Functional Annotation Clustering Research Articles

Abstract P1-06-05: DNA methylation-based classification are mostly concordant with intrinsic subtypes of breast cancer

Abstract Backgrounds: Breast cancer (BC) is a heterogeneous disease and usually divided into 5 subtypes according to clinicopathologic features. These subtypes have concordance with mRNA-based subtypes called as "intrinsic subtypes". Further gene expression analysis categorized triple negative BCs into 7 sub-subtypes which relate to clinical behaviors. DNA methylation is one of the epigenetic systems to regulate gene transcription. DNA methylation in CpG islands (CGIs) associated with the gene promoter region affects gene expression levels. Recently genome-wide analyses suggested that methylation status not only in CGIs, but also in CpG shores (within 2000 bps from CGIs) and CpG shelves (2000-4000 bps from CGIs) contribute to gene silencing. Thus, we hypothesized that intrinsic subtypes may possess specific methylation profiles especially in regions surrounding CGIs (islands, shores and shelves). To date, several reports demonstrated each intrinsic subtype has specific methylation patterns in CGIs. They indicate that DNA methylation is promising markers for cancer detection, prediction of therapeutic response and clinical outcomes. However, there were no report that conducted a genome-wide high-resolution epigenetic analysis for BC. Objectives: This study aims to determine whether specific DNA methylation patterns exist in CGIs, shores and shelves in BC cells, and whether the DNA methylation-based classification correlates with intrinsic subtypes, furthermore, to identify the subtype specific epigenetic markers using high-resolution methylation array. Materials and Methods: DNA was extracted from 31 samples of 28 BC cell lines (8 luminal, 18 basal, 2 unclassified) and 3 samples of 3 non-BC cell lines. We performed genome-wide methylation analysis using Illumina Infinium HumanMethylation450 BeadChip. We did unsupervised hierarchical clustering analysis of cell lines using top 3000 variably methylated (VM) loci among cancer cell lines. We also analyzed top 3000 VM CGI-related loci. Gene functional annotation clustering was performed using DAVID. Results: In top 3000 VM loci, CGIs were most frequently observed (43.0%). According to clustering analysis using top 3000 VM loci, cell lines were roughly classified into 4 clusters. There were trends that cell lines of same subtype fell into the same cluster, but not definitely. In case of clustering using top 3000 VM CGI-related loci, cell lines were clearly divided into 2 clusters, high and low methylated clusters. High methylated cluster contained almost all luminal cell lines and low methylated cluster contained most of basal cell lines and all non-BC cell lines. In low methylated cluster, cell lines of the same sub-subtype, such as Basal-like 1 and 2, were clustered closely. Gene functional annotation clustering suggested that genes related to "transcription" and "differentiation" were more frequently regulated by epigenetics. Conclusions: High-resolution methylation analysis revealed methylation-based clusters were concordant with intrinsic subtypes in BC cells. Transcriptional factors and differentiation-related genes could be differently regulated by epigenetics among different subtypes of BC. Citation Format: Natsue Uehiro, Fumiaki Sato, Sunao Tanaka, Fengling Pu, Junji Itou, Shigehira Saji, Masakazu Toi. DNA methylation-based classification are mostly concordant with intrinsic subtypes of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-05.

Genome-wide DNA methylation study of hip and knee cartilage reveals embryonic organ and ‎skeletal system morphogenesis as major pathways involved in osteoarthritis

