GO Pathway Analysis Research Articles

Systematic Understanding of the Mechanism of Salvianolic Acid A via Computational Target Fishing.

Salvianolic acid A (SAA) is one of the most abundant water-soluble and potent anti-oxidative compounds isolated from Danshen, a traditional Chinese medicine. A systematic overview of its mechanism of action is yet to be performed. In the present study, the druggability of SAA was measured using the TCMSP server, and potential targets of SAA were identified by PharmMapper and DRAR-CPI. Intersecting targets were then assessed by GeneMANIA and GO pathway analysis, and drug-target-pathway networks were constructed to give a visual view. The results showed that SAA has good druggability, and 13 putative protein targets were identified. Network analysis showed that these targets were associated with cancer, metabolism and other physiological processes. In summary, SAA is predicted to target multiple proteins and pathways to form a network that exerts systematic pharmacological effects.

STEM CELL MODELS OF BIPOLAR DISORDER - A DEVELOPMENTAL PERSPECTIVE

A major challenge in understanding human neuropsychiatric disorders is the lack of viable cells and tissues for analysis. Patient-derived induced pluripotent stem cells (iPSC) offer the opportunity to examine the full complement of neural tissues to identify underlying disease mechanisms. Increasingly considered to have a neurodevelopmental basis, to study Bipolar Disorder (BP), we have derived and characterized iPSC from fibroblasts obtained from undiagnosed Controls (C) and patients with BP, and have coaxed them to form neurons and glial cells. Using microarray analysis of BP and unaffected control iPSC neurons, we previously identified differences in neuronal lineage allocation between groups, with BP neurons favoring differentiation of ventral MGE derivatives and Control neurons forming dorsal cortical precursors. To determine the developmental basis of these observations we employed microarray analysis, identifying 59 miRNAs that were significantly dysregulated in BP iPSC neurons. We have validated 5 miRNAs that were elevated and 3 miRNAs that were lower in BP derived neurons. GO pathway analysis of miRNAs significantly elevated in BP derived neurons indicate that these miRNAs regulate a wide variety of cellular processes, including proliferation, cell junction and extracellular matrix organization, chromatin modification, mRNA processing, and carbohydrate metabolism. We use a luciferase assay to validate the targets of these miRNAs, which will then be overexpressed in iPSC derived cells to determine their effects on neuronal behavior.To determine if our results are restricted to neurons we examined differences in the behavior of astrocytes derived from BP and C individuals. After differentiation as astrospheres, precursors were passaged and expanded in FGF-2 and EGF producing a population of astrocytes which were positive for the glial precursor marker CD44. Expression of S100beta, and the glutamate transporters EAAT1 and EAAT2 were markedly lower in BP astrocytes, and cell doubling times at early passage number were greater in BP cells. These differences in gene expression and behavior suggest that BP astrocytes are functionally distinct from control astrocytes. Current investigations are in progress to examine gliotransmitter expression and behaviors in co-culture.We have identified differences in calcium signaling in BP vs C neurons; BP neurons are more active than those derived from Control patients. Remarkably, lithium pre-treatment significantly reduced calcium transients and wave amplitude in BP neurons to Control levels, providing a tractable model system to examine the response of iPSC-derived neurons to pathway perturbagens and treatments involved in BP. One of those is ketamine, which we are applying to brain organoids derived from iPSC to determine their response to this therapeutic. Additional work is examining the effects of increased CACNA1C expression associated with the AA allele of rs1006737 -- the strongest and most replicated association with bipolar disorder -- in neuronal differentiation and function. AA carrier BP cells are undergoing gene editing to revert the mutant genotype to GG, and their calcium signaling and differentiation potential assessed. The overarching goal of our research is to identify novel disease phenotypes and mechanisms involved in bipolar disorder, with the long term goal of improving treatment.

The Role and Mechanism Study of Lncrnas Involved in Glucocorticoid Resistance in Paediatric Acute Lymphoblastic Leukemia

