Gene Ontology Cellular Component Research Articles

Efficacy of Danlou tablet on myocardial ischemia/ reperfusion injury assessed by network pharmacology and experimental verification.

To investigate the potential pharmacological mechanism of Danlou tablet (, DLT) with a long-term clinical application in the treatment of myocardial ischemia/reperfusion (I/R) injury through network pharmacology, molecular docking and experimental verification. The main chemical ingredients in DLT were retrieved from the Traditional Chinese Medicine (TCM) System Pharmacology Database, the TCM information database, the bioinformatics analysis tool for molecular mechanism of TCM, and HERB database. Disease targets of I/R were accessed from the databases of Online Mendelian Inheritance in Man, GeneCards, Therapeutic Target Database, and DisGeNET database. The overlaying genes of DLT and I/R were obtained from the Venny online platform. The core targets and protein-protein interaction network were constructed and analyzed via the Search Tool for the Retrieval of Interacting Genes Proteins database and Cytoscape software. Furthermore, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by the Metascape platform. Based on the results, the component-target-pathway network was constructed and drafted via the Cytoscape software and the platform of Bioinformatics. Furthermore, we performed molecular docking to predict the binding information between chemical molecules and target proteins. Finally, oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocytes were used to validate the results of network pharmacology in vitro. A total of 189 active chemical components in DLT and 849 correlative targets of I/R were screened. Of note, 133 overlaying genes found from the Venny online platform were concentrated into 28 core genes. Furthermore, the GO and KEGG pathway enrichment analysis presented that DLT might participate in 42 types of GO molecular functions, 747 types of GO biological processes, 19 types of GO cellular components, and 140 kinds of pathways to treat I/R. In the component-target-pathway network, the indirect relationship between herbs and their possible effective pathways was clarified. Based on the molecular docking, we speculated that Baicalein-prostaglandin G/H synthase 2 (PTGS2) with -3.24 kcal/mol, Luteolin-heat shock protein 90 alpha family class A member 1 (HSP90AA1) with -3.22 kcal/mol, Baicalein-HSP90AA1 with -3.13 kcal/mol, and Quercetin-HSP90AA1 with -3.05 kcal/mol possessed the strongest binding force of less than -3 kcal/mol, sequentially. Experimental verification showed that Quercetin, Luteolin, and Baicalein could increase the relative cell viability of OGD/R-stimulated cardiomyocytes, probably by suppressing PTGS2, and activating HSP90AA1 and estrogen receptor 1 expression. We predicted the potential active compounds as the material basis of DLT that may provide a new approach to elucidate the novel pharmacological mechanism underlying the treatment of cardiac I/R damage.

Transcriptomic analysis of the submandibular gland under psychological stress condition.

Psychological stress is associated with changes in salivary flow and composition. However, studies to show the effect of psychological stress on the transcriptome of the salivary gland are limited. This study aims to perform a transcriptomic analysis of the submandibular gland under psychological stress using a chronic restraint stress model of rats. Sprague-Dawley rats were divided into stress groups and control groups. Psychological stress was induced in the stress group rats by enclosing them in a plastic tube for 4 h daily over 6 weeks. RNA sequencing was performed on RNA extracted from the submandibular gland. The differentially expressed genes were identified, and the genes of interest were further validated using qRT-PCR, immunofluorescence, and western blot. A comparison between control and stress groups showed 45 differentially expressed genes. The top five altered genes in RNA sequencing data showed similar gene expression in qRT-PCR validation. The most downregulated gene in the stress group, FosB, was a gene of interest and was further validated for its protein-level expression using immunofluorescence and western blot. The genesets for gene ontology cellular component, molecular function, and KEGG showed that pathways related to ribosome biosynthesis and function were downregulated in the stress group compared to the control. Psychological stress showed transcriptomic alteration in the submandibular gland. The findings may be important in understanding stress-related oral diseases.

Unsupervised Machine Learning Identify Cis‐Regulatory Network Involved in Neurodegeneration

