Pathway Annotation Research Articles

Gene transcriptome analysis of nasal epithelial cells in chronic rhinosinusitis with nasal polyps

Objective: To identify the differentially expressed genes in nasal epithelial cells from chronic rhinosinusitis with nasal polyps (CRSwNP), and to analyze related genes which are involved in deficiency of nasal epithelial barrier in CRSwNP patients by analyzing the datasets download from the gene expression omnibus(GEO) database. Methods: The mRNA expression microarray data numbered GSE107624 (7 CRSwNP and 7 controls) and GSE69093 (13 CRSwNP and 11 controls) were downloaded from the publicly available GEO database. These two datasets were jointly analyzed to screen the differentially expressed genes in nasal epithelial cells of controls and CRSwNP patients. In the meanwhile, we further evaluated the function annotation and regulatory pathways of the differentially expressed genes. To further confirmed what we have observed, sinus tissues were collected from patients with CRSwNP (14 cases, 46.8±17.9 years) and uncinate process tissues were collected from patients with nasal septum deviation (7 cases, 23.4±2.3 years) as control group. The primary epithelial cells of nasal mucosa were cultured and the mRNA level of screened genes were measured by Q-PCR. SPSS 22.0 software was used to for statistical analysis. Results: GSE107624 dataset showed that there were 3 856 differentially genes in nasal epithelial cells between CRSwNP and control group, while there were 771 differentially expressed genes in GSE69093 dataset. Finally, 55 up-regulated genes and 3 down-regulated genes were noticed in nasal epithelial cells of CRSwNP patients in the two datasets. GO gene functional annotation analysis showed that SPTBN1, FNBP1L, VAPB and SNX1 were involved in cell adhesion function, MAP1B was participated in the formation of microtubule related complex. KEGG pathway enrichment analysis indicated that BAMBI and SIAH1 were involved in regulation of Wnt pathway, COL6A1 and EIF4E were involved in the regulation of PI3K-AKT pathway. String protein interaction network analysis assumed that MAP1B and VAPB were the core functional proteins. Among top 3 differentially expressed genes COL6A1, MAP1B and BAMBI, only MAP1B gene was increased in nasal epithelial cells of CRSwNP patients in comparison to controls. Conclusion: The increased MAP1B gene in epithelial cells of CRSwNP, as well as abnormal regulation of Wnt and PI3K-AKT signal pathways may mediate the barrier dysfunction in CRSwNP.

CPLM 4.0: an updated database with rich annotations for protein lysine modifications.

Here, we reported the compendium of protein lysine modifications (CPLM 4.0, http://cplm.biocuckoo.cn/), a data resource for various post-translational modifications (PTMs) specifically occurred at the side-chain amino group of lysine residues in proteins. From the literature and public databases, we collected 450 378 protein lysine modification (PLM) events, and combined them with the existing data of our previously developed protein lysine modification database (PLMD 3.0). In total, CPLM 4.0 contained 592 606 experimentally identified modification events on 463 156 unique lysine residues of 105 673 proteins for up to 29 types of PLMs across 219 species. Furthermore, we carefully annotated the data using the knowledge from 102 additional resources that covered 13 aspects, including variation and mutation, disease-associated information, protein-protein interaction, protein functional annotation, DNA & RNA element, protein structure, chemical-target relation, mRNA expression, protein expression/proteomics, subcellular localization, biological pathway annotation, functional domain annotation, and physicochemical property. Compared to PLMD 3.0 and other existing resources, CPLM 4.0 achieved a >2-fold increase in collection of PLM events, with a data volume of ∼45GB. We anticipate that CPLM 4.0 can serve as a more useful database for further study of PLMs.