Purpose: The objectives of this study were to describe the genome wide DNA methylation changes in hip ‎and knee osteoarthritis (OA) and to identify novel genes and pathways involved in OA by ‎comparing the DNA methylome of the hip and knee osteoarthritic cartilage with those of healthy ‎individuals. ‎ Methods: OA cartilage samples were collected from the tibial plateau and femoral head articular surfaces of ‎patients with severe OA undergoing total hip/knee replacement surgery. Healthy cartilage ‎samples were obtained from individuals with femoral neck fractures, but no OA. Chondrocyte ‎DNA methylation was assayed by Illumina Infinium HumanMethylation450 BeadChip array, ‎allowing for the analysis of >480,000 CpG sites. A sub-analysis was also done to compare hip ‎and knee OA. An independent sample T-test was conducted for each CpG site and those sites ‎with at least 10% methylation difference and a P-value of <0.0001 were selected as differentially ‎methylated regions (DMR). DAVID v6.7 was used for the functional annotation clustering of ‎the DMR genes. ‎ Results: The study population comprises 5 patients with hip OA, 6 patients with knee OA and 7 hip ‎samples from healthy individuals. The comparisons of hip, knee and combined hip/knee OA ‎patients with controls resulted in 26, 72, and 103 DMRs, respectively. The comparison between ‎hip and knee OA revealed 67 DMRs. The overall number of the sites after considering the ‎overlaps was 239, among which 151 sites were annotated to 145 genes. 28 of these genes were ‎reported in previous methylation studies of OA. The functional annotation clustering of the ‎identified genes revealed clusters significantly enriched in skeletal system morphogenesis ‎‎(bonferroni corrected p=8.4e-9) and development (bonferroni corrected p=9.0e-6), embryonic ‎organ (bonferroni corrected p=1.4e-5) and skeletal system morphogenesis (bonferroni corrected ‎p=9.9e-7), as well as Homeobox genes (bonferroni corrected p=3.1e-4). A sub-analysis after the ‎removal of the genes differentially methylated between hip and knee OA highlighted the same ‎pathways excluding Homeobox genes.‎ Conclusions: We demonstrated a number of CpG sites and genes across the genome differentially methylated ‎among OA patients and controls, a remarkable portion of which seem to be involved in potential ‎etiologic mechanisms of OA. Genes involved in skeletal developmental pathways and embryonic ‎organ morphogenesis may be a potential area of concentration for future osteoarthritis ‎genetic/epigenetic research. ‎

Mo2037 Phosphodiesterase 2A Mediate Neonatal Inflammation-Induced Visceral Hypersensitivity in a Mouse Model of Irritable Bowel Syndrome

G A A b st ra ct s in each disease type. Figure 1 shows the number of differentially regulated transcripts in various comparisons. IBS vs CD showed the highest number of deregulated transcripts (N= 686). Table 1 shows GO terms, associated genes and p values inferred from functional annotation clustering for all the comparisons. Clustering revealed upregulation of inflammatory genes (e.g., CD38molecule; TRGC2: T-cell receptor gamma locus; IGHG4: Immunoglobulin heavy constant gamma 4 (G4M marker) for both CD and UC compared to IBS. There were 4 transcripts deregulated between IBS and HCs. microRNA miR-4461 was significantly downregulated (FC: -2.2, p=0.006) in IBS compared to HCs. However, at a lower fold change cutoff (FC>1.3), inflammation and defense response associated genes, such as NFKB1A, S100 Calcium Binding Protein A12 (S100A12), NCF1B, were upregulated in IBS patients vs. HCs. miR-27A, reported as a class of adipogenic inhibitors, was upregulated in IBS patients. Most genes that had significantly different expressions in IBS vs. HCs were unannotated. Conclusions:Our findings suggest that while inflammatory genes were modestly upregulated in IBS vs. HCs, they were much lower compared to IBD. Interestingly, miR-27, which is upregulated in obesity, is also upregulated in IBS, suggesting that there may be a shared pathophysiologic mechanism. Thus, comparing mRNA profiles of IBS to that of GI disease controls such as IBD and healthy controls may lead to a better understanding of the pathophysiology of IBS and potentially a diagnostic biomarker. Gene Ontology (GO) Terms Associated with IBS vs Healthy and Disease Controls

Pharmacologic inhibition of the CK2-mediated phosphorylation of B23/NPM in cancer cells selectively modulates genes related to protein synthesis, energetic metabolism, and ribosomal biogenesis.

