Process Gene Ontology Research Articles

Timing for success: expression phenotype and local adaptation related to latitude in the boreal forest tree, Populus balsamifera

Timing of seasonal dormancy is among the most important traits allowing forest tree populations to span latitudinal gradients of temperature and growing season length. We used RNA-sequencing (RNA-seq) data to identify gene expression differences between latitude-adapted populations and search for elevated genetic differentiation, a signature of adaptation, between northern and southern populations of Populus balsamifera. RNA was extracted from leaf tissues collected from trees originating from two northern (65° N) and two southern (50° N) populations and growing in a common garden. Leaves for RNA-seq were collected on the same day in late July, when southern trees were actively growing and northern trees had set bud. Transcripts of 2,594 genes were more abundant in actively growing leaves, whereas 1,424 were more abundant in leaves of trees that had set bud. The actively growing southern trees exhibited overexpression of genes in the biological process gene ontology (GO) domain, whereas dormant northern trees exhibited overexpression of genes in the cellular component domain. We identified relatively few genes (641) bearing signatures consistent with local adaptation to latitude for three or more SNPs. Exons from differentially expressed (DE) genes were not more likely than non-DE genes to exhibit patterns of latitudinal differentiation consistent with local adaptation. These results indicate that genes associated with differences in the timing of bud set, which is correlated with initiation of dormancy, were not more likely than other categories to exhibit latitudinal differentiation in exonic regions.

Investigation of De Novo Unique Differentially Expressed Genes Related to Evolution in Exercise Response during Domestication in Thoroughbred Race Horses

Previous studies of horse RNA-seq were performed by mapping sequence reads to the reference genome during transcriptome analysis. However in this study, we focused on two main ideas. First, differentially expressed genes (DEGs) were identified by de novo–based analysis (DBA) in RNA-seq data from six Thoroughbreds before and after exercise, here-after referred to as “de novo unique differentially expressed genes” (DUDEG). Second, by integrating both conventional DEGs and genes identified as being selected for during domestication of Thoroughbred and Jeju pony from whole genome re-sequencing (WGS) data, we give a new concept to the definition of DEG. We identified 1,034 and 567 DUDEGs in skeletal muscle and blood, respectively. DUDEGs in skeletal muscle were significantly related to exercise-induced stress biological process gene ontology (BP-GO) terms: ‘immune system process’; ‘response to stimulus’; and, ‘death’ and a KEGG pathways: ‘JAK-STAT signaling pathway’; ‘MAPK signaling pathway’; ‘regulation of actin cytoskeleton’; and, ‘p53 signaling pathway’. In addition, we found TIMELESS, EIF4A3 and ZNF592 in blood and CHMP4C and FOXO3 in skeletal muscle, to be in common between DUDEGs and selected genes identified by evolutionary statistics such as FST and Cross Population Extended Haplotype Homozygosity (XP-EHH). Moreover, in Thoroughbreds, three out of five genes (CHMP4C, EIF4A3 and FOXO3) related to exercise response showed relatively low nucleotide diversity compared to the Jeju pony. DUDEGs are not only conceptually new DEGs that cannot be attained from reference-based analysis (RBA) but also supports previous RBA results related to exercise in Thoroughbred. In summary, three exercise related genes which were selected for during domestication in the evolutionary history of Thoroughbred were identified as conceptually new DEGs in this study.

Gene expression profile of cytokines and receptors of inflammation from cultured keratinocytes of burned patients

IntroductionAt all stages of wound healing, growth factors and cytokines play a particularly important role in the interaction with keratinocytes cellular receptors. Keratinocytes have received little attention about their potential to act as a source and target of cytokines. Changes in the cytokine levels after the burning occur prior to the metabolic abnormalities. Thus, it may be possible to develop therapeutic interventions that can mitigate the acute inflammatory response and modulating expression of these cytokines. The objective was to evaluate the expression of 84 genes mediators of the inflammatory response by using PCR array in a primary human epidermal cultured keratinocytes from patients with burns. MethodsKeratinocytes cultured from normal skin around injury from small and large burn patient were treated for DNA synthesis. The samples were analyzed by the PCR Superarray® assay and curve analyses were performed for 84 relevant human genes and their involvement in the inflammatory cytokines pathway and receptors. These genes were checked for the up or down regulation. And it was used MetaCore™ for the analysis of networks and Gene Ontology (GO) processes. ResultsChemokines of the CXC family were more expressed in the large burn group, except CXCL12. The C, CC and CX3C chemokine family were downregulated, especially in the small burn group. The interleukins IL8 and IL1B were more expressed in large burn than in small burn; except IL13RA1, IL13 and IL5RA that were downregulated, mainly in the small burn group. ConclusionsThe cytokine profile showed some important differences between the large and small burn patients, and from this original database, we can create new interventional trials in acute inflammation in burns.

