DAVID Functional Annotation Clustering Research Articles

Comparison of the transcriptome in circulating leukocytes in early lactation between primiparous and multiparous cows provides evidence for age-related changes

BackgroundPrevious studies have identified many immune pathways which are consistently altered in humans and model organisms as they age. Dairy cows are often culled at quite young ages due to an inability to cope adequately with metabolic and infectious diseases, resulting in reduced milk production and infertility. Improved longevity is therefore a desirable trait which would benefit both farmers and their cows. This study analysed the transcriptome derived from RNA-seq data of leukocytes obtained from Holstein cows in early lactation with respect to lactation number.ResultsSamples were divided into three lactation groups for analysis: i) primiparous (PP, n = 53), ii) multiparous in lactations 2–3 (MP 2–3, n = 121), and iii) MP in lactations 4–7 (MP > 3, n = 55). Leukocyte expression was compared between PP vs MP > 3 cows with MP 2–3 as background using DESeq2 followed by weighted gene co-expression network analysis (WGCNA). Seven modules were significantly correlated (r ≥ 0.25) to the trait lactation number. Genes from the modules which were more highly expressed in either the PP or MP > 3 cows were pooled, and the gene lists subjected to David functional annotation cluster analysis. The top three clusters from modules more highly expressed in the PP cows all involved regulation of gene transcription, particularly zinc fingers. Another cluster included genes encoding enzymes in the mitochondrial beta-oxidation pathway. Top clusters up-regulated in MP > 3 cows included the terms Glycolysis/Gluconeogenesis, C-type lectin, and Immunity. Differentially expressed candidate genes for ageing previously identified in the human blood transcriptome up-regulated in PP cows were mainly associated with T-cell function (CCR7, CD27, IL7R, CAMK4, CD28), mitochondrial ribosomal proteins (MRPS27, MRPS9, MRPS31), and DNA replication and repair (WRN). Those up-regulated in MP > 3 cows encoded immune defence proteins (LYZ, CTSZ, SREBF1, GRN, ANXA5, ADARB1).ConclusionsGenes and pathways associated with lactation number in cows were identified for the first time to date, and we found that many were comparable to those known to be associated with ageing in humans and model organisms. We also detected changes in energy utilization and immune responses in leukocytes from older cows.

MALDI-TOF mass spectrometric protein profiling of THP-1 cells and their microvesicles

Extracellular vesicles that are shed from the plasma membranes take an active part in intercellular communication, transporting a wide range of molecules, including proteins, lipids, nucleic acids and carbohydrates, being of great functional importance. One of the steps to better understanding of distant communications of cells and their regulatory mechanisms is a proteomic study of various extracellular vesicles, including microvesicles and exosomes. Pro-inflammatory cytokines produced by monocytes and individual complement system components play a key role in their specific functioning. The aim of this work was to study proteomic composition of THP-1 monocyte-like cells and their microvesicles. The MALDI-mass spectrometric analysis of electrophoretic protein fractions of cell lysates and microvesicles allowed for identifying 107 proteins that perform various functions. Among 19 determined functional groups, the largest ones comprise transcription regulators and proteins with unknown functions. The smallest functional groups include regulators of cell differentiation and development, proteins participating in immune response and inflammation, cellular receptors and their regulators, transporter and transport regulatory proteins, as well as cell proteins mediating adhesion and matrix structures, processing regulators, proteins of ubiquitin-proteasome system, intracellular signaling, autophagy and exocytosis regulators, chromatin structural proteins, hemostatic regulators, and peptide hormones. An intermediate position is occupied by cytokines and growth factors, enzymes, cytoskeleton and motor proteins, as well as RNA processing and translation regulators. The subsequent DAVID Functional Annotation Clustering analysis allowed for identifying the most common groups distributed by their molecular function, biological processes, and cellular component. Separately, in the microvesicles derived from THP-1 monocyte-like cells, proteins of the immune response and inflammation, cytokines and growth factors, intracellular signaling proteins, cell differentiation regulators and developmental proteins, as well as cell adhesion and matrix proteins were identified among other protein molecules. The data obtained on the partial proteome of THP-1 monocyte-like cells and their microvesicles extend the existing knowledge on distant communications between the cells and suggest new mechanisms of interaction between monocytes/macrophages and their microenvironment.