AbstractBackgroundCis‐Regulatory Modules (CRMs) are crucial to ensure the precise spatiotemporal gene expression, but we still lack a complete catalog of mammalian CRMs.MethodsWe developed the Mammalian Regulatory Module Detector (MrMOD), that accurately predicts a comprehensive set of high‐resolution CRMs in mouse and human genomes including enhancers, promoters, silencers, locus control regions, and microRNA regulatory modules not limited to any tissue, cell type, developmental stage, or stimuli. We validated our predictions using 19 orthogonal experimental data sets, including thousands of experimental defined CRMs and millions of putative regulatory elements from multiple databases. Next, we used an unsupervised machine learning method to annotate CRM functions. We scanned the mouse genome for transcription factor binding sites (TFBS) using position weight matrices of known transcription factors (TF) from the CIS‐BP database and obtained TFBS abundance in each CRM for every TF. Unsupervised clustering was performed using Seurat on CRMs from chromosome 17. Each CRM cluster represents a set of CRMs with similar TFBS compositions. Cluster marker genes represent TFs whose binding sites are enriched in the CRM cluster. Genes associated with the CRMs in the cluster represent the putative target genes. Functional enrichment analyses of the TFs and CRM associated genes were performed using ClusterProfiler and disgenet2r.ResultsSeurat identified a total of 43 clusters. In particular, CRM cluster #17 is linked to neurodegeneration based on five supporting evidence: 1) TF marker genes of cluster #17 include TFs known to be involved in neurodegeneration such as Spi1, Yy1, Nr4a2, Nr4a3, and Tfeb. 2) TF marker genes of cluster #17 were significantly enriched for Alzheimer’s Disease, Parkinson’s Disease, Multiple Sclerosis, among others. 3) Genes associated with cluster #17 CRMs were significantly enriched for neurodegeneration: Multiple Sclerosis and neurodegenerative disorders. Gene Ontology Cellular Components terms significantly enriched for synapse, presynapse, axon, and synaptic membrane. 4) Genes associated with cluster #17 CRMs significantly overlap the known TF target genes in the ChIP‐Atlas database defined by human ChIP‐seq experiments. 5) Top TF marker genes of cluster #17 known to activate transcription in neurons cooperatively.ConclusionsWe developed a novel approach to elucidate mechanisms involved in neurodegeneration.

219 Identification of Functional Single Nucleotide Polymorphisms Responsible for Alternative Splicing Affecting Gastrointestinal Nematodes Resistance in Grazing Sheep

Abstract Sheep infected with gastrointestinal nematodes (GIN) can show weight loss, anemia, diarrhea, decrease appetite, and hypoproteinemia, which can sometimes lead to death. These signs of infection decrease productivity and cause economic losses. Economic losses can also be associated with treatment without efficacy due to GIN resistance to most anthelmintic classes. Combined approaches need to be used to reduce the reliance on anthelmintic as the only control method to GIN. The aim of this study is the identification of potential functional single nucleotide polymorphisms (SNPs) responsible for alternate splicing in Rideau X Dorset crossbred sheep with different immune profiles using RNA-Sequencing (RNA-Seq). Animals were exposed to GIN while grazing, after that those with the greatest and least gastrointestinal tract parasite burdens were selected for liver RNA extracting. These animals can be separated into groups of high (H; n = 5) and medium (M; n = 6) innate immune responses. In addition, liver RNA was extracted for GIN-unexposed control lambs (U; n = 4). RNA-Seq analysis was performed using the CLC genomics workbench software, version 20.0.4 (CLC Bio, Aarhus, Denmark). Quality control analyses, including guanine-cytosine (GC) content, ambiguous base content, Phred score, base coverage, nucleotide contributions, and over-represented sequences parameters, were performed on fastq files. Sequence reads were aligned to the annotated Oar_rambouillet_v.1 (release 109) ovine reference genome. After filtering steps, mRNA isoforms with multiple (³ 2) expressed mRNA isoforms with at least one mRNA isoform differently expressed (DE) between H- and M-innate immune response and GIN-unexposed control groups (FDR < 0.05 and |FC| > 2) were keep. A total of 1, 75, and 58 DE mRNA isoforms were identified for M versus H, U versus M, and U versus H comparisons, respectively. Gene ontology (GO) analysis including the biological process, molecular function, and cellular component GO categories and metabolic pathways analysis were performed using STRING-db software. Enriched biological processes and metabolic pathways related to the immune system, as well as cholesterol and other sterol metabolism (e.g., sterol biosynthesis, steroid metabolism, cholesterol biosynthesis, cholesterol metabolism, ascorbate and aldarate metabolism, drug metabolism, and terpenoid biosynthesis) were identified (FDR < 0.05). Next, the list of DE mRNA isoforms identified will be deeply studied by performing SNP discovery analysis to validate if specific functional SNPs are responsible for the alternative splicing affecting GIN resistance in grazing sheep. The complete results will help to better understand the biological mechanisms that may be regulating the immune response to GIN. Candidate functional SNPs have the potential to be included in a commercial SNP panel for selection of more resistant animals to improve GIN resistance in sheep flocks.