Mechanism exploration of Gouqi-wentang formula against type 2 diabetes mellitus by phytochemistry and network pharmacology-based analysis and biological validation

BackgroundThe Gouqi-wentang formula (GQWTF) is a herbal formula used by Academician Xiao-lin Tong for the clinical treatment of T2DM. GQWTF is beneficial to qi, nourishes Yin, clears heat, and promotes fluid production, but the effective components and their mechanism of action remain unclear.MethodsThe main components of GQWTF were detected by LC–MS, and the multi-target mechanisms of GQWTF in T2DM were elucidated using network pharmacology analysis, including target prediction, protein–protein interaction network construction and analysis, Gene Ontology (GO) terms, Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway annotation, and other network construction. Finally, the efficacy of the GQWTF was verified using biological experiments.ResultsFirst, the “herb-channel tropism” network suggested that GQWTF focuses more on treating diseases by recuperating the liver, which is considered as an important insulin-sensitive organ. Subsequently, a total of 16 active ingredients in GQWTF were detected and screened, and their biological targets were predicted. Then, “compound-target” network was constructed, where enrichment analysis of GQWTF targets reflected its potential pharmacological activities. After T2DM-related target identification, 39 cross targets of GQWTF and T2DM were obtained, and 30 key targets highly responsible for the beneficial effect of GQWTF on T2DM were identified by PPI analysis. GO analysis of these key targets showed that many biological processes of GQWTF in treating T2DM are key in the occurrence and development of T2DM, including components related to inflammatory/immune response, insulin, and metabolism. KEGG analysis revealed the regulation of multiple signalling pathways, such as insulin resistance, PPAR signalling pathway, FoxO signalling pathway, Fc epsilon RI signalling pathway, and pathways that influence diabetes primarily by regulating metabolism as well as other T2DM directly related pathways. Furthermore, a “formula-compound-pathway-symptom” network was constructed to represent a global view of GQWTF in the treatment of T2DM.ConclusionsThis study explored the mechanism of action of GQWTF in T2DM by multi-component and multi-target multi pathways, which could provide a theoretical basis for the development and clinical application of GQWTF.

Sulfuric Acid and Gibberellic Acid (GA3) Treatment Combined with Exposure to Cold Temperature Modulates Seed Proteins during Breaking of Dormancy to Germination in Tilia miqueliana.

Tilia miqueliana produces woody seeds that exhibit deep dormancy. In this study, we used cell biology methods, including Paraffin section determination and Coomassie brilliant blue staining, as well as proteomics-based methods, including two-dimensional electrophoresis with matrix-assisted laser desorption/ionisation tandem time-of-flight mass spectrometry (2DE-MALDI-TOF/TOF), to examine the effects of H2SO4-GA3 and cold stratification (3°C) treatment on proteins during dormancy release and germination in T. miqueliana seeds. The results revealed that during cold stratification, the area and density of proteins in the endosperm cells of H2SO4-GA3-treated seeds were significantly altered. Total protein content was continuously consumed and utilised. Storage proteins (albumin, globulin, prolamin, and glutelin) were degraded to varying degrees. Sixteen differential proteins were identified using mass spectrometry. Kyoto encyclopedia of genes (KEGG) pathway analysis revealed that the glycolysis/gluconeogenesis, secondary metabolite biosynthesis, glyoxylate and dicarboxylate metabolism, amino acid biosynthesis, and metabolic pathways were critical during dormancy release and germination. Gene ontology analysis and KEGG pathway annotation of differential proteins in the co-expression network indicated that the differential proteins are implicated in photosynthesis, glucose metabolism, biosynthesis of plant hormones, and glycolysis/gluconeogenesis. Synergistic interactions among these proteins accelerated dormancy release and germination. Therefore, H2SO4-GA3 cold stratification treatment is the best method for achieving rapid dormancy release and increasing the germination rate of T. miqueliana seeds.

Relationship between HSPA1A-regulated gene expression and alternative splicing in mouse cardiomyocytes and cardiac hypertrophy.