B23/NPM is a multifunctional nucleolar protein frequently overexpressed, mutated, or rearranged in neoplastic tissues. B23/NPM is involved in diverse biological processes and is mainly regulated by heteroligomer association and posttranslational modification, phosphorylation being a major posttranslational event. While the role of B23/NPM in supporting and/or driving malignant transformation is widely recognized, the particular relevance of its CK2-mediated phosphorylation remains unsolved. Interestingly, the pharmacologic inhibition of such phosphorylation event by CIGB-300, a clinical-grade peptide drug, was previously associated to apoptosis induction in tumor cell lines. In this work, we sought to identify the biological processes modulated by CIGB-300 in a lung cancer cell line using subtractive suppression hybridization and subsequent functional annotation clustering. Our results indicate that CIGB-300 modulates a subset of genes involved in protein synthesis (ES = 8.4, p < 0.001), mitochondrial ATP metabolism (ES = 2.5, p < 0.001), and ribosomal biogenesis (ES = 1.5, p < 0.05). The impairment of these cellular processes by CIGB-300 was corroborated at the molecular and cellular levels by Western blot (P-S6/P-4EBP1, translation), confocal microscopy (JC-1, mitochondrial potential), qPCR (45SrRNA/p21, ribosome biogenesis), and electron microscopy (nucleolar structure, ribosome biogenesis). Altogether, our findings provide new insights on the potential relevance of the CK2-mediated phosphorylation of B23/NPM in cancer cells, revealing at the same time the potentialities of its pharmacological manipulation for cancer therapy. Finally, this work also suggests several candidate gene biomarkers to be evaluated during the clinical development of the anti-CK2 peptide CIGB-300.

T-cell activation and early gene response in dogs.

T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR), and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA) (5μg/ml), including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), early growth response 1 (EGR1), growth arrest and DNA damage-inducible gene (GADD45B), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS), early growth response 2 (EGR2), hemogen (HEMGN), polo-like kinase 2 (PLK2) and polo-like kinase 3 (PLK3). Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in cell cycle regulation. This study provides a comprehensive analysis of the early T-cell gene response to activation in dogs.

Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.

A specific type of cyclin-like F-box domain gene is involved in the cryogenic autolysis of Volvariella volvacea

Cryogenic autolysis is a typical phenomenon of abnormal metabolism in Volvariella volvacea. Recent studies have identified 20 significantly up-regulated genes via high-throughput sequencing of the mRNAs expressed in the mycelia of V. volvacea after cold exposure. Among these significantly up-regulated genes, 15 annotated genes were used for functional annotation cluster analysis. Our results showed that the cyclin-like F-box domain (FBDC) formed the functional cluster with the lowest P-value. We also observed a significant expansion of FBDC families in V. volvacea. Among these, the FBDC3 family displayed the maximal gene expansion in V. volvacea. Gene expression profiling analysis revealed only one FBDC gene in V. volvacea (FBDV1) that was significantly up-regulated, which is located in the FBDC3 family. Comparative genomics analysis revealed the homologous sequences of FBDV1 with high similarity were clustered on the same scaffold. However, FBDV1 was located far from these clusters, indicating the divergence of duplicated genes. Relative time estimation and rate test provided evidence for the divergence of FBDV1 after recent duplications. Real-time RT-PCR analysis confirmed that the expression of the FBDV1 was significantly up-regulated (P < 0.001) after cold-treatment of V. volvacea for 4 h. These observations suggest that the FBDV1 is involved in the cryogenic autolysis of V. volvacea.

Functional Gene Networks: R/Bioc package to generate and analyse gene networks derived from functional enrichment and clustering.

Summary: Functional Gene Networks (FGNet) is an R/Bioconductor package that generates gene networks derived from the results of functional enrichment analysis (FEA) and annotation clustering. The sets of genes enriched with specific biological terms (obtained from a FEA platform) are transformed into a network by establishing links between genes based on common functional annotations and common clusters. The network provides a new view of FEA results revealing gene modules with similar functions and genes that are related to multiple functions. In addition to building the functional network, FGNet analyses the similarity between the groups of genes and provides a distance heatmap and a bipartite network of functionally overlapping genes. The application includes an interface to directly perform FEA queries using different external tools: DAVID, GeneTerm Linker, TopGO or GAGE; and a graphical interface to facilitate the use.Availability and implementation: FGNet is available in Bioconductor, including a tutorial. URL: http://bioconductor.org/packages/release/bioc/html/FGNet.htmlContact: jrivas@usal.esSupplementary information: Supplementary data are available at Bioinformatics online.

Sex-related gene expression profiles in the adrenal cortex in the mature rat: microarray analysis with emphasis on genes involved in steroidogenesis.