P-155 MicroRNA-155 (miR-155) expression profile and developmental characteristics of bovine early embryos are altered when cultured in group versus individually

Abstract Study question Culturing bovine preimplantation embryos in group versus individually has an impact on the expression profile of miR-155 and developmental characteristics. Summary answer MiR-155 was significantly greater in its expression when embryos were cultured in groups versus singly. Developmental characteristics of blastocysts were similar regardless of culture condition. What is known already MiRNAs are short non-coding RNAs that play important roles in many biological pathways including embryogenesis and are potent effectors of post-transcriptional gene silencing. It has been estimated that approximately 60% of all proteins-coding genes are regulated by miRNAs. MiR-155 is involved in early events of embryogenesis; however, it has not been characterized in bovine embryos and this knowledge has not yet been clinically translated. Existing literature indicates that group culture yields superior embryo development as embryos can benefit from autocrine signaling and paracrine communication but embryos in groups may be exposed to negative effects of dying or delayed embryos. Study design, size, duration The study consisted of two groups: zygotes cultured to blastocysts in group and those cultured individually. Each sample consists of 3 blastocysts. Droplet digital PCR (dd-PCR) was used to examine the expression of miR-155. Gene Set Enrichment Analysis (GSEA) of miRNAs was carried out to identify functional Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are enriched for the targets of one or more miRNAs. Participants/materials, setting, methods Oocytes were aspirated from antral follicles of bovine ovaries and matured in vitro. Twenty-four hours post retrieval, matured oocytes were inseminated with sperm from similar bull. Presumptive zygotes were cultured either in groups or individually until blastocyst stage, Blastocyst were collected and purified for RNA. Analysis for KEGG and GO Biological Process, Cellular Component and Molecular Functions were carried out on the identified target genes. Results were plotted according to level of enrichment for miR-155. Main results and the role of chance Five hundred and seventy-five oocytes were retrieved from 120 bovine ovaries. Cleavage rate and blastocyst formation after IVF were compared between those blastocysts cultured in groups versus individually. There was no significant difference among the two groups with regards to cleavage rate (69% vs. 54%) and blastocyst formation (42% vs.35%). ddPCR assessment showed significantly greater MiR-155 expression in those blastocysts cultured in groups (p = 0.002). The highest level of miR-151 expressed was 20 copies/sample. Every sample, regardless of the culture conditions, had detectable miR-155 level. The interaction of predicted miR-155 target clusters based on the GO Biological Processes shows that miR-155 plays a role in DNA binding, transcription factor binding, regulates transcription and affects cell adhesion. KEGG analysis revealed an association between miR-155 to steroidogenesis and controls apoptosis pathways. Limitations, reasons for caution The number of copies expressed for this miRNA was relatively low, which could suggest that miR-155 may not have a functional role at the blastocyst stage as the effectiveness of a miRNA depends on the relative number of binding site and target molecules. Blastocysts were pooled based on morphological assessment. Wider implications of the findings MiRNAs offer a novel mean to assess embryonic fitness. Further validation and more complex study designs are required to implement the profiling of embryonic miRNAs as a diagnostic test for embryo selection. Group culture increased miR-155 expression, possibly reflecting differences in cellular behavior between group cultured embryos and individually ones. Trial registration number Not applicable

PPIGCF: A Protein-Protein Interaction-Based Gene Correlation Filter for Optimal Gene Selection.

Biological data at the omics level are highly complex, requiring powerful computational approaches to identifying significant intrinsic characteristics to further search for informative markers involved in the studied phenotype. In this paper, we propose a novel dimension reduction technique, protein-protein interaction-based gene correlation filtration (PPIGCF), which builds on gene ontology (GO) and protein-protein interaction (PPI) structures to analyze microarray gene expression data. PPIGCF first extracts the gene symbols with their expression from the experimental dataset, and then, classifies them based on GO biological process (BP) and cellular component (CC) annotations. Every classification group inherits all the information on its CCs, corresponding to the BPs, to establish a PPI network. Then, the gene correlation filter (regarding gene rank and the proposed correlation coefficient) is computed on every network and eradicates a few weakly correlated genes connected with their corresponding networks. PPIGCF finds the information content (IC) of the other genes related to the PPI network and takes only the genes with the highest IC values. The satisfactory results of PPIGCF are used to prioritize significant genes. We performed a comparison with current methods to demonstrate our technique's efficiency. From the experiment, it can be concluded that PPIGCF needs fewer genes to reach reasonable accuracy (~99%) for cancer classification. This paper reduces the computational complexity and enhances the time complexity of biomarker discovery from datasets.

Exploring the active ingredients and potential mechanisms of action of sinomenium acutum in the treatment of rheumatoid arthritis based on systems biology and network pharmacology.