BackgroundCardiac hypertrophy may be classified as either physiological or pathological. Pathological hypertrophy has a complex etiology and is genetically regulated. In this study, we used a mouse model of cardiac hypertrophy to explore the mechanisms of gene regulation, in particular, modulation of the expression of target genes through transcription factor activity, regulation of immune and inflammation-associated genes and regulation of the alternative splicing of transcription factors.MethodsMouse models of pathological cardiac hypertrophy were established by transverse aortic constriction (TAC). We overexpressed HSPA1A in mouse cardiac HL-1 cells. GO and KEGG pathway annotation database was used to analyze all DEGs.ResultsThe expression of HSPA1A differed significantly between TAC + dantrolene vs. sham + dantrolene (Sham was the non-TAC group, and DMSO was the contrast agent), and TAC + DMSO vs. sham + DMSO. The RNA-binding protein Zfp36 was found to be differentially expressed between both TAC + dantrolene vs. sham + dantrolene and TAC + DMSO vs. sham + DMSO. The expression of mki67 and gm5619 was significantly different between TAC + dantrolene and TAC + DMSO. HSPA1A was found to selectively regulate the expression of non-coding RNAs related to cardiac hypertrophy, including Rn7sk and RMRP. The downregulated genes were mainly related to inflammation and the immune response. HSPA1A negatively regulated alternative splicing of Asxl2 and positively regulated alternative splicing of Runx1.ConclusionsHSPA1A was closely related to cardiac hypertrophy. Zfp36 was also related to cardiac hypertrophy. Dantrolene may delay cardiac hypertrophy and ventricular remodeling by regulating the expression of the RNA-binding protein genes mki67 and gm5619. HSPA1A positively regulated the expression of the non-coding RNAs RN7SK and RMRP while negatively regulating the expression of inflammation- and immune response-related genes. HSPA1A can play a role in cardiac hypertrophy by regulating the alternative splicing of asxl2 and runx1.

Dietary Polyphenol and Methylsulfonylmethane Supplementation Improves Immune, DAMP Signaling, and Inflammatory Responses During Recovery From All-Out Running Efforts.

Nutritional ingredients with defined mechanisms of action can be useful in the recovery of the body from the physical demands of a habitual training plan. The purpose of this study was to determine the effect of dietary supplementation with optimized curcumin, pomegranate ellagitannins, and MSM (R + MSM) on immune-associated mRNA during early recovery (i.e., up to 8 h post-exercise) following all-out running efforts (5-km, 10-km, and 21.1-km). Subjects (N = 14) were randomized to either a supplement (R + MSM) or a control group using an open label design. The study was completed over a period of 31-day prior to a scheduled half-marathon race. Venous blood samples were collected into PAXgene tubes at baseline, subsequent samples were collected at 2, 4, and 8 h after each running effort. A 574-plex mRNA Immunology Array (NanoString) was measured for each sample and ROSALIND® Advanced Analysis Software was used to examined changes in 31 annotated immune response pathways and specific mRNA changes. The greatest change in immune pathways occurred at 2 h (GSS > 3) followed by 4 h (GSS 2–3) and 8 h (GSS 1–2). R + MSM was associated with an increase in innate immunity (CAMP, LTF, TIRAP, CR1, IL1R1, CXCR1, PDCDILG2, and GNLY) and a blunted/smaller increase in damage-associated molecular pattern (DAMP) signaling/inflammation (TLR4, TLR5, S100A8, S100A9, and IFP35). We also found changes in immune-associated mRNA that have not been previously linked to exercise recovery (SOCS1, SOCS2, MME, CECAM6, MX1, IL-1R2, KLRD1, KLRK1, and LAMP3). Collectively these results demonstrate that supplementation with a combination of optimized curcumin, pomegranate ellagitannins, and methylsulfonylmethane resulted in changes that may improve biological recovery from all-out running efforts.

Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines

Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.

The Transcriptome of Schistosoma mansoni Developing Eggs Reveals Key Mediators in Pathogenesis and Life Cycle Propagation.

Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by parasite eggs becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species-Schistosoma mansoni, S. japonicum and S. haematobium-the transcriptomic profiles of developing eggs remain under unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis, days 1-3 and 3-6 post-oviposition, respectively. Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq datasets from S. mansoni eggs collected from the livers of rodent hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidial development and life cycle progression of schistosomes.

Statistical Approach of Gene Set Analysis with Quantitative Trait Loci for Crop Gene Expression Studies.