Notable sex-related differences exist in mammalian adrenal cortex structure and function. In adult rats, the adrenal weight and the average volume of zona fasciculata cells of females are larger and secrete greater amounts of corticosterone than those of males. The molecular bases of these sex-related differences are poorly understood. In this study, to explore the molecular background of these differences, we defined zone- and sex-specific transcripts in adult male and female (estrous cycle phase) rats. Twelve-week-old rats of both genders were used and samples were taken from the zona glomerulosa (ZG) and zona fasciculata/reticularis (ZF/R) zones. Transcriptome identification was carried out using the Affymetrix® Rat Gene 1.1 ST Array. The microarray data were compared by fold change with significance according to moderated t-statistics. Subsequently, we performed functional annotation clustering using the Gene Ontology (GO) and Database for Annotation, Visualization and Integrated Discovery (DAVID). In the first step, we explored differentially expressed transcripts in the adrenal ZG and ZF/R. The number of differentially expressed transcripts was notably higher in the female than in the male rats (702 vs. 571). The differentially expressed genes which were significantly enriched included genes involved in steroid hormone metabolism, and their expression levels in the ZF/R of adult female rats were significantly higher compared with those in the male rats. In the female ZF/R, when compared with that of the males, prevailing numbers of genes linked to cell fraction, oxidation/reduction processes, response to nutrients and to extracellular stimuli or steroid hormone stimuli were downregulated. The microarray data for key genes involved directly in steroidogenesis were confirmed by qPCR. Thus, when compared with that of the males, in the female ZF/R, higher expression levels of genes involved directly in steroid hormone synthesis were accompanied by lower expression levels of genes regulating basal cell functions.

BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation.

The SH-SY5Y culture system is a convenient neuronal model with the potential to elaborate human/primate-specific transcription networks and pathways related to human cognitive disorders. While this system allows for the exploration of specialized features in the human genome, there is still significant debate about how this model should be implemented, and its appropriateness for answering complex functional questions related to human neural architecture. In view of these questions we sought to characterize the posttranscriptional regulatory structure of the two-stage ATRA differentiation, BDNF maturation protocol proposed by Encinas et al. (2000) using integrative whole-genome gene and microRNA (miRNA) expression analysis. We report that ATRA-BDNF induced significant increases in expression of key synaptic genes, brain-specific miRNA and miRNA biogenesis machinery, and in AChE activity, compared with ATRA alone. Functional annotation clustering associated BDNF more significantly with neuronal terms, and with synaptic terms not found in ATRA-only clusters. While our results support use of SH-SY5Y as a neuronal model, we advocate considered selection of the differentiation agent/s relative to the system being modeled.

Functional relevance for multiple sclerosis-associated genetic variants.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of central nervous system. Many genetic variants associated with MS have been identified by genome-wide association studies, but functional mechanism underlying the associations is largely unclear. Utilizing the publically available datasets, we carried out gene relationships among implicated loci (GRAIL) analyses to search for MS-associated SNPs/genes. Expression quantitative trait loci (eQTLs) analyses were conducted to identify eQTL SNPs/target genes. Further, functional prediction for SNP, differential gene expression, and functional annotation clustering analyses for gene were conducted to explore their functional relevance to MS. Among the 284 identified MS-associated SNPs (P < 10(-4)), eQTL analysis showed that 45 SNPs act as cis-effect regulators on 19 MS-associated genes. Among the 19 eQTL target genes, 14 showed significantly differential expressions in MS-related cells. Among the 45 SNPs, 15 were predicted most likely located in transcription factor (TF) binding sites, and five predicted SNPs (rs3095329 of TUBB, rs9469220/rs2647046 of HLA-DQB1, rs11154801 of AHI1, and rs1062158 of NDFIP1) have corresponding target genes with significantly differential expressions in multiple cell groups, while rs7194 of HLA-DRA was predicted in the has-miR-6507-3p binding site. The functional evidence, taken together, highlighted the functional relevance of the six SNPs to MS. The present findings provide novel insights into the functional mechanisms underlying the MS-associated genetic variants, which improve our understanding of the genetic association for MS.