Objective: To investigate and predict the targets and signaling pathways of sinomenium acutum (SA) in the treatment of rheumatoid arthritis (RA) through systems biology and network pharmacology, and to elucidate its possible mechanisms of action. Methods: We screened the active ingredients and corresponding target proteins of SA in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Traditional Chinese Medicines Integrated Database (TCMID) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN); and obtained the targets of rheumatoid arthritis diseases in a database of gene-disease associations (DisGeNET), Online Mendelian Inheritance in Man (OMIM) database. The two targets were mapped by Venn diagram and the intersection was taken. The intersecting targets were used to construct protein-protein interaction (PPI) network maps in the String database, and Metascape was used for Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Finally, the molecular docking technique was applied to validate and further clarify the core target of SA for the treatment of rheumatoid arthritis. Results: A total of six active ingredients and 217 potential targets were obtained after screening; 2,752 rheumatoid arthritis-related targets and 66 targets common to RA and SA. GO function and KEGG pathway enrichment analysis yielded 751 GO function entries (652 GO biological processes, 59 GO molecular functions and 40 GO cellular components) and 77 KEGG signaling pathways. It mainly involves pathways related to neural activity ligand-receptor interaction pathways, cancer pathways, calcium signaling channels, Th17 cell differentiation and others, which are mainly classified into four categories, including regulation of immunity, anti-inflammation, regulation of cell growth and apoptosis, and signaling. The molecular docking results showed that the binding energy of PTGS2, CASP3, JUN and PPARG to the key components beta-sitosterol, 16-epi-Isositsirikine, Sinomenine and Stepholidine were ≤ -6.5kcal/mol, suggesting the existence of molecular binding sites. Conclusion: SA acts on key targets such as PTGS2, CASP3, JUN, and PPARG to modulate signaling pathways such as neural activity ligand-receptor interaction, cancer, calcium ion, NF-κB, and Th17 cell differentiation to regulate immunity, anti-inflammation, modulation of cell cycle, bone metabolism, and signaling for the treatment of RA. It was also confirmed that the treatment of RA with SA has multi-component, multi-target, multi-pathway and multi-mechanism characteristics.

Novel insights into the somatic proteome of Strongyloides stercoralis infective third-stage larvae

BackgroundStrongyloidiasis is a neglected tropical disease affecting an estimated 600 million people, particularly in resource-limited settings. The infection can persist lifelong due to unusual auto-infective cycle of Strongyloides stercoralis. The lack of a diagnostic gold standard and limited knowledge of the mechanisms underpinning this chronic infection are key issues in disease management. To date, only a few proteomics studies have been conducted to elucidate the molecular mechanisms associated with Strongyloides parasitism or to highlight novel immunological markers, with the result that our knowledge of S. stercoralis proteome remains limited. This study aims at expanding the characterization of S. stercoralis infective larvae (iL3) in order to further explore the mechanisms of parasitism and to highlight possible novel targets for serodiagnosis.MethodsiL3 obtained from an infected subject were analysed by high-throughput tandem mass spectrometry. To achieve a more comprehensive characterization of the iL3 proteome we analysed the experimental dataset using an automatic search strategy combined with manual annotation, which included gene ontology (GO) analysis, InterPro annotation, assessment of the homology with Homo sapiens and other pathogens of clinical importance and B-cell epitope prediction.ResultsOur pipeline identified 430 S. stercoralis proteins, 187 (43%) of which were uncharacterized. Oxidoreductases and peptidases were amongst the most represented protein categories, as highlighted by molecular function GO analyses, while membrane and mitochondrial proteins were the most represented cellular component GO categories. A high proportion of proteins bearing the CAP, SCP or thioredoxin domain or belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families were also identified. Additionally, we highlighted nine proteins displaying low homology with H. sapiens or other related pathogens and bearing amino acid sequences with immunogenic properties.ConclusionsOur comprehensive description and annotation of the S. stercoralis iL3 proteome contribute to expanding the ‘omics characterization of this parasite and provide experimental evidence on the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Moreover, novel candidate immunogenic proteins to be evaluated as novel serological diagnostic markers are highlighted.Graphical

Obesity Modifies the Proteomic Profile of the Periodontal Ligament.

This study aimed to assess the obesity effects on the proteomic profile of the periodontal ligament of rats submitted to obesity induction by a high-fat diet. Eight Holtzman rats were divided into control (n = 3) and obese (n = 5) groups. The maxillae were histologically processed for laser capture microdissection of the periodontal ligament of the first maxillary molars. Peptide mixtures were analyzed by LC-MS/MS. A total of 1379 proteins were identified in all groups. Among them, 335 (24.30%) were exclusively detected in the obese group, while 129 (9.35%) proteins were uniquely found in the control group. Out of the 110 (7.98%) differentially abundant proteins, 10 were more abundant and 100 had decreased abundance in the obese group. A gene ontology analysis showed some proteins related to obesity in the "extracellular exosome" term among differentially identified proteins in the gene ontology cellular component terms Prelp, Sec13, and Sod2. These three proteins were upregulated in the obese group (p < 0.05), as shown by proteomic and immunohistochemistry analyses. In summary, our study presents novel evidence that the proteomic profile of the periodontal ligament is altered in experimental obesity induction, providing a list of differentially abundant proteins associated with obesity, which indicates that the periodontal ligament is responsive to obesity.