Genome-wide expression study is a powerful genomic technology to quantify expression dynamics of genes in a genome. In gene expression study, gene set analysis has become the first choice to gain insights into the underlying biology of diseases or stresses in plants. It also reduces the complexity of statistical analysis and enhances the explanatory power of the obtained results from the primary downstream differential expression analysis. The gene set analysis approaches are well developed in microarrays and RNA-seq gene expression data analysis. These approaches mainly focus on analyzing the gene sets with gene ontology or pathway annotation data. However, in plant biology, such methods may not establish any formal relationship between the genotypes and the phenotypes, as most of the traits are quantitative and controlled by polygenes. The existing Quantitative Trait Loci (QTL)-based gene set analysis approaches only focus on the over-representation analysis of the selected genes while ignoring their associated gene scores. Therefore, we developed an innovative statistical approach, GSQSeq, to analyze the gene sets with trait enriched QTL data. This approach considers the associated differential expression scores of genes while analyzing the gene sets. The performance of the developed method was tested on five different crop gene expression datasets obtained from real crop gene expression studies. Our analytical results indicated that the trait-specific analysis of gene sets was more robust and successful through the proposed approach than existing techniques. Further, the developed method provides a valuable platform for integrating the gene expression data with QTL data.

Abstract 363: Investigating the potential clinical predictive value of virus genotype, menopausal status and mutational landscape in cervical cancer tissue using a NGS based human papillomavirus (HPV) assay and whole exome sequencing (WES)

Abstract Purpose: Persistent HPV infections are associated with nearly all cervical cancers and some head & neck and genitourinary cancers. In cervical cancer, 14 high risk HPV genotypes have been previously identified with varying geographical prevalence and carcinogenicity. HPV16 and 18 account for >70% of cervical cancer incidence worldwide. There is an unmet need to explore HPV-associated cancer pathology, HPV infection status and molecular profiles to detect virus load and identify predictive gene signatures. We investigated the association between HPV genotypes and clinicopathological features and analyzed the mutational landscape using the ImmunoID NeXT Platform®. Methods: Early stage cervical cancer FFPE tissue samples (n=38) were analyzed. We detected and quantified oncoviral DNA, identified likely somatic mutations, and quantified genome-wide mutational burden. Any sample with at least one filtered read of HPV evidence was considered positive; zero such evidential reads were reported across a negative control cohort of > 439 non-cervical cancer samples. Oncovirus DNA counts and HPV genotype status were then evaluated against covariates (age; menopausal status) for statistical significance. Finally, mutation frequency was assessed in genes comprising major cervical cancer driver pathways from the KEGG and Biocarta databases. Results: Oncoviral HPV infections were detected using a NGS based approach in all tumor tissue with demonstrated robust assay performance. A statistically significant difference in viral DNA counts was observed between high risk genotypes HPV16 and 18 (n=27) and all other genotypes (n=10), (p=0.028, Student's t). Genotypes were also significantly associated with menopausal status (p=0.01, Fisher's Exact Test) and age at surgical resection (p=0.011, Student's t). Among the pathways considered, TGF-β and MAP-kinase were the most frequently mutated across 38 samples and multiple pathway annotations. Conclusions: We applied a powerful NGS based WES approach that enables accurate detection of HPV genotypes with quantification of HPV viral DNA. This quantitative approach represents a possible promising clinical benefit over existing qualitative HPV genotyping assays and may have the potential for further development to define cancer type and treatment specific cutoff values for cancer prognosis. Furthermore, our results demonstrate that a WES based approach provides clinically valuable insights into the constitutive molecular mechanisms of cervical cancer. The association of HPV genotypes with clinical patient characteristics and/or mutational profiles has the potential to identify prognostic and predictive clinical biomarkers and to provide critical information for treatment defining strategies. Citation Format: Jean-Christophe Pignon, Heidi Giese, Dorothee Foernzler, Lee D. McDaniel, Gabor Bartha, Juergen Scheuenpflug, Zheng Feng. Investigating the potential clinical predictive value of virus genotype, menopausal status and mutational landscape in cervical cancer tissue using a NGS based human papillomavirus (HPV) assay and whole exome sequencing (WES) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 363.