Gene expression in the skin of dogs sensitized to the house dust mite Dermatophagoides farinae.

Atopic dermatitis is a multifactorial allergic skin disease in humans and dogs. Genetic predisposition, immunologic hyperreactivity, a defective skin barrier, and environmental factors play a role in its pathogenesis. The aim of this study was to analyze gene expression in the skin of dogs sensitized to house dust mite antigens. Skin biopsy samples were collected from six sensitized and six nonsensitized Beagle dogs before and 6 hr and 24 hr after challenge using skin patches with allergen or saline as a negative control. Transcriptome analysis was performed by the use of DNA microarrays and expression of selected genes was validated by quantitative real-time RT-PCR. Expression data were compared between groups (unpaired design). After 24 hr, 597 differentially expressed genes were detected, 361 with higher and 226 with lower mRNA concentrations in allergen-treated skin of sensitized dogs compared with their saline-treated skin and compared with the control specimens. Functional annotation clustering and pathway- and co-citation analysis showed that the genes with increased expression were involved in inflammation, wound healing, and immune response. In contrast, genes with decreased expression in sensitized dogs were associated with differentiation and barrier function of the skin. Because the sensitized dogs did not show differences in the untreated skin compared with controls, inflammation after allergen patch test probably led to a decrease in the expression of genes important for barrier formation. Our results further confirm the similar pathophysiology of human and canine atopic dermatitis and revealed genes previously not known to be involved in canine atopic dermatitis.

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.

Overexpression of FABP7 promotes cell growth and predicts poor prognosis of clear cell renal cell carcinoma

ObjectivesRenal cell carcinoma (RCC) is one of the most lethal urologic malignancies; however, the molecular events supporting RCC carcinogenesis remain poorly understood. The aim of the present study was to determine the differential expression of genes between normal kidney and clear cell RCC (ccRCC) samples and investigate the biological function of the most frequently altered gene in RCC cells. Materials and methodsThe gene expression profiles of 60 ccRCC and matched normal kidney samples from The Cancer Genome Atlas were analyzed. The altered genes were subjected to functional annotation clustering and integrative pathway analysis. The expression of one of the most frequently altered gene, fatty acid–binding protein (FABP) 7, in ccRCC and matched normal kidney samples was verified by immunohistochemistry and the association between FABP7 level and patient survival was investigated. Furthermore, FABP7 DNA copy number alteration, methylation, and mutation status in ccRCC from The Cancer Genome Atlas were analyzed. Finally, FABP7-overexpressing RCC cells were generated to determine the function of FABP7 in cell growth and the potential mechanisms of action. ResultsFABP7 was significantly up-regulated in ccRCC, and the expression of FABP7 positively correlated with advanced clinical stage and poor survival of patients with ccRCC. FABP7 DNA copy number alteration was not frequently detected in ccRCC, and no mutation of FABP7 was found. FABP7 messenger RNA expression inversely correlated with its DNA methylation. Overexpression of FABP7 in RCC cells enhanced cell growth, clonogenicity, cell cycle progression and activated both extracellular-signal-regulated kinases (ERK) and signal transducer and activator of transcription 3 (Stat3) signaling. ConclusionFABP7 is overexpressed in ccRCC and promotes cell growth by the activation of ERK and Stat3 signaling pathways. Evidence from the clinical observations and experimental data suggests that FABP7 is a novel prognostic marker and potential therapeutic target for ccRCC.

Neuroproteomics in the auditory brainstem: candidate proteins for ultrafast and precise information processing.

In the mammalian auditory brainstem, the cochlear nuclear complex (CN) and the superior olivary complex (SOC) feature structural and functional specializations for ultrafast (<1 ms) and precise information processing. Their proteome, the basis for structure and function, has been rarely analyzed so far. Here we identified and quantified the protein profiles of three major auditory brainstem regions of adult rats, the CN, the SOC, and the inferior colliculus (IC). The rest of the brain served as a reference. Via label-free quantitative mass spectrometry and 2-D DIGE/MALDI-MS, we identified 584 and 297 proteins in the plasma membrane/synaptic vesicle proteome and the cytosolic proteome, respectively. 'Region-typical' proteins, i.e., those with higher abundance in one region than in the other three, were considered candidates for functional specializations. Key proteins were validated via Western blots and immunohistochemistry. Functional annotation clustering revealed an overrepresentation of neurofilament proteins among the CN+SOC-typical proteins. These are related to regulation of axon diameter and, thereby, conduction velocity. Interestingly, the sets of synapse-associated proteins differed between regions. For example, synaptotagmin-2 (Syt2), a Ca2+ sensor for fast exocytosis, was CN+SOC+IC-typical, whereas Syt1 was CN+SOC+IC-atypical. Together, our quantitative comparison of protein profiles has revealed several interesting candidate proteins for ultrafast and precise information processing.

