborative Innovation Team Project of Guangzhou University of Chinese Medicine (Grant No.2021XK16), Guangdong Provincial Department of Education Innovation Team Project (Grant No: 2018KCXTD007), the Key-Area Research and Development Program of Guangdong Province (Grant No. 2020B1111100002), the National Natural Science Foundation of China (Grant No. 81973814 and No. 81904132), the Natural Science Foundation of Guangdong Province (Grant No. 2017A030310129), the Natural Science Foundation of Guangdong Province (Grant No. 2020A1515010589), the Liu Xiaohong Famous Traditional Chinese Medicine Inheritance Studio from the Traditional Chinese Medicine Bureau of Guangdong Province (Grant No. 201805), the Construction Project of Respiratory Department National Clinical Medical Research Center (Grant No. 2110200309), 2018 Guangzhou University of Chinese Medicine National University Student Innovation and Entrepreneurship Training Project (Grant No. 201810572038), 2020 National College Student Innovation and Entrepreneurship Training Project of Guangzhou University of Chinese Medicine (Grant No. 202010572001), the Student Learning Team Incubation Project of Innovation Academy from The First Affiliated Hospital of Guangzhou University of Chinese Medicine (Grant No. 2018XXTD003), and the Technology Research of COVID-19 Treatment and Prevention and Special Project of Traditional Chinese Medicine Application-Research on the platform construction for the prevention and treatment of viral infectious diseases with traditional Chinese medicine (Grant No. 2020KJCX-KTYJ-130).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.


Anisomeles indica (L.) Kuntze, using in silico molecular docking tools and compared it to specific inhibitors of the members of the MTSCPPC signature. Single-cell transcriptomic analysis of the public databases revealed high expression levels of the MTSCPPC signature in immune cells of adult human hearts during an MI event. The MTSCPPC signature was significantly associated with the cytokine–cytokine receptor interactions, chemokine signaling, immune and inflammatory responses, and metabolic dysregulation in MI. Analysis of a micro (mi)RNA regulatory network of the MTSCPPC signature suggested post-transcriptional activation and the roles of miRNAs in the pathology of MI. Our molecular docking analysis suggested a higher potential for ovatodiolide to target MAN2A2, CSNK1D, and TNFRSF12A. Collectively, the results derived from the present study further advance our understanding of the complex regulatory mechanisms of MI and provide a potential MI theranostic signature with ovatodiolide as a therapeutic candidate.Myocardial infarction (MI) is a multifactorial global disease, recognized as one of the leading causes of cardiovascular morbidity and mortality. Timely and correct diagnoses and effective treatments could significantly reduce incidence of complications and improve patient prognoses. In this study, seven unconventional differentially expressed genes (DEGs)  were identified through integrating DEGs from six MI microarray datasets. The pathological and theranostic roles of the MTSCPPC signature in MI were subsequently analyzed. We evaluated interactions of the MTSCPPC signature with ovatodiolide, a bioactive compound isolated from  Myocardial infarction (MI) is a multifactorial global disease, recognized as one of the leading causes of cardiovascular morbidity and mortality in both men and women . It is cTimely and correct diagnoses and effective treatments can significantly reduce incidence of complications and improve the prognosis of patients with MI . The curAlthough myocardial injury  is a prerequisite for the diagnosis of MI, the presence or absence of nonischemic myocardial injury must be confirmed in order to establish a reliable diagnosis . Hence, Despite increased understanding of the causes and treatments of MI, high rates of morbidity and mortality still remain . AlthougBioinformatics or in silico analysis of clinical data, novel targets, and drug candidate identification are indispensable parts of modern research strategies for disease prevention and treatment . TargetsAnisomeles indica (L.) Kuntze, that is commonly and traditionally used for treating inflammation-associated diseases [Natural products are rich sources of health-promoting bioactive compounds for treating numerous diseases ,25,26,27diseases . The