Integrated mRNA and miRNA Profiling Revealed Deregulation of the Cellular Stress Response in Bone Marrow Mesenchymal Stem Cells Derived from Patients with Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet destruction and decreased platelet production. Our understanding of its pathogenesis remains limited due to the complexity of the immune system and the heterogeneity of the disease (Rivière et al. Br J Haematol. 2015, 170). Recently, we found that the bone marrow mesenchymal stem cells (MSCs) derived from ITP patients (MSC-ITP) exhibited growth defects and functional abnormalities, which may be implicated in the breakdown of self-tolerance in ITP (Zhang et al. Stem Cells Transl Med.2016, 5). The underlying mechanism of these defects remains unclear. In this study, by utilizing microarray technique and bioinformatics analysis, we aimed to profile both the mRNA and microRNA expression and decipher the underlying mechanism involved in the impairment of MSC-ITP.Methods: MSCs were derived from 4 ITP patients and age-, sex- and BMI-matched healthy individuals served as control. Microarray was performed on total RNA specimens using Affymetrix mRNA and miRNA microarray.Results: Overall, we identified 740 genes and 32 microRNAs that were differently expressed in ITP patients compared with controls. Function and disease analysis revealed that the differentially expressed genes were most significantly enriched in the processes of cell death and survival, cellular development, cell growth and proliferation and cell cycle, which is in agreement with our previous study demonstrating the excessive apoptosis and senescence of MSCs from ITP patients. Ingenuity canonical pathway annotation enabled us to assign differentially expressed genes to 57 pathways. Most of these top enriched pathways have functions in cell proliferation, cell cycle and cell death. Of note, unfolded protein response (UPR), which is a defense response to protect cells from damage induced by endoplasmic reticulum stress, was identified as the most significantly changed. Furthermore, GSEA identified that UPR was negatively correlated with MSC-ITP. These data suggested that deficient UPR may play key roles in MSC-ITP. Upstream analysis revealed a significantly decreased overall DNA transcription, and many stress response transcriptional factors such as NUPR1 and TGF-b were down-regulated, indicating a potential inability of the cellular stress response. Further, we utilized gene network analysis in an attempt to target the key “cross-road” regulator in MSC-ITP. Network analysis demonstrated that JUN and CDKN1A were two key cross regulators with multiple roles in the biological process of MSC-ITP. JUN and CDKN1A were critical downstream regulators in UPR and the p53 pathway, respectively, implying a potential cross-talk between UPR and the p53 pathway, the latter of which has been verified in MSC-ITP in our previous study. Finally, we utilized microRNA-mRNA interactions analysis to improve our understanding of the regulated genes. Reactome enrichment of the predicted targeted genes of differentially expressed microRNA indicated that the cellular response to stress was deregulated in MSC-ITP. Then, the predicted target genes were cross-referenced against the mRNAs significantly regulated in MSC-ITP and MSC-control, resulting in 163 overlap genes. Again, UPR was identified as the most related process in MSC-ITP by Reactome enrichment, which further confirmed the association between UPR and the defect of MSC-ITP.Conclusion: Taken together, our results illustrate that the mRNA and microRNA profiles change in MSC-ITP, which revealed a weakened ability to initiate the cellular stress response to protect cells from damage and restore homeostasis. Considering MSCs as an important immunoregulator, these results in MSC-ITP may shed light on the complex ITP bone marrow niche and on the breakdown of self-tolerance. DisclosuresNo relevant conflicts of interest to declare.

Differentiation Response to Quizartinib, a Potent 2nd Generation FLT3 Inhibitor, in Relapsed FLT3 -Mutant AML Patients Correlates with Specific DNA Methylation Signature Prior to Therapy