Indoleamine 2, 3-Dioxygenase Inhibition Shifts Gene Expression From Coding to Non-Coding Sequences Following Ischemia-Reperfusion Injury of the Kidney.

Background: Indoleamine 2,3 dioxygenase (IDO) minimizes rejection in rat renal transplantation. Ischemia-reperfusion injury (IRI) is unavoidable in renal transplantation and correlates with graft loss. Despite its effects on rejection, IDO may facilitate renal IRI. To address this paradox, we queried for shifts in the gene expression signatures of renal IRI with and without IDO blockade. Methods: Male SD rats (N=5/group) underwent IRI (30 min ischemia, 60 min recovery) or sham surgery (SHA). To test IDO blockade, pretreatment with 1-methyl-D-tryptophan (1MT) or vehicle (VEH) was given. Expression of the renal cortical transcriptome was determined using the GeneChip®Rat Gene 1.0 ST Array (Affymetrix). Gene annotations were provided by Affymetrix. DAVID v6.7 was used for functional annotation clustering. Two comparisons (CMP) were made: IRI vs SHA with VEH (CMP1) and 1MT vs VEH in IRI (CMP2). CMP1 is a positive control for IRI, and CMP2 assessed the effect of IDO blockade in IRI. Results: The array contains over 27,000 gene-level probe sets, of which 8711 exhibited an interquartile range of 0.25, and were thus included in the analysis. Coding sequences altered: CMP1=108, CMP2=18, no overlap. Non-coding sequences altered: CMP1=3, CMP2=66, no overlap. The coding sequences changed in CMP1 (number of transcripts from various categories) included genes most commonly from apoptosis (17), cell biosynthesis (20-23), RNA associated processes (17-26) and transcriptional processes (17-29), none of which were altered with 1MT pre-treatment. The non-coding sequences changed in CMP2 were predominantly snoRNA (50). Conclusions: Blocking IDO in IRI induces a shift in gene expression from coding to non-coding transcripts. Loss of apoptosis activating coding sequences and/or activation of non-coding snoRnA sequences may contribute to improved recovery from renal IRI in 1MT treated rats.

CGG repeats in RNA modulate expression of TDP-43 in mouse and fly models of fragile X tremor ataxia syndrome

Determining the molecular mechanism(s) leading to Purkinje neuron loss in the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS) is limited by the complex morphology of this cell type. Purkinje neurons are notoriously difficult to isolate and maintain in culture presenting considerable difficultly to identify molecular changes in response to expanded CGG repeat (rCGG)-containing mRNA that induces neurotoxicity in FXTAS. Several studies have uncovered a number of RNA-binding proteins involved in translation that aberrantly interact with the CGG-containing RNA; however, whether these interactions alter the translational profile of cells has not been investigated. Here we employ bacTRAP translational profiling to demonstrate that Purkinje neurons ectopically expressing 90 CGG repeats exhibit a dramatic change in their translational profile even prior to the onset of rCGG-induced phenotypes. This approach identified ∼500 transcripts that are differentially associated with ribosomes in r(CGG)₉₀-expressing mice. Functional annotation cluster analysis revealed broad ontologies enriched in the r(CGG)₉₀ list, including RNA binding and response to stress. Intriguingly, a transcript for the Tardbp gene, implicated in a number of other neurodegenerative disorders, exhibits altered association with ribosomes in the presence of r(CGG)₉₀ repeats. We therefore tested and showed that reduced association of Tardbp mRNA with the ribosomes results in a loss of TDP-43 protein expression in r(CGG)₉₀-expressing Purkinje neurons. Furthermore, we showed that TDP-43 could modulate the rCGG repeat-mediated toxicity in a Drosophila model that we developed previously. These findings together suggest that translational dysregulation may be an underlying mechanism of rCGG-induced neurotoxicity in FXTAS.