Transcriptome profile of the anterior pituitary gland in pigs during maternal recognition of pregnancy

The activity of the pituitary gland determines the success of female reproduction. The knowledge regarding the reproductive-status-related changes in the transcriptome of the porcine pituitary is limited. This study aimed to determine and compare the transcriptome profile of the pituitary gland collected from pigs during maternal recognition of pregnancy, i.e. on days 12–13 of pregnancy and during the respective days of the estrous cycle. Analysis indicated 482 differentially expressed genes (DEGs) with an FC > 1.5 (P < 0.05) in the pituitary of pregnant vs. estrous-cyclic pigs. Among them, 68 were up-regulated and 414 were down-regulated. The evaluated DEGs were annotated into 39 gene ontology (GO) biological process terms, 13 GO cellular component terms, and 10 GO molecular function terms. Among the evaluated DEGs were selected genes coding for proteins potentially involved in the regulation of early pregnancy in pigs and used for gene interaction analysis and validation of microarray results. An analysis of the relationships among DEGs in pituitaries collected during maternal recognition of pregnancy showed that some of them are connected with, for example, TGFβ signaling pathway, PRL synthesis, adipocytokines pathway and immune response during maternal recognition of pregnancy. These findings expand the knowledge regarding the molecular mechanisms appearing in the porcine pituitary during the maternal recognition period of pregnancy.

Ral GTPases are critical regulators of spinal cord myelination and homeostasis.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

SubcellulaRVis: a web-based tool to simplify and visualise subcellular compartment enrichment.

Cells contain intracellular compartments, including membrane-bound organelles and the nucleus, and are surrounded by a plasma membrane. Proteins are localised to one or more of these cellular compartments; the correct localisation of proteins is crucial for their correct processing and function. Moreover, proteins and the cellular processes they partake in are regulated by relocalisation in response to various cellular stimuli. High-throughput ‘omics experiments result in a list of proteins or genes of interest; one way in which their functional role can be understood is through the knowledge of their subcellular localisation, as deduced through statistical enrichment for Gene Ontology Cellular Component (GOCC) annotations or similar. We have designed a bioinformatics tool, named SubcellulaRVis, that compellingly visualises the results of GOCC enrichment for quick interpretation of the localisation of a group of proteins (rather than single proteins). We demonstrate that SubcellulaRVis precisely describes the subcellular localisation of gene lists whose locations have been previously ascertained. SubcellulaRVis can be accessed via the web (http://phenome.manchester.ac.uk/subcellular/) or as a stand-alone app (https://github.com/JoWatson2011/subcellularvis). SubcellulaRVis will be useful for experimental biologists with limited bioinformatics expertise who want to analyse data related to protein (re)localisation and location-specific modules within the intracellular protein network.

MO619: Landscape RNA Profiling of Urinary Extracellular Vesicles in Patients with Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Recent studies have revealed that urinary extracellular vesicles (uEV) are secreted with rich biological information useful for understanding the pathophysiological conditions and identifying potential biomarkers for kidney disease. Diabetic nephropathy (DN) has become the major cause of CKD worldwide, while the features of uEV in DN were poorly understood. The present study aims to provide the landscape and in-depth analysis of the transcriptome characteristics of uEV in DN. METHOD Urine was collected from a total of 40 subjects, including healthy controls (HC, n = 14), type 2 DM (T2DM, n = 16), and renal biopsy-confirmed DN (n = 10). uEV was isolated by differential centrifugation and total RNA was extracted for long RNA sequencing. A model based on CIBERSORT was calculated to trace the genetic source of uEV toward resident cells of urinary system. Differentially expressed genes (DEG) between DN and HC and DN and T2DM were applied to gene ontology (GO), Reactome pathway enrichment, as well as gene set enrichment analysis (GSEA). Correlation of the transcriptome profiling between uEV and kidney tissue in DN was also explored. RESULTS About 67%∼68% of transcripts in RNA-seq were mRNA, 11%∼12% were LncRNA and 7%∼10% were CircRNA, and the abundance of mRNA was greater than other RNA types. By CIBERSORT model applying marker genes of podocytes, epithelial cells of tubule of different segments, loop of Henle, and collecting duct, and bladder transitional epithelial cells. The uEV was shown to originate mainly from the collecting duct epithelial cells of the kidney and bladder transitional epithelial cells (Fig. 1a). Confocal laser microscopy showed the co-localization of CD63 and AQP2 (Fig. 1b) in kidney tissues. GO and Reactome analysis were further performed in upregulated mRNA between DN and T2DM and DN and HC to explore the mRNA profiling in terms of the biological significance. Impressively, ‘extracellular exosome’ as well as other terms associated with constitution of EV, ‘plasma membrane’ and ‘extracellular space’ were enriched by GO cellular component (CC) analysis in DN. Besides, Reactome analysis identified pathways of ‘immune system’, ‘extracellular matrix organization’ and ‘degradation of extracellular matrix’ reflecting the pathogenesis mechanism of DN (Fig. 1c). Additionally, microarray data of DN glomeruli and tubule from the GEO database were analyzed in comparison to uEV RNA-seq data. GSEA showed that great similarities were found in the top 10 terms of Reactome pathways (Fig. 2). CONCLUSION In conclusion, this study provides a landscape transcriptome profile, the genetic trace and the biological function terms of uEV in DN. uEV holds potentials in reflecting the transcriptome characteristics of renal tissue, which is of significance for understanding the pathogenesis of DN and biomarker discovery.