Activating mutations in Fms-like tyrosine kinase 3 receptor (FLT3 ) gene occur in 30% of AML patients, and are correlated with a high risk for early relapse. The second generation FLT3 inhibitor AC-220 (quizartinib), has shown significant responses in patients with relapsed and refractory FLT3 mutant AML. For those patients who respond, two different types of pathologic responses have been observed: a cytotoxic response or a differentiation response. Given these two different responses to therapy, we hypothesized that pre-existing epigenetic programs may be responsible for determining response type, and that DNA methylation analysis of AML blasts before and after treatment may help better understand the molecular underpinnings of the varied roles of FLT3 in AML pathogenesis. For this purpose, we analyzed a cohort of 13 FLT3 -ITD mutant AML patients relapsed after standard chemotherapy and treated with quizartinib as part of the Phase II ACE trial (NCT00989261). Patients were evaluated prior to therapy and after at time of maximal response and classified as having either cytotoxic (6 patients) or differentiation (7 patients) responses as previously described. Bone marrow or peripheral blood specimens were collected from 13 patients at baseline, prior to the administration of quizartinib, and 10 matched, post-treatment specimens were available for 9 patients. DNA was extracted from mononuclear cell fractions and analyzed using multiplexed Enhanced Reduced Representation Bisulfite Sequencing (mERRBS), which captures quantitative DNA methylation status at ~3 million CpG sites across the human genome. Supervised analysis between the different groups was performed using a beta binomial model as implemented in MethylSig, with significant differentially methylated regions (DMRs) being called with methylation differences ≥25% and false discovery rates (FDR) < 0.1. Notably, paired analysis of pre- and post-treatment samples revealed very little impact of quizartinib on the DNA methylation landscape of patients with cytotoxic responses, with only 21 DMRs detected. Similarly, those who exhibited a differentiation response, showed moderate methylation changes with 84 DMRs enriched at introns compared to whole coverage of mERRBS (Background [BG] 32% vs DMRs 56%; exact binomial test [EBT] p-value 5.053e-06). Thus, FLT3 inhibition did not appear to modify DNA methylation in these patients. However, and importantly, a direct comparison at baseline, prior to the administration of quizartinib, between patients who developed either differentiation or cytotoxic response, revealed robust epigenetic differences between these two groups of patients, with 580 DMRs. The majority of these (414 DMRs, 71%) were hypermethylated in patients with cytotoxic response. These DMRs were strongly depleted from promoter regions (BG 24% vs DMRs 9%; EBT p-value 2.2x10E-16) while they were enriched at intergenic regions (BG 35% vs DMRs 49%; EBT p-value = 1.064e-10). Given the preferential location of DMRs at these distal intergenic regions, we analyzed their relationship to enhancer elements. A total of 46.2% of DMRs (n=268) overlapped with ENCODE-defined enhancers (EBT p-value 3.57x10E-13 compared to 31.8% of BG). Pathway annotation of DMRs to the nearest genes revealed that they were associated with ion transmembrane transporter activity and extracellular matrix binding. In addition, amongst the DMRs were several annotated to HOX genes, including HOXB3 , HOXA7 and HOXD13 genes, which were hypomethylated in patients with a differentiation response. In summary, our analyses reveal that pre-existing epigenetic programs are encoded in FLT3 -mutant AML cells that correlate with the type of response they will undergo upon quizartinib treatment. The precise consequences of these epigenetic differences at the transcriptional level are still being explored and will be discussed at the meeting. We hypothesize that the pathologic role of FLT3 in AML may be modified by the epigenetic state of the transformed cells. DisclosuresPerl:Arog Pharmaceuticals: Consultancy; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Pfizer: Other: Advisory Board; Astellas: Consultancy; Asana Biosciences: Other: Scientific advisory board; Daiichi Sankyo: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

Using Multilayer Heterogeneous Networks to Infer Functions of Phosphorylated Sites.

Mass spectrometry-based quantitative phosphoproteomics has become an essential approach in the study of cellular processes such as signaling. Commonly used methods to analyze phosphoproteomics datasets depend on generic, gene-centric annotations such as Gene Ontology terms, which do not account for the function of a protein in a particular phosphorylation state. Analysis of phosphoproteomics data is hampered by a lack of phosphorylated site-specific annotations. We propose a method that combines shotgun phosphoproteomics data, protein–protein interactions, and functional annotations into a heterogeneous multilayer network. Phosphorylation sites are associated to potential functions using a random walk on the heterogeneous network (RWHN) algorithm. We validated our approach against a model of the MAPK/ERK pathway and functional annotations from PhosphoSitePlus and were able to associate differentially regulated sites on the same proteins to their previously described specific functions. We further tested the algorithm on three previously published datasets and were able to reproduce their experimentally validated conclusions and to associate phosphorylation sites with known functions based on their regulatory patterns. Our approach provides a refinement of commonly used analysis methods and accurately predicts context-specific functions for sites with similar phosphorylation profiles.