Abstract P5-04-13: Splicing factor ESRP1 controls ER-positive breast cancer progression by altering metabolic pathway genes

Abstract Background Epithelial Splicing Regulatory Protein (ESRP1)is a key splicing factor that regulates Epithelial-to-Mesenchymal Transition (EMT) splicing program. Our previous study demonstrated that high levels of ESRP1 are associated with poor prognosis in human ER-positive (ER+) breast tumors in an independent manner of EMT process. We next explored the potential mechanisms that contribute to the role ESRP1 in endocrine therapy-resistant breast cancer. Methods Probe based-Human Transcriptome Array 2.0 (HTA; Applied Biosystems/Thermo Fisher) was performed using RNAs from control and ESRP1 knockdown cells (LCC2 versus 2C3 ESRP1 and LCC9 versus 9C2 ESRP1) of endocrine resistant breast cancer. Functional enrichment analyses were performed using the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/). To confirm the functional importance of ESRP1 on regulation of cellular metabolism, we have performed experiments that analyze the metabolic substrate flux in response to ESRP1 knockdown in resistant cells (The Seahorse XFp Cell Energy Phenotype Assay). Differentially expressed genes were validated using Western blotting assay. Results Transcriptome profiling of ESRP1in 2C3 and 9C2 knockdown models revealed differentially expressed genesusing HTA 2.0 platform. In LCC2 versus 2C3 ESRP1 knockdown, expression of 1186 genes (1263 transcripts) have been altered significantly, while 413 genes (432 transcripts) have been significantly regulated in LCC9 versus 9C2 ESRP1 knockdown with FDR<0.1 Of these significant genes, 34 downregulated and 68 upregulated (102 genes total) were shared by both 2C3 and 9C2 ESRP1 knockdowns. Using the DAVID Functional Annotation Clustering Tool, we identified the biological processes altered significantly in response to ESRP1 knockdown. The most significant annotation clusters downregulated in ESRP1 knockdown consists of fatty acid metabolism/lipid metabolism (SCD, ACACA, FASN, ACAT2, PLCH1, and HPGD), and oxireductase processes (SCD, PHGDH, FASN, DHTKD1 and HPGD. We confirmed the altered metabolic function using the Seahorse analyzer. These analyses confirmed that ESRP1 knockdown altered the glycolysis rate (extracellular acidification rate; ECAR) in both tamoxifen-resistant and fulvestrant-resistant models. In addition, ESRP1 knockdown decreased the mitochondrial respiration in tamoxifen-resistant cells, but not fulvestrant resistant cells. Using Western blotting, we validated the altered levels of fatty acid synthase (FASN) and Stearoyl-CoA desaturase 1 (SCD1), key enzymes in fatty acid metabolism. Phosphoglycerate Dehydrogenase (PHGDH), a poor prognosis marker in cancers including breast cancer, was also downregulated in response to ESRP1 knockdown. Taken together, we have demonstrated a novel functional impact of ESRP1 on the regulation of tumor growth at the functional and molecular level independent of EMT. Conclusions For the first time, we show the role of ESRP1 in altering the cellular metabolism thereby contributing to tumor growth. The study provides a molecular evidence for the role of altered metabolism in determining adverse prognosis of ER+ breast cancer via the control of ESRP1, a splicing factor. Further studies to determine the therapeutic value are underway. Citation Format: Gokmen-Polar Y, Gu Y, Gu X, Badve SS. Splicing factor ESRP1 controls ER-positive breast cancer progression by altering metabolic pathway genes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-13.

Distinct molecular pathways in ovarian endometrioid adenocarcinoma with concurrent endometriosis.

Women with endometriosis, a benign growth of endometrial tissue outside the uterine cavity, are at increased risk of specific histotypes of epithelial ovarian cancer, such as ovarian endometrioid adenocarcinoma (OEA). Women with OEA who have endometriosis at time of surgical staging demonstrate improved clinical prognosis compared to women with OEA without evidence of endometriosis. However, the molecular contributions of the endometriotic tumor microenvironment to these ovarian cancers remain poorly understood. As a starting point, we used a platform for genome-wide transcriptomic profiling to compare specimens of OEA from women with and without concurrent endometriosis and benign reproductive tract tissues, including proliferative endometrium and typical and atypical endometrioma samples (n = 20). Principle component analysis revealed distinct clustering between benign and malignant samples as well as malignant samples with and without concurrent endometriosis. Examination of gene signatures revealed that OEA with concurrent endometriosis contained a unique molecular signature compared to OEA without concurrent endometriosis, distinguished by 682 unique genes differentially expressed (fold change < or >1.5, p < 0.01). Bioinformatic analysis of these differentially expressed gene products using ingenuity pathway analysis revealed activation of NFkB signaling, an inflammatory signaling pathway constitutively active in endometriosis. DAVID functional annotation clustering further revealed enrichment in RAS signaling as both cytoskeleton organization and GTPase regulator activity relied heavily on RAS protein signal transduction. Gene set enrichment analysis highlighted immune and inflammatory nodes involved in OEA with concurrent endometriosis. These observations provide novel resources for understanding molecular subtleties potentially involved in OEA within the context of the endometriotic tumor microenvironment.