thediseases ,32,33,34diseases  and can diseases . Howeverdiseases ,38. Hencp-value combination analysis, respectively , CAV1 (23 node degrees), EGF (22 node degrees), CD44 (22 node degrees), and ANXA2 (19 node degrees)  B. On thedegrees) C and genGenomic aberrations are major drivers of disease progression and therapy response. We queried the gene–disease association network of the DEGs. Our analysis suggested the involvement of DEGs in several diseases including ischemic stroke, myocardial ischemia, atherosclerosis, acute coronary syndrome, carotid atherosclerosis, diabetic maculopathy, and so on A. To invIn order to further understand the mechanisms and pathological roles of the MTSCPPC signature, we queried the miRNA regulation network targets for this signature A. Our anWe queried the MTSCPPC signature for the available clinical/preclinical drug targets A–D and iAnnual cases of myocardial infarction  are steadily increasing. Exploring molecular mechanisms of MI is critical to controlling the number of patients with MI and developing precision therapeutic strategies. This study analyzed DEGs from six independent microarray datasets and identified seven overlapping genes (termed the MTSCPPC signature), subsequently analyzed for their pathogenic roles in MI and as therapeutic targets. GO biological and function enrichment analyses identified that the MTSCPPC signature was mainly enriched in several metabolic- (particularly carbohydrate metabolic pathways), hormonal-, immune-, and inflammation-related pathways. Similarly, our KEGG pathway analysis identified cytokine–cytokine receptor interactions, Toll-like receptor signaling, GnRH secretion, gap junctions, ECM-receptor interactions, complement and coagulation cascades, HIF-1, AMPK, Apelin, Hippo, Hedgehog, chemokine signaling, and various sugar-metabolism pathways. Results of these enrichment analyses are in line with higher levels of the MTSCPPC signature in the immune cells than in other cells of the human heart during an MI event, as revealed by our analysis from single-cell transcriptomic databases A. These Results of our analysis may provide future directions for better diagnoses and treatments of AMI. Innate immune responses and inflammatory reactions are essential regulators of tissue damage and repair after an MI . MyocardWe further investigated interactions between proteins encoded by the MTSCPPC signature via a PPI network and found a significantly higher degree of interactions with EGFR, CAV1, EGF, CD44, and ANXA2. Therefore, we speculated that these genes, which were core genes in the PPI network, might play important roles in regulating cardiac remodeling and be more closely related to MI. Our literature survey revealed that these genes were implicated in immune differentiation, regulation of inflammation, and MI ,46,47, wIntegrating these genes with the MTSCPPC signature of MI would produce a significantly pronounced pathological phenotype compared to phenotypes of individual genes ,50. Per Increasing evidence has revealed that dysfunction of 3′- and 5′-untranslated regions (UTRs) of mRNAs is often associated with the pathophysiology of several diseases . miRNAs In silico receptor–ligand interaction studies are an important stage in drug discovery and development pipelines. By studying receptor–ligand interactions, behaviors of therapeutic agents around the accommodating cavity of a target protein can be known, and hence we can speculate on the biological activities of drug candidate ,60 InterHowever, the limitations of the present study merit discussion. The sample size was relatively small as only a few datasets of MI from the GEO database qualified for inclusion. Therefore, larger cohorts of patients with MI are required to confirm the diagnostic relevance of the identified genes. In addition, no validation experiment was performed. In future studies, we plan to perform experiments such as qPCR and Western blot to verify the expression changes of MAN2A2, TNFRSF12A, SPP1, CSNK1D, PLAUR, PFKFB3, and CXCL16, as well as microRNAs that might target the DEGs in experimental models of MI and clinical samples. As we described, the DEGs were associated with metabolic and immune-inflammatory dysregulation. Whether these screened biomarkers are linked to the above indicators in MI patients also requires confirmation. In-depth functional studies including gene over-expressing/silencing