MO620: A LC-MS/MS Analysis of Proteins from Urinary Exosomes in Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Urinary exosomes are small extracellular vesicles mainly secreted by epithelial cells of urinary system, reflecting the composition and characteristics of bioactive molecules in physiology and disease conditions of kidney. Diabetic nephropathy (DN) has become the leading cause of CKD and ESRD in the world. The purpose of this study was to describe the proteomic characteristics by LC-MS/MS, and clarify the protein composition and functional changes of urinary exosomes in DN. METHOD A total of 50 mL of first morning urine were collected from 24 subjects, including healthy controls group (Ctrl, n = 8), type 2 diabetes mellitus (T2DM, n = 8) and renal biopsy confirmed DN (n = 8). Exosomes were isolated by differential centrifugation and proteins were extracted and analyzed by LC-MS/MS. The types and expression abundance of proteins in urinary exosome were identified. Differential expression proteins (DEPs) were screened, and gene ontology (GO) and Reactome pathway enrichment analysis were performed to reveal the composition and function changes of DEPs in urinary exosomes of DN. RESULTS Under transmission electron microscope, scattered vesicles with diameters of 40–100 nm were observed in the urine. Nanoparticle tracking analysis showed that the number of urine exosomes was about 10^ 11 to 10^ 12 in 24-h urine with a peak diameter of about 100 nm. Western blot showed the expression of CD63, TSG101 and CD9, which were regarded as the markers of exosome. An average of 3110 (2506∼3380) proteins were identified in the urinary exosomes of the 24 subjects. In each group, the numbers of co-expressed proteins in urinary exosomes of eight subjects were 1680, 1309 and 1416, respectively. GO Cellular Component (CC) analysis showed that extracellular exosomes ranked first in the functional annotation of co-expressed proteins. Subcellular localization showed that the proportions of proteins annotated as extracellular exosome in the three groups were 77%, 81% and 76%, respectively. In upregulated proteins of urinary exosome in DN, GOCC annotation showed that ‘Extracellular exosome’ ranks first. GO molecular function (MF) enrichment analysis showed that the top 10 terms identified that pathways associated with complement activation such as ‘Regulation of Complement activation’, ‘Complement activation,’ ‘Complement activation, classical Pathway’ and ‘Complement activation, alternative Pathway’. Reactome pathway analysis showed that ‘Complement cascade’ and ‘Regulation of complement cascade’ were significantly activated in the top 10 pathways. CONCLUSION Exosomes can be steadily isolated from urine of healthy adults, T2DM and DN, and about 2506 to 3380 proteins can be identified by LC-MS/MS technique, of which about 76%–83% are related to the secretion and composition of exosomes. DEP analysis showed significant enrichment of exosome production-related annotations in urinary exosomes in DN patients compared with HC or T2DM; GOMF and Reactome pathway enrichment analysis showed that pathways related to complement activation were significantly enriched in the urinary exosomes of DN. This study provides proteomics characters of urinary exosome for healthy adults, T2DM and DN patients, suggesting that the production of urinary exosome is increased during DN, and complement activation may be one of the main characteristics of urinary exosome in DN, of significance of reference for studying the pathogenesis and developing new biomarkers of DN.

Identification of novel therapeutic targets for Fuchs' endothelial corneal dystrophy based on gene bioinformatics analysis.

Fuchs’ endothelial corneal dystrophy (FECD) is a disease where progressive visual impairment occurs by the thickening of the Descemet’s membrane and the gradual degeneration and loss of corneal endothelial cells. This study aimed to investigate the key changes in gene expression associated with FECD and explore potential biomarkers and new therapeutic strategies for FECD. To explore the potential therapeutic targets of FECD, we downloaded the gene expression dataset GSE171830 from the Gene Expression Omnibus (GEO) database. A total of 303 differentially expressed genes (DEGs) were identified by the limma package. The enriched Gene Ontology (GO) annotations and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs mostly included the extracellular matrix organization, collagen-containing extracellular matrix, and the structural constituents of the extracellular matrix. Fifteen hub genes from the most significant module were ascertained by Cytoscape. Both collagen-containing extracellular matrix and extracellular matrix hit to ANXA1, VCAN, GPC3, TNC, IGFBP7, MATN3, and SPARCL1 genes in the GO cellular components. Among these genes, the expression of SPARCL1 was down-regulated in the FECD samples, whereas the expression of GPC3, MATN3, IGFBP7, TNC, VCAN, and ANXA1 was up-regulated in the FECD samples. Gene set enrichment analysis (GSEA) plots showed that among the 20,937 genes, SPARCL1 played an important role in three pathways, cytokine-cytokine receptor interaction, the TGF-beta signaling pathway, and antigen processing and presentation. The top three pathways enriched by the GPC3, MATN3, IGFBP7, TNC, VCAN, and ANXA1 genes were those for cytokine-cytokine receptor interaction, TGF-beta signaling, and RIG-I-like receptor signaling. In conclusion, the DEGs identified here might assist clinicians in understanding the pathogenesis of FECD. Furthermore, these identified biomarkers might serve as potential therapeutic targets for the treatment of FECD.