Differences of RNA Expression in the Tendon According to Anatomic Outcomes in Rotator Cuff Repair

Background: Despite increased understanding of the pathophysiology of rotator cuff tears and the evolution of rotator cuff repair, healing failure remains a substantial problem. The critical roles played by biological factors have been emphasized, but little is known of the implications of gene expression profile differences at the time of repair. Purpose: To document the relationship between the perioperative gene expression of healed and unhealed rotator cuffs by RNA microarray analysis. Study Design: Case-control study; Level of evidence, 3. Methods: Superior (supraspinatus involvement) and posterosuperior (supraspinatus and infraspinatus involvement) tears were included in the study. Samples of rotator cuff tendons were prospectively collected during rotator cuff surgery. Three samples were harvested at the tendon ends of tears from the anterior, middle (apex), and posterior parts using an arthroscopic punch. Seven patients with an unhealed rotator cuff were matched one-to-one with patients with a healed rotator cuff by sex, age, tear size, and fatty degeneration of rotator cuff muscles. mRNA microarray analysis was used to identify genetic differences between healed and unhealed rotator cuff tendons. Gene ontology and gene association files were obtained from the Gene Ontology Consortium, and the Gene Ontology system in DAVID was used to identify enhanced biological processes. Results: Microarray analyses identified 262 genes that were differentially expressed by at least 1.5-fold between the healed and unhealed groups. Overall, in the healed group, 103 genes were significantly downregulated, and 159 were significantly upregulated. DAVID Functional Annotation Cluster analysis showed that in the healed group, the genes most upregulated were related to the G protein–coupled receptor protein signaling pathway and to the neurological system. On the other hand, the genes most downregulated were related to immune and inflammatory responses. BMP5 was the gene most upregulated in the healed group, and the majority of downregulated genes were involved in the immune/inflammatory response. Conclusion: The downregulation of inflammatory response genes and the upregulation of cell differentiation genes in torn rotator cuffs at the time of surgery are related to rotator cuff healing. These results provide useful baseline information for future biological studies on rotator cuff healing.

Microarray Analysis of microRNAs Expression Profiles in Adult and Aged Mice Hippocampus

Purpose: To analyze the miRNA expression profiles in C57 mice ageing hippocampus in detail, and investigate the functional information of these hippocampus specific miRNAs and the related regulatory networks. Methods: Microarrays were used to analyze miRNA expression profiles in adult and aged hippocampi, and bioinformatics analysis methods, such as three public datasets (Mirbase, Miranda, and Mirdb), DAVID online tools and KEGG pathway tools were used to study in detail the target genes of the differentially expressed miRNAs. Results: 26 differentially expressed miRNAs were identified by greater than 1.5-fold change (intersection of two sets), of which 16 were up-regulated and 10 were down-regulated. DAVID Functional Annotation Cluster (FAC) analysis of the 132 predicted target genes of up-regulated miRNAs revealed confident enrichment scores for synaptic function and apoptosis etc. (Figure 1), indicating the functional significance and importance of these miRNAs during hippocampal ageing. Conclusions: Bioinformatic analyses of the differentially expressed miRNAs have identified a number of miRNAs with putative involvement in the hippocampus ageing process. This study lays a solid foundation for further studies to clarify the important regulation function of miRNAs in ageing process of brain tissue.

Identification and Predicted Biological Significance of MicroRNAs Involved in the Hippocampal Development of the Rat by Microarray and Bioinformatics Analysis

microRNAs (miRNAs) are functional small non-coding RNA that play essential roles in regulation of gene expression affecting development, differentiation and proliferation. In order to investigate the function of miRNAs in hippocampal development, newborn and adult hippocampi of Lewis rat were selected for analysis of the miRNA expression profiles by microarray in this study. Microarray analyses identified 25 differentially expressed miRNAs (＞1.5 fold, intersection of three sets). Of these, 14 were down-regulated and 11 were upregulated during hippocampal development. DAVID Functional Annotation Cluster (FAC) analysis of the 317 predicted target genes of the down-regulated miRNAs revealed confident enrichment scores for cell adhesion and neuron development, indicating the functional significance and importance of these miRNAs during hippocampal development. Similarly, 311 predicted target genes of the up-regulated miRNAs by DAVID FAC analysis produced 26 enriched functional clusters. The top 8 included transmembrane region, response to hormone stimulus, symport and transport, nucleotide metabolic process, lipid metabolism, GTPase activity, cellular homeostasis, inorganic cation transport. Bioinformatics analysis of the differentially expressed miRNAs has identified a number of miRNAs with putative involvement in the hippocampus development process. This study lays a solid foundation for further studies to clarify important regulatory functions of miRNAs in hippocampal development.