and clinical studies such as correlations analysis between gene expression and clinical indicators in MI patients will also be conducted. Furthermore, the full therapeutic potential of ovatodiolide for targeting the DEGs awaits our experimental validation with in vitro and in vivo models.p-value adjustment of the false discovery rate (FDR) as reported previously [p-value analysis was adjusted for multiple testing by the FDR (<0.05) method. All the necessary codes and files to repeat the main analysis are presented in the In total, six datasets  [eviously . The coreviously . Therefoeviously . All ourWe used the Human Protein Atlas (HPA) database to acquire the subcellular localization of the DEGs based on immunofluorescence staining of the proteins within the nucleus, endoplasmic reticulum (ER), and microtubules . In addip < 0.05. Protein–protein interaction (PPI) enrichment of the DEGs was analyzed using the Multiple Protein modules of the String server [A DEG enrichment analysis including the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) enrichment analyses of the DEGs was conducted using the Enrichr server ,72, withg server , while gg server . In ordeg server .A drug-sensitivity analysis of the DEGs was conducted through the GSCALite server . We explp-value of 0.05 and a minimum required hit of four miRNAs.miRNA regulatory networks of the DEGs were collected from experimentally verified databases  and predicted databases (miRanda and targetscan). The miRNA regulatory network was visualized using the visNetwork R package. We used the miRNA Enrichment Analysis and Annotation (miEAA) tool to conduct a functional enrichment analysis of sets of miRNA targets . AnalyseWe used the DGIDB database, a drug–gene interaction database  and ConnThe present study identified MAN2A2/TNFRSF12A/SPP1/CSNK1D/PLAUR/PFKFB3/CXCL16 as a novel and potential signature of cytokine–cytokine receptor interactions, chemokine signaling, immune and inflammatory responses, and metabolic dysregulation associated with MI. Based on the molecular docking of receptor–ligand interaction, ovatodiolide demonstrated the potential for targeting this signature and could be considered for subsequent experimental validation for the development of a new drug.


Immune checkpoint inhibitors (ICIs) have changed the treatment paradigm of metastatic urothelial carcinoma (mUC), a dominant type of bladder cancer (BC). Previous studies have shown an association between gene mutations in the DNA damage response (DDR) pathway and the immunotherapy response in mUC but have neglected the effect of the activation level of the DDR pathway on the ICI response in mUC. A published immunotherapy cohort with genome, transcriptome and survival data for 348 mUC patients was used. An external cohort (The Cancer Genome Atlas Bladder Cancer) and the GSE78220 cohort were used for validation. The activation level of the DDR pathway was quantified using single-sample gene set enrichment analysis (ssGSEA). Further analysis on the genome, immunogenicity, and the immune microenvironment was conducted using the DDR ssGSEA enrichment score-high (DSSH) group and the DDR ssGSEA enrichment score-low (DSSL) group. In the mUC cohorts, the DSSH group was associated with longer overall survival times . The DSSH group was also associated with higher tumor mutation burden, neoantigen load, immune-activated cell patterns, and immune-related gene expression levels. The GSEA results indicated an immune activation state in DSSH group, which correlated with a down-regulation in the transforming growth factor β receptor signaling pathway. Our study suggests that the activation level of the DDR pathway may be a novel predictive marker for immunotherapy efficacy in patients with mUC. Bladder cancer (BC) originates from the urinary tract system and is the 10th most common tumor worldwide; its incidence in men is significantly higher than that in women . The firIn previous studies, a series of corresponding biomarkers, including TMB, tumor neoantigen load (NAL), PD-L1 expression level, microsatellite instability (MSI), tumor microenvironment (TME) markers and T cell gene expression profiles (GEPs) , 10, havThe DNA damage response (DDR) pathway is a monitoring mechanism in the cell that is activated and plays a corresponding role when DNA damage occurs to maintain the integrity and stability of the cell genome . GeneralThe DDR