Screening criteria of mRNA indicators for wound age estimation

Wound age estimation is a crucial and challenging problem in forensic pathology. Although mRNA is the most commonly used indicator for wound age estimation, screening criteria are lacking. In the present study, the feasibility of screening criteria using mRNA to determine injury time based on the adenylate-uridylate-rich element (ARE) structure and gene ontology (GO) categories were evaluated. A total of 78 Sprague-Dawley male rats were contused and sampled at 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h after inflicting injury. The candidate mRNAs were classified based on with or without ARE structure and GO category function. The mRNA expression levels were detected using qRT-PCR. In addition, the standard deviation (STD), mean deviation (MD), relative average deviation (d%), and coefficient of variation (CV) were calculated based on mRNA expression levels. The CV score (CVs) and the CV of CV (CV’CV) were calculated to measure heterogeneity. Finally, based on classic principles, the accuracy of combination of candidate mRNAs was assessed using discriminant analysis to construct a multivariate model for inferring wound age. The results of homogeneity evaluation of each group based on CVs were consistent with the MD, STD, d%, and CV results, indicating the credibility of the evaluation results based on CVs. The candidate mRNAs without ARE structure and classified as cellular component (CC) GO category (ARE–CC) had the highest CVs, showing the mRNAs with these characteristics are the most homogenous mRNAs and best suited for wound age estimation. The highest accuracy was 91.0% when the mRNAs without ARE structure were used to infer the wound age based on the discrimination model. The accuracy of mRNAs classified into CC or multiple function (MF) GO category was higher than mRNAs in the biological process (BP) category. In all subgroups, the accuracy of the composite identification model of mRNA composition without ARE structure and classified as CC was higher than other subgroups. The mRNAs without ARE structure and belonging to the CC GO category were more homogenous, showed higher accuracy for estimating wound age, and were appropriate for rat skeletal muscle wound age estimation. Supplemental data for this article is available online at https://doi.org/10.1080/20961790.2021.1986770 .

Exploring the Pharmacological Mechanisms of Tripterygium wilfordii Hook F against Cardiovascular Disease Using Network Pharmacology and Molecular Docking

Background Tripterygium wilfordii Hook F (TwHF) has been used in traditional Chinese medicine (TCM) for treating cardiovascular disease (CVD). However, the underlying pharmacological mechanisms of the effects of TwHF on CVD remain elusive. This study revealed the pharmacological mechanisms of TwHF acting on CVD based on a pharmacology approach. Materials and Methods The active compounds were selected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database according to the absorption, distribution, metabolism, and excretion (ADME). The potential targets of TwHF were obtained from the SwissTargetPrediction database. The CVD-related therapeutic targets were collected from the DrugBank, the GeneCards database, and the OMIM database. Protein–protein interaction (PPI) network was generated by the STITCH database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed by R package. The network of drug-targets-diseases-pathways was constructed by the Cytoscape software. Results The 41 effective ingredients of TwHF and the 178 common targets of TwHF and CVD-related were collected. Furthermore, AKT1, amyloid precursor protein (APP), mitogen-activated protein kinase 1 (MAPK), phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA), and cellular tumor antigen p53 (TP53) were identified as the core targets involved in the mechanism of TwHF on CVD. Top ten GO (biological processes, cellular components, and molecular functions) and KEGG pathways were screened with a P value ≤0.01. Finally, we constructed the network of TwHF-targets-CVD-GO-KEGG. Conclusions These findings demonstrate that the main active compound of TwHF, the core targets, and pathways maybe provide new insights into the development of a natural therapy for the prevention and treatment of CVD.

Bioinformatic analysis for the identification of potential gene interactions and therapeutic targets in atrial fibrillation.