MP72-15 GENE EXPRESSION IN THE BLOOD OF INTERSTITIAL CYSTITIS PATIENTS AND CONTROLS DISTINGUISHES PATHWAYS ASSOCIATED WITH INTERSTITIAL CYSTITIS AND PREDICTS CLINICAL RESPONSE TO CYCLOSPORINE THERAPY

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis1 Apr 2016MP72-15 GENE EXPRESSION IN THE BLOOD OF INTERSTITIAL CYSTITIS PATIENTS AND CONTROLS DISTINGUISHES PATHWAYS ASSOCIATED WITH INTERSTITIAL CYSTITIS AND PREDICTS CLINICAL RESPONSE TO CYCLOSPORINE THERAPY Daniel Shoskes, Karen Keslar, Barbara Tucky, and Robert Fairchild Daniel ShoskesDaniel Shoskes More articles by this author , Karen KeslarKaren Keslar More articles by this author , Barbara TuckyBarbara Tucky More articles by this author , and Robert FairchildRobert Fairchild More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1628AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Interstitial cystitis (IC) is a heterogeneous condition with urinary and systemic symptoms. Clinically relevant phenotyping has been a challenge, but some patients, especially those with Hunner's Ulcers, have evidence for immune activation. We hypothesize that gene expression signatures can be used as a biomarker to phenotype patients with IC and predict response to specific therapies. METHODS As part of a clinical trial of oral cyclosporine A (CyA), blood was collected from 26 IC patients prior to treatment and after 3 months of CyA at 3 mg/kg/d as well as from 20 asymptomatic controls. A training set of 6 controls and 6 IC patients including those who did or did not respond to CyA was initially run. RNA was isolated from whole blood (Tempus tubes) and analyzed using Illumina HT-12 v4 BeadChip arrays. PANTHER pathway analysis and DAVID functional classification were used for annotation and grouping of differently expressed genes. RESULTS Using the criteria of fold-changes less than 0.5 or greater than 2 and p<.05, we found 155 unique genes that were differently expressed in the blood of IC patients vs controls representing 55 pathways including inflammation mediated by chemokines and cytokines, T cell activation, integrin signaling, angiogenesis, and apoptosis. Comparing patients who subsequently had significant clinical improvement with CyA therapy to those who didn't improve, there were 37 genes with at least two fold differential expression. Using DAVID functional annotation clustering, these genes represented responses in inflammation, wound healing, and innate immunity. Finally, following CyA therapy, interleukin signaling changed in all patients but other pathways that changed were distinct between responders and non-responders. In CyA responders, the altered gene expression pathways were related to matrix remodeling (plasminogen activating cascade) and pain modulation (cannabinoid and dopamine receptor signaling). CONCLUSIONS Gene expression signatures in whole blood distinguished between IC patients and controls as well as between patients who would subsequently improve with CyA therapy. Pathways altered following CyA therapy in the responders could serve as potential targets for new drug development. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e958 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Daniel Shoskes More articles by this author Karen Keslar More articles by this author Barbara Tucky More articles by this author Robert Fairchild More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mood stabilizing drugs regulate transcription of immune, neuronal and metabolic pathway genes in Drosophila.

Lithium and valproate (VPA) are drugs used in the management of bipolar disorder. Even though they reportedly act on various pathways, the transcriptional targets relevant for disease mechanism and therapeutic effect remain unclear. Furthermore, multiple studies used lymphoblasts of bipolar patients as a cellular proxy, but it remains unclear whether peripheral cells provide a good readout for the effects of these drugs in the brain. We used Drosophila culture cells and adult flies to analyze the transcriptional effects of lithium and VPA and define mechanistic pathways. Transcriptional profiles were determined for Drosophila S2-cells and adult fly heads following lithium or VPA treatment. Gene ontology categories were identified using the DAVID functional annotation tool with a cut-off of p < 0.05. Significantly enriched GO terms were clustered using REVIGO and DAVID functional annotation clustering. Significance of overlap between transcript lists was determined with a Fisher's exact hypergeometric test. Treatment of cultured cells and adult flies with lithium and VPA induces transcriptional responses in genes with similar ontology, with as most prominent immune response, neuronal development, neuronal function, and metabolism. (i) Transcriptional effects of lithium and VPA in Drosophila S2 cells and heads show significant overlap. (ii) The overlap between transcriptional alterations in peripheral versus neuronal cells at the single gene level is negligible, but at the gene ontology and pathway level considerable overlap can be found. (iii) Lithium and VPA act on evolutionarily conserved pathways in Drosophila and mammalian models.