Network pharmacology-based and molecular docking prediction of the active ingredients and mechanism of ZaoRenDiHuang capsules for application in insomnia treatment

BackgroundThe ZaoRenDiHuang (ZRDH) capsule is widely used in clinical practice and has significant therapeutic effects on insomnia. However, its active ingredients and mechanisms of action for insomnia remain unknown. In this study, network pharmacology was employed to elucidate the potential anti-insomnia mechanisms of ZRDH. MethodsThe potential active ingredients of ZRDH were obtained from the Traditional Chinese Medicine Systems Pharmacology Database. Possible targets were predicted using SwissTargetPrediction tools. The insomnia-related targets were identified using the therapeutic target database, Drugbank database, Online Mendelian Inheritance in Man database, and gene-disease associations database. A compound-target-disease network was constructed using Cytoscape for visualization. Additionally, the protein functional annotation and identification of signaling pathways of potential targets were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the Metascape platform. ResultsIn this study, 61 anti-insomnia components and 65 anti-insomnia targets of ZRDH were filtered through database mining. The drug-disease network was constructed with five key components. Sixty-five key targets were identified using topological analysis. Docking studies indicated that bioactive compounds could stably bind to the pockets of target proteins. Through data mining and network analysis, the GO terms and KEGG annotation suggested that the neuroactive ligand-receptor interaction, serotonergic synapse CAMP signaling, HIF−1a signaling, and toll-like receptor signaling pathways play vital roles against insomnia. ConclusionThe potential mechanisms of ZRDH treatment for insomnia involve multiple components, targets, and pathways. These findings provide a reference for further investigations into the mechanisms underlying ZRDH treatment of insomnia.

Proteomic Analysis of Histone Crotonylation Suggests Diverse Functions in Myzus persicae.

Myzus persicae is one of the most important economic pests of cultivated crops. In the present study, we used an integrated approach involving high-performance liquid chromatography fractionation, affinity enrichment, and mass spectrometry-based proteomics to carry out a comprehensive proteomic analysis of lysine crotonylation in M. persicae. Altogether, 7530 lysine crotonylation sites were identified in 2452 protein groups. Intensive bioinformatic analyses were then carried out to annotate those lysine crotonylated targets identified in terms of Gene Ontology annotation, domain annotation, subcellular localization, Kyoto Encyclopedia of Genes and Genomes pathway annotation, functional cluster analysis, etc. Analysis results showed that lysine-crotonylated proteins were involved in many biological processes, such as the amino acid metabolism, aminoacyl-tRNA biosynthesis, spliceosomes, ribosomes, and so forth. Notably, the interaction network showed that there were 199 crotonylated proteins involved in the amino acid metabolism and numerous crotonylation targets associated with fatty acid biosynthesis and degradation. The results provide a system-wide view of the entire M. persicae crotonylome and a rich data set for functional analysis of crotonylated proteins in this economically important pest, which marks an important beginning for the further research.

Multidimensional molecular differences between artificial cultivated and wild Artemisia rupestris L. based on metabolomics–transcriptomics integration strategy

Various ecological environments affect the active ingredients and molecular content of medicinal plants. Artemisia rupestris L. is a traditional medicinal plant, and shortages of the wild resource has led to the increased use of artificial cultivated varieties. However, few investigations have studied the molecular differences between the cultivated and wild varieties in a systematic manner. In this study, two A. rupestris varieties were collected in the Altay–Fuyun region, Xinjiang, China. We used a liquid chromatography-mass spectrometry based untargeted metabolomics approach to profile the metabolome in the flower, stem, and leaf samples respectively, and simultaneously analyzed the levels of a panel of representative known metabolites in the plant. The genetic basis of these samples was explored using a de novo transcriptomics approach to investigate differentially expressed genes and their pathway annotations. The results indicated that the metabolic differences between the two varieties were mainly reflected in flavonoids and chlorogenic acid/caffeic acid derivatives. Thirty-four chemical markers, including 19 potentially new compounds, of these two structural categories were identified after validation using another batch of samples. Correlation analysis combined with quantitative real-time polymerase chain reaction revealed that six differentially expressed genes in different organs were closely correlated with 24 chemical markers. This study provides novel insight into the molecular landscape of this medicinal plant.

WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization.