pathway plays an important role in the occurrence and evolution of UC and is closely related to the clinical prognosis of UC patients. Teo et al. found that in patients with advanced BC treated with cisplatin, gene mutations in the DDR pathway are associated with a favorable clinical prognosis . In receIn view of the information above, we mainly analyzed how the activation level of the DDR pathway affects the efficacy of ICI therapy in UC patients from the aspects of the tumor genome, tumor immunogenicity, and the tumor immune microenvironment. According to the results of this study, a high activation level of the DDR pathway indicates a favorable prognosis and immune response in UC patients receiving ICI treatment and may serve as a biomarker for predicting immunotherapy response.http://research-pub.gene.com/IMvigor210CoreBiologies/ were downloaded via the IMvigor210CoreBiologies package  Xena database  group and the DDR ssGSEA ES-low (DSSL) group in terms of TMB and NAL. We also performed the above comparison in the sub-pathway-related groups. TMB was calculated according to the practice of Chalmers et al. , which dimmune characteristics of UC, we first used the DESeq2 package to perform differential gene analysis between the DSSH and DSSL groups in the TCGA-BLCA cohort  database . The genhttps://www.r-project.org/; version 4.0.0).For comparisons of factors such as overall survival (OS), TMB, NAL, immune cell fraction and abundance between the DSSH and DSSL groups, we used the Mann-Whitney U test. Fisher’s exact test and Chi-square test were used to analyze the contingency table. We utilized Spearman’s correlation coefficient for the correlation analysis. The KM method and the log-rank test were applied in the survival analysis. When performing the survival analysis, the survminer package was used to calculate the cutoff of each cohort according to the relationship between the survival result and the ES of each pathways with the maximally selected rank statistics . The medThe flowchart of our work is presented in To evaluate whether the activation level of the DDR pathway can predict the prognosis of mUC patients and the efficacy of ICI treatment, we first grouped the mUC cohorts according to the ES of the DDR-related pathways and performed KM survival analysis. mUC patients with a high level of the DDR pathway activation showed a significantly longer OS time than those with a low activation level  4A. MultAccording to the response to treatment, mUC patients were divided into responder  and non-responder  groups. In the analysis of clinicopathological characteristics, there were more ICI responders, and better best overall response (BOR) in the DSSH group. Higher PD-L1 expression in tumor cells (TC) or immune cells (IC), and more inflamed phenotypes were also allocated in the DSSH group. These characteristics indicate that patients in DSSH group may show better anti-tumor immune response   fraction (p=0.024) and a lower stromal fraction (p=0.0071) than the DSSL group  (Although the discovery of ICIs has changed the treatment of mUC patients, only a small number of patients completely or partially respond to ICIs and benefit from them. Moreover, immunotherapy use is accompanied by a risk of irAEs. Therefore, to achieve better antitumor immune responses from ICI treatment and obtain better prognostic results, predictive markers are urgently required. In this study, through survival analysis, we found that in the mUC cohort treated with ICIs, a high activation level of the DDR pathway can improve the outcome of ICI treatment and the prognosis of mUC patients. This result indicated that the activation level of the DDR pathway can function as a biomarker for ICI treatment in mUC patients to some extent. In the TCGA-BLCA samples, the information was insufficient to classify patients according to treatment. It may be that the activation of the DDR pathway can predict the prognosis of mUC samples treated with atezolizumab but has no predictive effect on the prognosis of primary tumor samples in the TCGA-BLCA cohort receiving conventional chemotherapy and radiotherapy. Notably, the DSSH group had more TP53 mutations than the DSSL group; Andrea Necchi et al. noted that TP53 mutations are associated with higher levels of leukocyte infiltration at the pancancer level , suggestmUC with activation of the DDR pathway was characterized by higher