Objective: Glucocorticoids (GCs) are key components in the treatment of childhood acute lymphoblastic leukemia (ALL) and most ALL therapeutic failures can be explained by cellular resistance to GCs. However, the mechanisms of GC resistance are poorly understood. LncRNAs are involved in normal hematopoiesis and leukemia development, whereas the roles of lncRNAs in GC resistance are still unknown. Our goal was to investigate the role of lncRNAs involved in glucocorticoid resistance in paediatric acute lymphoblastic leukemia, and also elucidate the mechanism preliminarily.Methods: In this study, lncRNA microarray was performed on GC-resistant cell line CEM-C1 and GC-sensitive cell line CEM-C7 to screen the differential expression of lncRNAs. Five up-regulated and five down-regulated lncRNAs were randomly chosen for validation by Real-time PCR. GO-Pathway analysis was done to investigate potential signaling pathways regulated by the lncRNAs.Results: The microarray revealed that 4286 lncRNAs differed (p<0.05 and fold change>2.0) in GC-sensitive cell from those in GC-resistant cell. 826 lncRNAs changed more than 5-fold, while 356 lncRNAs changed more than 10-fold. Among them, 203 lncRNAs were up-regulated and 153 lncRNAs were down-regulated. Expression of ten selected lncRNAs was validated by Real-time PCR. GO analysis indicated that differentially expressed lncRNAs were involved in apoptotic process related GO terms. Pathway analysis revealed that these lncRNAs were involved in apoptosis, cell cycle, mTOR and some other signaling pathways.Conclusions: Our study showed that lncRNA expression profile was altered in GC-sensitive and GC-resistant cells, indicating that differentially expressed lncRNAs may play important functional roles in GC resistance of ALL. And these lncRNAs may be involved in GC resistance by regulating signaling pathways associated with cell proliferation, differentiation and apoptosis. Our study provides new biological foundations for further mechanism study in GC resistance and also provides a new strategy for therapeutic development of ALL. DisclosuresNo relevant conflicts of interest to declare.

The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases.

Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases.

Toxic responses in rat embryonic cells to silver nanoparticles and released silver ions as analyzed via gene expression profiles and transmission electron microscopy

After exposing rat embryonic cells to 20 μg/mL of silver nanoparticle (NP) suspension and their released ions for different time periods, silver nanoparticles were found in cellular nuclei, mitochondria, cytoplasm and lysosomes by transmission electron microscopy (TEM). We also observed mitochondrial destruction, distension of endoplasmic reticulum and apoptotic bodies. Global gene expression analysis showed a total of 279 genes that were up-regulated and 389 genes that were down-regulated in the silver-NP suspension exposure group, while 3 genes were up-regulated and 41 genes were down-regulated in the silver ion exposure group. Further, the GO pathway analysis suggested that these differentially expressed genes are involved in several biological processes, such as energy metabolism, oxygen transport, enzyme activities, molecular binding, etc. It is possible that inhibition of oxygen transport is mediated by the significant down-regulation of genes of the globin family, which might play an important role in silver ion-induced toxicity. KEGG pathway analysis showed that there were 23 signal pathways that were affected in the cells after exposure to silver-NP suspension, but not silver ion alone. The most significant change concerned inflammatory signal pathways, which were only found in silver-NP suspension exposed cells, indicating that inflammatory response might play an important role in the mechanism(s) of silver-NP-induced toxicity. The significant up-regulation of matrix metalloproteinases 3 and 9 suggests that silver NPs could induce extracellular matrix degradation via an inflammatory signaling pathway. The significant up-regulation of secretory leukocyte peptidase inhibitor and serine protease inhibitor 2c was considered to be an embryonic cellular defense mechanism in response to silver-NP-induced inflammation.

Proper Interpretation of Molecular Changes Associated With Atrophy-Fibrosis Requires Correction For Time Post-Transplant.

We previously reported that fibrosis (ci-lesions) in renal transplant biopsies was associated with plasma and mast cells, and with immunoglobulin transcripts (IGTs) and mast cell transcripts (MCATs). Fibrosis is a feature of late biopsies, and these changes could reflect time post-transplant rather than fibrosis per se. We explored the molecular features associated with time and fibrosis in 703 indication biopsies taken 6 days to 36 years post-transplant. We wanted to find the molecular features of time post-transplant and to separate those from the changes associated with fibrosis. Time associated transcripts were identified by a classifier comparing late biopsies (>1year) to early biopsies (<1year). Fibrosis associated transcripts were identified by two classifiers: cigt1 - comparing biopsies with ci scores 2-3 (high fibrosis) to biopsies with ci scores 0-1 (low fibrosis), and cigt1time that had time as the covariate, i.e. time-corrected. The cigt1time classifier had the best AUC for predicting fibrosis compared to cigt1 classifier, injury response (AKI score), IGTs, MCATs and time alone (Table 1).Table: No Caption available.The time classifier used mostly IGTs and MCATs (Figure 1A).Table: No Caption available.The Cigt1 classifier used a high proportion of time-associated IGTs and thus was actually predicting time rather than fibrosis. The Cigt1time classifier selected injury-response transcripts (AKI Score) but did not select time-associated transcripts. Notably, the latter frequently included transcripts whose expression was decreased in biopsies with high fibrosis. IPA and GO pathway analyses of time corrected gene expression revealed that increase in acute phase signaling and ILK signaling (related to tissue injury), and decrease in amino acid metabolism and mitochondrial transcripts (related to atrophy) characterizes biopsies with high fibrosis (Figure 1B). We conclude that when interpreting kidney transplant molecular features, it is important to distinguish between time-dependent features (e.g. IGTs/MCATs) and features actually associated with atrophy-fibrosis and parenchymal injury. DISCLOSURES:Halloran, P.: Speaker's Bureau, Astellas, Novartis, OneLamba.