In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology, and the response to therapeutic drugs. Visualization of the metabolic pathways that comprise the metabolic network is extremely useful for interpreting a wide variety of experiments. Detailed annotated metabolic pathway maps for C. elegans are mostly limited to pan-organismal maps, many with incomplete or inaccurate pathway and enzyme annotations. Here, we present WormPaths, which is composed of two parts: (1) the careful manual annotation of metabolic genes into pathways, categories, and levels, and (2) 62 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on the WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the future, we envision further developing these maps to be more interactive, analogous to road maps that are available on mobile devices.

Signalling pathways and key genes involved in regulation of ovarian development of Sepia esculenta

The golden cuttlefish (Sepia esculenta Hoyle) which has one-time reproduction and die after reproduction, what changes have taken place in the ovarian development in the whole life cycle is a very noteworthy issue. In this study, we performed RNA-Seq technology of ovary tissue from female golden cuttlefish collected at three key developmental periods: growth period (IG), spawning period (IS) and post-reproductive death period (IA). Then, we obtained 66.65 Gb of clean data, with Q20 > 96%. Comparison of the transcriptome data for the IG vs. IS, IG vs. IA and IS vs. IA revealed 10,290, 17,107 and 380 differentially expressed genes (DEGs) respectively. Gene ontology (GO) enrichment analysis of DEGs revealed that oxidative phosphorylation, NADH dehydrogenase (ubiquinone) activity, respiratory chain and other GO entries were significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database alignment and annotation revealed that 317 metabolic pathways differed between IG vs. IS, 317 differed between IG vs. IA; and 161 differed between IS vs. IA. Significantly enriched pathways included oxidative phosphorylation, cell cycle, mTOR signalling pathway, AMPK signalling pathway and ubiquitin-mediated proteolytic pathway were associated and interacted, and the post-reproductive death of golden cuttlefish was regulated by a complex signalling network. This study clarified the regulation of transcripts expression in the ovary of golden cuttlefish at different developmental periods, which not only filled the blank of molecular mechanism of cephalopod ovarian development, but also provided research ideas and experimental models for solving the complex biological proposition of post-reproductive death.

PathBIX-a web server for network-based pathway annotation with adaptive null models.

Pathway annotation is a vital tool for interpreting and giving meaning to experimental data in life sciences. Numerous tools exist for this task, where the most recent generation of pathway enrichment analysis tools, network-based methods, utilize biological networks to gain a richer source of information as a basis of the analysis than merely the gene content. Network-based methods use the network crosstalk between the query gene set and the genes in known pathways, and compare this to a null model of random expectation. We developed PathBIX, a novel web application for network-based pathway analysis, based on the recently published ANUBIX algorithm which has been shown to be more accurate than previous network-based methods. The PathBIX website performs pathway annotation for 21 species, and utilizes prefetched and preprocessed network data from FunCoup 5.0 networks and pathway data from three databases: KEGG, Reactome, and WikiPathways. https://pathbix.sbc.su.se/. erik.sonnhammer@scilifelab.se. Supplementary data are available at Bioinformatics Advances online.

Predicting the Molecular Mechanism of “Angong Niuhuang Pills” in the Treatment of COVID-19 Based on Network Pharmacology

Introduction Angong Niuhuang Pills (AGNH), a Chinese patent medicine recommended in the “Diagnosis and Treatment Plan for COVID-19 (8th Edition),” may be clinically effective in treating COVID-19. The active components and signal pathways of AGNH through network pharmacology have been examined, and its potential mechanisms determined. Methods We screened the components in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) via Drug-like properties (DL) and Oral bioavailability (OB); PharmMapper and GeneCards databases were used to collect components and COVID-19 related targets; KEGG pathway annotation and GO bioinformatics analysis were based on KOBAS3.0 database; “herb-components-targets-pathways” (H-C-T-P) network and protein-protein interaction network (PPI) were constructed by Cytoscape 3.6.1 software and STRING 10.5 database; we utilized virtual molecular docking to predict the binding ability of the active components and key proteins. Results A total of 87 components and 40 targets were screened in AGNH. The molecular docking results showed that the docking scores of the top 3 active components and the targets were all greater than 90. Conclusion Through network pharmacology research, we found that moslosooflavone, oroxylin A, and salvigenin in AGNH can combine with ACE2 and 3CL, and then are involved in the MAPK and JAK-STAT signaling pathways. Finally, it is suggested that AGNH may have a role in the treatment of COVID-19.