immunogenicity. This feature was mainly manifested as a significant increase in TMB and NAL in mUC. In addition, a positive correlation between the activation level of the DDR pathway and the number of segments, the fraction altered, the aneuploidy score, and the HR defect score indicated the instability of the tumor genome. The GSEA results also showed that cell cycle-related pathways and DNA synthesis pathways, especially those that occur during DNA repair, were significantly enriched in the DSSH group. Therefore, a higher activation level of the DDR pathway was highly associated with a higher mutation burden. Further studies showed that activation of the DDR pathway induces an antitumor immune microenvironment. In the DSSH group, which has a higher TIL fraction, some immune-activating genes were upregulated, while some immunosuppressive genes were downregulated. Moreover, a high activation level of the DDR pathway was accompanied by a significant increase in the abundance of activated CD4 cells and a significant decrease in the abundance of Treg cells. Based on previous studies, CD4+ T cells are related to the production of effective antitumor immune responses, while Treg cells inhibit the antitumor immune microenvironment by secreting cytokines IL10 and TGFβ in the tumor microenvironment . TherefoAdditionally, our study demonstrated that the DSSH group had a significantly reduced response to TGFβ compared with that in the DSSL group and also had downregulated expression of TGFB1. Through GSEA of the two cohorts, we found that the activation level of the TGFβ receptor signaling pathway and other related pathways was significantly downregulated in the DSSH group. The TGFβ signaling pathway plays a regulatory role in many biological processes, including cell growth, cell differentiation, apoptosis, metastasis, and cancer evolution . AlthougCollectively, our study demonstrates the specific value of DDR pathway in predicting the efficacy of ICI in patients with mUC from the perspective of transcriptome. Compared with previous studies, which predict the efficacy of ICI from mutations in the DDR pathway , our stuin vivo and in vitro validation. Because cohorts of mUC patients that have received immunotherapy and have available follow-up data, gene expression data, and genome data are rather rare, it was difficult for us to verify our findings in mUC patients who have received ICI treatment in our hospital. Therefore, we used the metastatic melanoma cohort to observe whether DDR ES can predict the outcome of ICI treatment in other cancers. We hope to perform corresponding cell experiments or animal experiments in the future to determine the influence of the activation level of the DDR pathway on the immune microenvironment of mUC and to clarify the detailed mechanism underlying the efficacy of ICI treatment. Finally, this study did not find a uniform cutoff value, but used the median of the ES of the DDR pathway and DDR related pathways to group patients; we hope to find a more consistent cutoff in future research.Although we successfully noted that the activation level of the DDR pathway may be an effective predictor of ICI treatment response in mUC patients, our research still has some limitations. First, we have not validated our findings in mUC patients who have received immunotherapy. Second, the results of functional enrichment analysis need further Our study provided evidence that the activation level of the DDR pathway is associated with longer OS times and with known immunotherapy response markers, including the TMB, NAL, immune-related genes, and the abundance of specific immune cells. In addition, the TGFβ pathway is in a state of low expression when DDR pathway is activated. All of these factors contribute to a better response to ICIs in mUC. Therefore, the state of the DDR pathway could be a predictive biomarker for ICIs. A series of prospective clinical studies and molecular mechanistic explorations are needed to support our findings.The original contributions presented in the study are included in the article/Conceptualization: JZ and PL. Formal analysis: CZ, AL, and MC. Resources: CZ, JZ, and PL. Software: CZ, AL, WD, and MC. Supervision: JZ and PL. Visualization: CZ, AL, and WD. Writing–original draft: CZ, AL, and MC. Writing–review and editing: CZ, AL, WD, WM, NG, ZC and MC. 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.