MicroRNA Expression in the Glaucomatous Retina.

MicroRNAs are small, endogenous noncoding RNAs that modulate posttranscriptional gene expression. Although the contribution of microRNAs to the pathogenesis of glaucomatous damage is unknown, supporting evidence from central nervous system (CNS) research suggests they may play a role. It was therefore hypothesized that microRNAs known to be altered in CNS injury are also altered in experimental glaucoma. Intraocular pressure (IOP) was elevated in rats by unilateral injection of hypertonic saline and IOP monitored for 5 weeks. After rats were killed, retrobulbar optic nerve sections were graded for damage. MicroRNA was extracted from whole retinae of eyes with advanced nerve damage (n = 8) and from normal, noninjected control eyes (n = 8). Quantitative PCRs were performed using a panel of 17 microRNAs, reported from CNS research to be implicated in mechanisms also linked to glaucomatous damage. Computationally and experimentally derived gene targets were identified for the differentially expressed microRNAs. These were then integrated with existing gene array data. Functional interpretation was performed using the Molecular Signatures Database and DAVID Functional Annotation Clustering. Eight microRNAs were significantly downregulated in glaucomatous retinae compared with controls (miR-181c, miR-497, miR-204, let-7a, miR-29b, miR-16, miR106b, and miR-25); miR-27a was significantly upregulated. Enrichment of targets associated with extracellular matrix/cell proliferation, immune system, and regulation of apoptosis were observed. Cholesterol homeostasis and mTORC-1 pathways showed reduced expression. MicroRNAs are differentially expressed in retinae of eyes with advanced glaucomatous damage compared with normal controls. Integrating microRNA with gene expression data may improve understanding of the complex biological responses produced by chronically elevated IOP.

Characterization and Isolation of Splenic Littoral Cells, a Possible Cellular Niche for Extramedullary Hematopoiesis in Myelofibrosis

Splenic littoral cells (LC) line the venous sinusoids of the human spleen and have been thought to act as blood cells filters. Little is known about SLCs beyond their preliminary characterization using immunochemistry and electron microscopy by others. Since SLCs comprise a significant portion of spleen, we hypothesized that SLC might be an important component of the splenic microenvironment that contributes to the development of extramedullary hematopoiesis in myelofibrosis (MF) patients.To further phenotypically characterize viable SLCs, surgically removed fresh spleens were treated with collagenase B and the hematopoietic cells were depleted using anti-CD45 micro-beads. The enriched CD45- cells were then stained and analyzed on a FACS analyzer. The SLCs were i, CD3-, CD45-, CD34-, CD8a+, CD31+ (Figure 1), ii, CD206+, CD21-, CD14-, FHOD1+, SIRP1a+, a phenotype identical to that previously reported based on IHC. SLCs and SECs were then identified by IHC in the red pulp of healthy individuals and MF patients using anti- CD8a and CD34 antibodies. SLCs were much more abundant than ECs in normal spleens. MF SLCs were however, much less condensed due to the expansion of hematopoietic cells than normal SLCs and the sinusoids encircled by SLCs were more elongated and had a more irregular shape as compared to normal spleen (Figure 2).To isolate the viable SLC and EC, fresh or cryopreserved spleen single cell suspensions were prepared as above and were FACS sorted for CD3-, CD45-, CD34-, CD8a+, CD31+ SLCs and CD3-, CD45-, CD34+, CD8a-, CD31+ SECs. The SSC/FSC profiles revealed two cell populations which could be distinguished by size and complexity, SLC being bigger and less uniform in size and shape. The CD31 signal intensity was greater in SEC than in SLC.The gene expression profiles of FACS sorted SLC, SEC and mononuclear cells (MNCs) were analyzed using human genome U133 Plus 2.0 arrays. DAVID Functional Annotation Clustering was applied to identify enriched gene clusters in selected lists. MNCs were significantly different from both SLC and EC, which expressed several clusters of genes involved in cell morphology, adhesion, and blood vessel formation. This indicated that SLCs were not closely related to myeloid cells but share features with SEC. SLC could however be differentiated from SEC by expression of genes involved in chromatin modification and regulation of RAS protein signaling, as well as intravesicle transportation genes, which may be related to their assumed capacity for phagocytosis. In addition SLC expressed many cytokines and adhesion molecules known to support hematopoiesis. Transcripts for various cytokines expressed by SEC and SLC were, however, distinct suggesting that they might serve as niches for different subpopulations of HSC/HPC. Preliminary microarray analysis of SLCs from an MF patient was also performed. Genes associated with apoptosis, intracellular lumens were upregulated as well as a cluster of genes in the cancer pathway. Cell cycle genes, genes of transcription regulation, and proteolysis were down-regulated in MF SLCs.Sorted SLC were also cultured in EC medium (ECM). The cultured SLCs were able to be repeatedly passaged. These cells were wide and spindle shaped. At a lower density, the cells tended to connect and organize into rings with a hollow space in the middle which resembled a splenic sinusoid. Immunostaining for CD8a and FHOD1 were conducted on these cultured cells, revealing that they continued to express these two markers. Interestingly, the expression of FHOD1, a stress fiber inducing protein was strongly polarized.To determine the relationship between SLC and the MF malignant clone SLCs from MF patients were isolated and assayed for JAK2V617F using allele specific NESTED PCR. Samples from three MF patients, were analyzed and no JAK2V617F was detected.In conclusion, we have isolated, cultured and characterized SLCs from normal and MF spleens for the first time. This will allow for further analysis of their function in normal individuals and individuals with blood diseases. [Display omitted] [Display omitted] DisclosuresSalama:Promedior: Consultancy.