An imprinted rheumatoid arthritis methylome signature reflects pathogenic phenotype

BackgroundA DNA methylation signature has been characterized that distinguishes rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) from osteoarthritis (OA) FLS. The presence of epigenetic changes in long-term cultured cells suggest that rheumatoid FLS imprinting might contribute to pathogenic behavior. To understand how differentially methylated genes (DMGs) might participate in the pathogenesis of RA, we evaluated the stability of the RA signature and whether DMGs are enriched in specific pathways and ontology categories.MethodsTo assess the RA methylation signatures the Illumina HumanMethylation450 chip was used to compare methylation levels in RA, OA, and normal (NL) FLS at passage 3, 5, and 7. Then methylation frequencies at CpGs within the signature were compared between passages. To assess the enrichment of DMGs in specific pathways, DMGs were identified as genes that possess significantly differential methylated loci within their promoter regions. These sets of DMGs were then compared to pathway and ontology databases to establish enrichment in specific categories.ResultsInitial studies compared passage 3, 5, and 7 FLS from RA, OA, and NL. The patterns of differential methylation of each individual FLS line were very similar regardless of passage number. Using the most robust analysis, 20 out of 272 KEGG pathways and 43 out of 34,400 GO pathways were significantly altered for RA compared with OA and NL FLS. Most interestingly, we found that the KEGG 'Rheumatoid Arthritis' pathway was consistently the most significantly enriched with differentially methylated loci. Additional pathways involved with innate immunity (Complement and Coagulation, Toll-like Receptors, NOD-like Receptors, and Cytosolic DNA-sensing), cell adhesion (Focal Adhesion, Cell Adhesion Molecule), and cytokines (Cytokine-cytokine Receptor). Taken together, KEGG and GO pathway analysis demonstrates non-random epigenetic imprinting of RA FLS.ConclusionsThe DNA methylation patterns include anomalies in key genes implicated in the pathogenesis of RA and are stable for multiple cell passages. Persistent epigenetic alterations could contribute to the aggressive phenotype of RA synoviocytes and identify potential therapeutic targets that could modulate the pathogenic behavior.

Genotoxicity and molecular response of silver nanoparticle (NP)-based hydrogel

BackgroundSince silver-nanoparticles (NPs) possess an antibacterial activity, they were commonly used in medical products and devices, food storage materials, cosmetics, various health care products, and industrial products. Various silver-NP based medical devices are available for clinical uses, such as silver-NP based dressing and silver-NP based hydrogel (silver-NP-hydrogel) for medical applications. Although the previous data have suggested silver-NPs induced toxicity in vivo and in vitro, there is lack information about the mechanisms of biological response and potential toxicity of silver-NP-hydrogel.MethodsIn this study, the genotoxicity of silver-NP-hydrogel was assayed using cytokinesis-block micronucleus (CBMN). The molecular response was studied using DNA microarray and GO pathway analysis.Results and discussionThe results of global gene expression analysis in HeLa cells showed that thousands of genes were up- or down-regulated at 48 h of silver-NP-hydrogel exposure. Further GO pathway analysis suggested that fourteen theoretical activating signaling pathways were attributed to up-regulated genes; and three signal pathways were attributed to down-regulated genes. It was discussed that the cells protect themselves against silver NP-mediated toxicity through up-regulating metallothionein genes and anti-oxidative stress genes. The changes in DNA damage, apoptosis and mitosis pathway were closely related to silver-NP-induced cytotoxicity and chromosome damage. The down-regulation of CDC14A via mitosis pathway might play a role in potential genotoxicity induced by silver-NPs.ConclusionsThe silver-NP-hydrogel induced micronuclei formation in cellular level and broad spectrum molecular responses in gene expression level. The results of signal pathway analysis suggested that the balances between anti-ROS response and DNA damage, chromosome instability, mitosis inhibition might play important roles in silver-NP induced toxicity. The inflammatory factors were likely involved in silver-NP-hydrogel complex-induced toxic effects via JAK-STAT signal transduction pathway and immune response pathway. These biological responses eventually decide the future of the cells, survival or apoptosis.