Gene expression profile of LPS-stimulated dendritic cells induced by a recombinant Sj16 (rSj16) derived from Schistosoma japonicum.

Sj16, a 16-kDa protein secreted from Schistosoma japonicum, has been demonstrated an anti-inflammatory effect in vitro and in vivo, but its mechanism is still not clear. In this study, microarray analysis was performed to investigate the effects of recombinant Sj16 (rSj16) on the gene expression of the lipopolysaccharide (LPS)-stimulated dendritic cells (DCs). Immature DCs were treated with LPS, LPS + recombinant Sj16 (rSj16), or rSj16 alone for 24 h, and the gene expression profiles were examined using complementary DNA (cDNA) microarrays. With the cutoff value of 2-fold change in the expression, 509 genes were affected, 226 genes upregulated, and 283 genes downregulated after adding rSj16. Analysis by functional annotation clustering tool showed that rSj16-affected genes mainly associated with inflammatory response, defense response, regulation of immune system process, apoptosis, and cell migration. The results revealed that rSj16 reduced the LPS-induced pro-inflammatory genes such as cytokines (e.g., IL6, IL18, IFN-γ, IL12a, IL1b), chemokines, and receptors (e.g., CXCL1, CXCL9, CCL5, CCR5, CCR1, CCR2, CXCR3) and increased the anti-inflammatory gene IL-10. Further data mining of these genes by pathway analysis showed that genes regulated by rSj16 were significantly involved in cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, antigen processing and presentation, and Jak-STAT signaling pathway. In addition, quantitative real-time PCR (qRT-PCR) and Western blot analysis showed that rSj16 downregulated the expression of inhibitor of nuclear factor kappa-β kinase subunit beta (IKKβ) and nuclear factor-kappa β p65 (NF-κβ) messenger RNA (mRNA) and inhibited the phosphorylation of IKKβ and the NF-κB p65 protein, which implied that rSj16 exerting immunomodulatory effects by suppressing NF-κB signaling pathway. These results provide useful information in further understanding of the immunoregulation mechanisms of Sj16, and it is indicated that Sj16 could be as a potential molecule for the immunosuppressant drug development.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database forAnnotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Riding the Spermatogenic Wave: Profiling Gene Expression Within Neonatal Germ and Sertoli Cells During a Synchronized Initial Wave of Spermatogenesis in Mice1

Continual sperm production relies on germ cells undergoing spermatogenesis asynchronously. As a result, the testis always contains a mixed population of germ cells at different stages of their differentiation process. The heterogeneous nature of the testis makes profiling gene expression within Sertoli cells or specific populations of germ cells impossible when a wild-type testis is assessed. We recently reported a unique method for synchronizing spermatogenesis without affecting fertility by manipulating RA levels within the neonatal testis. Using this protocol, combined with the RiboTag transgenic mouse line, we have mapped the Sertoli and germ cell translatome during the initial synchronized wave of spermatogenesis. Using microarray analysis, we identified 392 and 194 germ cell and Sertoli cells transcripts, respectively, that dynamically change during spermatogonial differentiation, division, and the onset of meiosis. Functional annotation clustering revealed that transcripts enriched in germ cells were mostly associated with meiosis (21 transcripts), chromatin organization (12 transcripts), and cell cycle (3 transcripts). In addition, glycoproteins (65 transcripts), cell adhesion (15 transcripts), and cell junction (13 transcripts) transcripts were overrepresented in the Sertoli cell-enriched list. These datasets represent the first transcriptional analysis of spermatogonial differentiation, division, and meiotic onset. These data suggest that several of the genes encoding meiotic proteins are expressed and are actively being translated well before germ cells enter meiosis. In addition, this study provides novel candidate genes, Asf1b and Esyt3, that may be involved in the regulation of spermatogonial chromatin reorganization, germ-Sertoli cell interactions, and/or blood-testis barrier formation.