Circulating tumour DNA profiling can cost-efficiently accelerate biomarker discovery within oncology trials. However, biomarker identification in metastatic castration-resistant prostate cancer is confounded by a heterogeneous genomic landscape with few commonly-perturbed genes and a large number of infrequently mutated, yet potentially biologically-relevant, cancer drivers. Hence, large sample sizes are required for the stratified evaluation of these infrequent perturbations. To circumvent this issue, we investigated whether grouping genomic alterations with other events within the same cellular pathways would offer increased precision for biomarker discovery. We undertook an individual patient-level pooled analysis of 342 patients with metastatic castration-resistant prostate cancer-initiating abiraterone acetate or enzalutamide. We found that the total number of altered pathways, which we termed the pathway complexity index (PCI) was associated with a poor prognosis. Since genomic profiling is now standard practice in interventional oncology trials, our findings highlight the importance of comprehensive genomic profiling for biomarker discovery and utilization.n = 342). We associated the detected hotspot, pathogenic, and/or high impact protein function-affecting perturbations in 39 genes into 13 pathways. Progression-free (PFS) and overall survival (OS) were analysed using Kaplan–Meier curves and multivariate Cox regression models. Driver gene alterations were detected in 192/342 (56.1%) evaluable patients. An increased number of affected pathways, coined pathway complexity index (PCI), resulted in a decremental PFS and OS, and was independently associated with prognosis once ≥3 pathway or gene classes were affected (PFS HR (95%CI): 1.7 (1.02–2.84), p = 0.04, and OS HR (95%CI): 2.5 (1.06–5.71), p = 0.04). Additionally, visceral disease and baseline PSA and plasma ctDNA levels were independently associated with poor prognosis. Elevated PCI is associated with poor ARSi outcome and supports comprehensive genomic profiling to better infer mCRPC prognosis.Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease, characterized by common and rare driver gene alterations that provide a selective growth advantage for progressing tumour cells. We hypothesized that the number of distinct gene driver alteration-affected pathways or gene classes was associated with poor prognosis in patients initiating androgen receptor signalling inhibitors (ARSi). We performed a post hoc analysis of an amalgamated baseline circulating tumour DNA (ctDNA) mutational landscape dataset of ARSi-treated men with mCRPC ( TP53 alterations can stratify mCRPC patients on androgen receptor signalling inhibitors (ARSi) into clinically relevant prognostic groups [Comprehensive genomic profiling of metastatic castration-resistant prostate cancer (mCRPC) has demonstrated extensive inter-patient heterogeneity, with the identification of relatively few commonly perturbed genes and a large number of infrequently mutated cancer drivers in the so-called “long tail” . Consequc groups ,5. Thus,TP53 perturbations and ARSi prognosis was the high (24.8%) frequency of TP53-altered patients [TP53 is the most frequently mutated gene in cancer, alterations in TP53 have the same basic consequence as uncommonly altered oncogenic genes; namely to provide a selective growth advantage. Most cancers harbour alterations in a handful of driver genes, but heterogeneity exists. The known drivers of cancer, commonly or uncommonly altered, are all associated with a limited number of cellular signalling pathways  which in turn affect core cellular processes, resulting in increased cancer cell fitness [A prerequisite for identifying an association between  fitness .Although the sizes of currently existing datasets prevent clinical evaluation of genes in the long tail, we hypothesized that an increase in the number of perturbed distinct pathways in an individual cancer, contributed to by common and uncommon driver genes, will lead to an increase in biological versatility. Therefore, we investigated if the number of altered pathways, coined the pathway complexity index (PCI), was associated with prognosis in men with mCRPC starting ARSi.n = 342), recruited between 2014 and 2017, was assembled using publicly available datasets  [n = 200) [p < 0.001) in the CORE/PROBIO cohort compared with the VANC cohort. However, when comparing clinical features within the subgroup of treatment-naive patients (n = 277) from both cohorts, no differences were observed (p = 0.23) and overall survival (OS)  estimates between the CORE/PROBIO and VANC cohorts respectively   and the observed . Overallectively . NonetheAR , TP53 , PTEN , FOXA1 , and SPOP  were the most commonly perturbed genes  , TP53 gene class , the PI3K pathway , the cell cycle signalling , and DNA repair  were the most commonly perturbed pathways or gene classes  evaluable patients at baseline . Overalled genes . For the= 0.001) . When th classes . No drivAR pathway, TP53, PI3K pathway, cell cycle signalling, and DNA repair driver perturbations    . For OS, < 0.05) . The maj < 0.05) . MultivaS and OS . This waS and OS . In bothC cohort .p < 0.0001) and OS  survival curves in comparison to patients without driver pathway hits  (p = 0.04) (p = 0.04) (p < 0.001) were independently associated with inferior outcomes. Importantly, incorporating and modelling the PSA and ctDNA fraction as continuous variables within the Cox regression model (No frequency differences were observed between both cohorts (Chi-square:  = 0.10) . In mult = 0.04) B and OS  = 0.04) D prognosn = 69), which revealed that 44/69 (63.8%) patients harboured combined AR pathway and TP53 gene class perturbations, of whom 24/44 (54.5%) carried an additional perturbation within the PI3K pathway  cases). Importantly, RB1 intronic sequencing was not carried out in the plasma samples from the Vancouver cohort, which may have led to a misclassification of a subset of patients as not carrying a driver RB1 event, due to an inability of detecting inactivating GSRs. Additionally, it is unclear if Abida et al. [RB1 bi-allelic inactivation and poor overall survival using the CORE/PROBIO-cohort data clonal mutations; which likely harbour prognostic information in the context of the disease trajectory and therapeutic selection pressure , nor to  of GSRs . Finally of GSRs  and resp of GSRs . It is, nd CHEK2 . The difutations  and bialons [RB1  to be ina et al.  incorpora et al. . Althouga et al. , we wereort data . To the tasertib . The reptasertib  and the tasertib , where ln = 142) [n = 200) [AR, where any gain was annotated as a driver hit in the AR gene; and (4) Only significant genomic structural rearrangements (GSRs), resulting in loss of- or gain of protein function were kept.A baseline ctDNA mutational landscape dataset from abiraterone- or enzalutamide-treated men was assembled using publicly available datasets  ) , TP53 class (TP53), PI3K pathway , cell cycle signalling , DNA repair deficiency , WNT/b-catenin pathway , SPOP class (SPOP), chromatin modifiers , CDK12 class (CDK12), transcription factors , MAPK pathway , IDH1 subtype (IDH1), and other . The ctDNA fraction was estimated as described previously [The driver genomic alterations were grouped into pathways except for  pathway , SPOP ansubtypes , as doess [CDK12 . The patp-value < 0.05 as being considered as statistically significant.The current post hoc analysis evaluated progression-free (PFS) and overall (OS) survival as clinical endpoints. PFS was defined as time to first confirmed PSA progression, clinical and/or radiologic progression, or death from disease. Overall survival (OS) outcome was defined as the time from baseline blood sampling to death from any cause. Survival curves for the individual and the number of affected pathways or gene classes  were estimated by Kaplan–Meier (KM) analysis. Survival differences were determined using the log-rank test. The effects of pathway/gene class alterations and PCI were quantified by hazard ratios (HRs) in cohort-stratified multivariate Cox regression models, adjusted for the following covariates: baseline PSA and ctDNA levels, prior chemotherapy, prior ARSi exposure, and presence of visceral metastases. The baseline ctDNA fraction and PSA concentration were incorporated in the multivariate model as continuous variables, and were modelled in the Cox regression model using restricted cubic splines. The association of baseline ctDNA fraction and PSA concentration as continuous variables with the HRs for time-to-event outcomes (PFS and OS) were graphically presented using their median values as reference points. Differences between the two cohorts were assessed using the Mann–Whitney U (continuous variable) and Chi-square tests . All tests were performed in R (v.1.1.463), with a two-sided We demonstrate for the first time that the elevated pathway mutational complexity is independently associated with poor prognosis, which warrants comprehensive genomic profiling for the prognostication of patients with advanced prostate cancer.