Salivary Pellicle vs Whole Saliva: The Response of Oral Fibroblasts Based on a Genome-Wide Microarray.

Whole saliva comprises components of the salivary pellicle that spontaneously forms on surfaces of implants and teeth. However, there are no studies that functionally link the salivary pellicle with a possible change in gene expression. This study examined the genetic response of oral fibroblasts exposed to the salivary pellicle and whole saliva. Oral fibroblasts were seeded onto a salivary pellicle and the respective untreated surface. Oral fibroblasts were also exposed to freshly harvested sterile-filtered whole saliva. A genome-wide microarray of oral fibroblasts was performed, followed by gene ontology screening with DAVID functional annotation clustering, KEGG pathway analysis, and the STRING functional protein association network. Exposure of oral fibroblasts to saliva caused 61 genes to be differentially expressed (P < .05). Gene ontology screening assigned the respective genes into 262 biologic processes, 3 cellular components, 13 molecular functions, and 7 pathways. Most remarkable was the enrichment in the inflammatory response. None of the genes regulated by whole saliva was significantly changed when cells were placed onto a salivary pellicle. The salivary pellicle per se does not provoke a significant inflammatory response of oral fibroblasts in vitro, whereas sterile-filtered whole saliva does produce a strong inflammatory response.

The over-expression of aquaporin-1 alters erythroid gene expression in human erythroleukemia K562 cells.

Aquaporin genes are differentially expressed in primitive versus definitive erythropoiesis. Our previous research results showed that over-expression of aquaporin-1 (AQP1) gene greatly promotes the erythroid differentiation of erythroleukemia K562 cells, using benzidine staining and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis for representative erythroid-related genes, including γ-globin. But the molecular mechanisms underlying erythroid-specific gene regulation remain unknown. In this study, we demonstrated that AQP1 induced hemoglobins expression and altered erythroid gene expression by microarray analysis in K562 cells. The retroviral expression vector of AQP1 (pBABE-puro-AQP1) was constructed and infected K562 cells to establish a stable AQP1 over-expression cell line (K562-AQP1). AQP1 over-expression effectively inhibited cell proliferation and induced cell growth arrest in G1 phase of K562 cells. Then microarray profile was applied to analyze the differentially expressed genes which involved the mechanism of AQP1 in erythroid differentiation induction. The DAVID functional annotation clustering tool was used to identify biological functions enriched with the differentially expressed genes (n = 466 genes) and to group genes into clusters based on their functional similarity. Significant enrichment of genes involved in "oxygen transporter activity" (p = 3.8E-7) including hemoglobins (HBD, HBG, HBB, HBE1, and HBQ1), HEMGN, and EBP42 were validated by qRT-PCR. Moreover, silencing of HEMGN by RNA interference in K562-AQP1 cells resulted in down-regulation of these genes. These data provide a better understanding of the role of AQP1 in erythroid differentiation, by promoting HEMGN induction and other potential signaling pathways associated with hemoglobin induction.