Atrial fibrillation (AF) is the most common type of tachycardia. The major injury caused by AF is a systemic embolism. Although AF therapies have evolved substantially, the success rate of sinus rhythm maintenance is relatively low. The reason is the incomplete understanding of the AF mechanisms. A Gene Expression Omnibus (GEO) dataset (GSE79768) was downloaded. Differentially expressed genes (DEGs) were identified by bioinformatic analysis. Enriched terms and pathways were identified by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A protein-protein interaction (PPI) network was constructed to determine regulatory genes. CytoHubba and the Molecular Complex Detection (MCODE) algorithm were used to identify potential hub genes and important modules. The Predicting Associated Transcription Factors From Annotated Affinities (PASTAA) method was used to predict transcription factors (TFs). Two hundred thirty-five upregulated DEGs and seventy-seven downregulated DEGs were identified. In the GO biological process, cellular component, and molecular function analyses, positive regulation of cell migration, anchoring junctions, and cell adhesion molecule binding were enriched significantly. The Hippo signalling pathway was the most significantly enriched pathway. In the PPI network analysis, we found that Class A/1 (rhodopsin-like receptors) may be the critical module. Ten hub genes were extracted, including 6 upregulated genes and 4 downregulated genes. CXCR2, TLR4, and CXCR4 may play critical roles in AF. In the TF prediction, we found that Irf-1 may be implicated in AF. We found that the CXCR4, TLR4, CXCR2 genes, the Hippo signalling pathway and the class A/1 (rhodopsin-like receptors) module may play critical roles in AF occurrence and maintenance, which may provide novel targets for AF treatment.

Differential DNA methylation in recovery from shift work disorder

The human DNA methylome is responsive to our environment, but its dynamics remain underexplored. We investigated the temporal changes to DNA methylation (DNAme) in relation to recovery from a shift work disorder (SWD) by performing a paired epigenome-wide analysis in an occupational cohort of 32 shift workers (25 men, age = 43.8 ± 8.8 years, 21 SWD cases). We found that the effect of vacation on DNAme was more prominent in the SWD-group as compared to controls, with respect to the amount of significantly differentially methylated positions (DMPs; Punadj < 0.05) 6.5 vs 3.7%, respectively. The vast majority (78%) of these DMPs were hypomethylated in SWD but not in controls (27%) during the work period. The Gene Ontology Cellular component “NMDA glutamate receptor” (PFDR < 0.05) was identified in a pathway analysis of the top 30 genes in SWD. In-depth pathway analyses revealed that the Reactome pathway “CREB phosphorylation through the activation of CaMKII” might underlie the recovery. Furthermore, three DMPs from this pathway, corresponding to GRIN2C, CREB1, and CAMK2B, correlated with the degree of recovery (Punadj < 0.05). Our findings provide evidence for the dynamic nature of DNAme in relation to the recovery process from a circadian disorder, with biological relevance of the emerging pathways.

Neuronal-Glial Interaction in a Triple-Transgenic Mouse Model of Alzheimer's Disease: Gene Ontology and Lithium Pathways.

Neuronal-glial interactions are critical for brain homeostasis, and disruption of this process may lead to excessive glial activation and inadequate pro-inflammatory responses. Abnormalities in neuronal-glial interactions have been reported in the pathophysiology of Alzheimer’s disease (AD), where lithium has been shown to exert neuroprotective effects, including the up-regulation of cytoprotective proteins. In the present study, we characterize by Gene Ontology (GO) the signaling pathways related to neuronal-glial interactions in response to lithium in a triple-transgenic mouse model of AD (3×-TgAD). Mice were treated for 8 months with lithium carbonate (Li) supplemented to chow, using two dose ranges to yield subtherapeutic working concentrations (Li1, 1.0 g/kg; and Li2, 2.0 g/kg of chow), or with standard chow (Li0). The hippocampi were removed and analyzed by proteomics. A neuronal-glial interaction network was created by a systematic literature search, and the selected genes were submitted to STRING, a functional network to analyze protein interactions. Proteomics data and neuronal-glial interactomes were compared by GO using ClueGo (Cytoscape plugin) with p ≤ 0.05. The proportional effects of neuron-glia interactions were determined on three GO domains: (i) biological process; (ii) cellular component; and (iii) molecular function. The gene ontology of this enriched network of genes was further stratified according to lithium treatments, with statistically significant effects observed in the Li2 group (as compared to controls) for the GO domains biological process and cellular component. In the former, there was an even distribution of the interactions occurring at the following functions: “positive regulation of protein localization to membrane,” “regulation of protein localization to cell periphery,” “oligodendrocyte differentiation,” and “regulation of protein localization to plasma membrane.” In cellular component, interactions were also balanced for “myelin sheath” and “rough endoplasmic reticulum.” We conclude that neuronal-glial interactions are implicated in the neuroprotective response mediated by lithium in the hippocampus of AD-transgenic mice. The effect of lithium on homeostatic pathways mediated by the interaction between neurons and glial cells are implicated in membrane permeability, protein synthesis and DNA repair, which may be relevant for the survival of nerve cells amidst AD pathology.