Identification of Genes Differentially Expressed in Myogenin Knock-Down Bovine Muscle Satellite Cells during Differentiation through RNA Sequencing Analysis

BackgroundThe expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd) in primary bovine muscle satellite cells (MSCs).ResultsAbout 61–64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC) and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated.ConclusionsThis study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L), Protein lyl-1 (LYL1), various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle development and reveal the vital regulatory role of MyoG in retaining muscle cell differentiation.

Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain

Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV sensitivity of tumor cells are poorly understood. The aim of the present study was to look for gene expression changes in PC12 pheochromocytoma cells infected with an attenuated NDV strain that may be related to NDV susceptibility. PC12 cells were infected with the NDV strain MTH-68/H for 12h at a titer corresponding to the IC50 value. Total cytoplasmic RNA samples isolated from control and MTH-68/H-infected cells were analyzed using a rat specific Affymetrix exon chip. Genes with at least 2-fold increase or decrease in their expression were identified. MTH-68/H-induced gene expression changes of 9 genes were validated using quantitative reverse transcriptase PCR. A total of 729 genes were up- and 612 genes were down-regulated in PC12 cells infected with MTH-68/H. Using the DAVID functional annotation clustering tool, the up- and down-regulated genes can be categorized into 176 and 146 overlapping functional gene clusters, respectively. Gene expression changes affecting the most important signaling mechanisms (Toll-like receptor signaling, RIG-I-like receptor signaling, interferon signaling, interferon effector pathways, apoptosis pathways, endoplasmic reticulum stress pathways, cell cycle regulation) are analyzed and discussed in detail in this paper. NDV-induced gene expression changes described in this paper affect several regulatory mechanisms and dozens of putative key proteins that may determine the NDV susceptibility of various tumors. Further characterization of these proteins may identify susceptibility markers to predict the chances of virotherapeutic treatment of human tumors.

N‐hexane inhalation during pregnancy alters DNA promoter methylation in the ovarian granulosa cells of rat offspring

The N-hexane-induced impact on the reproductive system of the offspring of animals exposed to n-hexane has caused great concern. Pregnant Wistar rats inhaled 500, 2 500 or 12 500 ppm n-hexane during gestational days 1-20. Clinical characteristics and developmental indices were observed. Ovarian granulosa cells were extracted from F1 rats, the number of follicles was determined in ovarian slices and promoter methylation was assessed using MeDIP-Chip. Several methods were used to analyze the scanned genes, including the Gene Ontology Consortium tools, the DAVID Functional Annotation Clustering Tool, hierarchical clustering and KEGG pathway analysis. The results indicated that the live pups/litter ratio was significantly lowest in the 12 500 ppm group. A significant decrease in secondary follicles and an increase in atresic follicles were observed in the 12 500 ppm group. The number of shared demethylated genes was higher than that of the methylated genes, and the differentially methylated genes were enriched in cell death and apoptosis, cell growth and hormone regulation. The methylation profiles of the offspring from the 500 ppm and control groups were different from those of the 2500 and 12 500 ppm groups. Furthermore, the methylation status of genes in the PI3K-Akt and NF-kappa B signaling pathways was changed after n-hexane exposure. The Cyp11a1, Cyp17a1, Hsd3b1, Cyp1a1 and Srd5a1 promoters were hypermethylated in the n-hexane-exposed groups. These results indicate that the developmental toxicity of n-hexane in F1 ovaries is accompanied by the altered methylation of promoters of genes associated with apoptotic processes and steroid hormone biosynthesis.

Differential Expression of miR-130a in Postmortem Prefrontal Cortex of Subjects with Alcohol Use Disorders.

Emerging evidence suggests that neuroadaptations to alcohol may result from chronic alcohol consumption-induced expression changes of microRNAs (miRNAs) and their target genes. Studies with animal or cell culture models have demonstrated that ethanol exposure leads to miRNA expression alterations. However, there is limited information on miRNA expression in the brains of subjects with alcohol use disorders (AUDs). The present study aimed to analyze expression changes of miRNAs and their target genes in postmortem prefrontal cortex (PFC) of AUD subjects. Genome-wide miRNA and mRNA expression was examined in postmortem PFC of 23 European Australia AUD cases and 23 matched controls using the Illumina HumanHT-12 v4 Expression BeadChip array, which targets 43,270 coding transcripts and 3,961 non-coding transcripts (including 574 miRNA transcripts). Multiple linear regression analysis and permutation test were performed to identify differentially expressed miRNAs and their target mRNAs. Target gene prediction, Gene Set Enrichment Analysis (GESA), and DAVID functional annotation clustering analysis were applied to identify AUD-associated gene sets and biological modules. Two miRNAs and 787 coding genes were differentially expressed in the PFC of AUD cases [miR-130a (downregulated): Ppermutation=0.023, miR-604 (upregulated): Ppermutation=0.019, coding genes: 1.6×10-5≤Ppermutation≤0.05; but all P values did not survive multiple-testing correction]. GESA showed that the 202 predicted target genes of miR-130a were highly enriched in differentially expressed genes (Pnominal<0.001), but not the 116 predicted target genes of miR-604 (Pnominal=0.404). DAVID functional clustering further revealed that the hub target genes (e.g., ITPR2 and ATP1A2) of miRNA130a were mainly responsible for regulating ion channel function. This study provides evidence that downregulation of miR-130a may lead to altered expression of a number of genes in the PFC of AUD subjects. Further studies are warranted to confirm these findings in replication samples and other reward-related brain regions.

Microarray analysis of gene expression profiles in ripening pineapple fruits

BackgroundPineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening.ResultsMicroarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested.ConclusionsThis is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

Distinct gene expression profiles characterize cellular responses to palmitate and oleate

Obese individuals are both insulin resistant and have high levels of circulating free fatty acids (FFAs). In cell culture, saturated but not unsaturated fatty acids induce endoplasmic reticulum (ER) stress. We hypothesized that chronic exposure to low dose fatty acids would significantly attenuate the acute stress response to a saturated fatty acid challenge and that unsaturated fatty acids (oleate) would be more protective than saturated fatty acids (palmitate). The ER stress response to palmitate was reduced after low dose fatty acid exposure in human hepatoma cells. Palmitate and oleate gave distinctive transcript responses, both acutely and after chronic low dose exposure. Differentially regulated pathways included lipid, cholesterol, fatty acid, and triglyceride metabolism, and IkappaB kinase and nuclear factor kappaB kinase inflammatory cascades. Oleate reduced palmitate-induced changes significantly more than low dose palmitate and completely blocked palmitate-induced phosphoinositide 3 kinase inhibitor (PIK3IP1) as well as induction of GADD45A and B. These changes are predicted to alter the PI3 kinase pathway and the pro-apoptotic p38 MAPK pathway. We recapitulated the oleate response by small interfering RNA-mediated block of PIK3IP1 stimulation with palmitate and significantly protected cells from palmitate-mediated ER stress. We show that transcriptional responses to oleate and palmitate are distinct, broad, and often discordant. We identified several potential candidates that may direct the transcriptional networks and demonstrate that PIK3IP1 partially accounts for the protective effects of oleate.

Abstract 4924: Gene-specific hyper and hypomethylation in follicular lymphoma progression

Abstract Epigenetic mechanisms involved in the origin and transformation of indolent lymphomas to aggressive forms are poorly understood. Based on the comparison of methylation states of follicular lymphoma (FL) pre and post- transformation, it has been suggested that DNA methylation changes occur during the early stages of lymphomagenesis. To obtain a more complete understanding of methylation aberrations responsible for progression and transformation of FL, we analyzed 20 untreated FL biopsies (10 low grade, 10 high grade), 5 diffuse large B cell lymphoma (DLBCL) biopsies, and 5 benign reactive lymph node (RLN) biopsies by using methyl-DNA immunoprecipitation coupled with analysis with Agilent 244K human promoter ChIP-on-chip microarrays. Our analysis revealed statistically significant differences in methylation status for 120 genes between FL samples and RLN samples and for 23 genes between low and high grade FL samples. To investigate the biological significance of these findings, we used DAVID functional annotation clustering algorithms and Gominer software. We found that the most methylation-enriched functional groups were comprised of genes with roles in cell cycle control, adhesion and motility, transcription regulation, homeobox, and developmental genes. In order to validate the DNA methylation differences of the significant gene regions, we utilized the MassARRAY EpiTYPER assay. Our data identified 13 genes (CYP26B1, GRM7, HS3ST2, DOCK5, GRB10, SST, TAC1, PALM2-AKAP, FLJ40125, ST6GAL2, WDR35, NCAM1, SATB2) with increased levels of DNA methylation in FL samples compared to controls; 3 of these genes (ST6GAL2, WDR35, NCAM1) exhibited increased methylation in high grades FLs and 1 gene (SATB2) in low grades FLs. We are currently investigating gene specific DNA hypomethylation in low versus high grade FL and FL versus DLBCL. Our data are the first to characterize the global changes in methylation that occur in FL progression. Our findings suggest that high grade FL are associated with hypermethylation of a specific subset of genes compared to low grade subtypes. Ongoing efforts include investigating the functional relevance of these methylation changes by treating, in vitro, FL cell lines with demethylating agents to evaluate changes in gene expression levels and correlating with phenotype alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4924.