Toxicogenomics-Based Identification of Mechanisms for Direct Immunotoxicity

Compounds with direct immunotoxic properties, including metals, mycotoxins, agricultural pesticides, and industrial chemicals, form potential human health risks due to exposure through food, drinking water, and the environment. Insights into the mechanisms of action are currently lacking for the majority of these direct immunotoxicants. Therefore, the present work aimed to gain insights into the molecular mechanisms underlying direct immunotoxicity. To this end, we assessed in vitro the effects of 31 test compounds on the transcriptome of the human Jurkat T-cell line. These compounds included direct immunotoxicants, immunosuppressive drugs with different mode of actions, and nonimmunotoxic control chemicals. Pathway analysis of the microarray data allowed us to identify canonical pathways and Gene Ontology processes that were transcriptionally regulated in common by immunotoxicants (1) with structural similarities, such as tributyltin chloride and tributyltin oxide that activated the retinoic acid/X receptor signaling pathway and (2) without structural similarities, such as As2O3, dibutyltin chloride, diazinon, MeHg, ochratoxin A (OTA), S9-treated OTA, S9-treated cyclophosphamide, and S9-treated benzo[a]pyrene, which activated unfolded protein response, and FTY720, lindane, and propanil, which activated the cholesterol biosynthesis pathway. In addition, processes uniquely affected by individual immunotoxicants were identified, such as the induction of Notch receptor signaling and the downregulation of acute-phase response genes by OTA. These findings were validated by quantitative real-time PCR analysis of genes involved in these processes. Our study indicated that diverse modes of action are involved in direct immunotoxicity and that a set of pathways or genes, rather than one single gene, can be used to screen compounds for direct immunotoxicity.

Abstract 5165: Pathway oriented phospho-proteomics analysis with reverse phase protein array (RPPA) technology in cell growth and cancer therapy.

Abstract A number of molecularly targeted drugs have been successfully launched as anti-cancer agents, and intensive efforts are underway to discover new targets and their modifiers. Nevertheless, many compounds selected by enzyme screening methodology demonstrated unexpected effects in intact cells or animal models. Since recent efforts from signaling pathway analysis have revealed that complexity of signaling networks, homeostatic controls and off-target effects can lead to unexpected cellular/in vivo consequences, profiling the effects of drug candidates on a wide range of intracellular signaling pathways is becoming an attractive strategy. Many proteomics approaches, especially in the area of phospho-proteomics, have been developed, and their applications are rapidly growing for the purpose of cellular signal profiling. Among these approaches, reverse phase protein array (RPPA) is one of the most powerful technologies because of its high sensitivity and reliability. We have developed a signal transduction oriented RPPA system that focuses on phospho-protein profiles in samples. Thus far, our system utilizes approximately 180 phospho-protein specific antibodies and can profile the phosphorylation status of more than 100 Gene Ontology processes covering more than 10 components in each process. Our current studies on modification of cellular signals induced by tyrosine kinase inhibitors (TKIs) in several cancer cell types are elucidating novel mechanisms for TKI actions. For example, our studies on Erlotinib and Gefitinib treated cells have resulted in very similar profiles as EGF-stimulated cancer cells. However, several differences in the signaling profiles between them may suggest differences of efficacy and toxicity. Independently, we have developed a nearly comprehensive in vitro kinase inhibitor profiling system, and we already have complete inhibition profiles for nine kinase inhibitors such as Erlotinib or Gefitinib. This allows us to combine in vitro profiling results with pathway intervention profiles to build a database integrating kinase-inhibition-potencies and cellular-signaling-profiles. Our aim is to expand this database to obtain new information to guide strategies for effective targeting in drug discovery. In this presentation, we present the novel targeting discovery strategy based on this database analysis. Citation Format: Takeomi Inoue, Naoko Iwata, Eiji Nishiwaki, Yasuyuki Kirii. Pathway oriented phospho-proteomics analysis with reverse phase protein array (RPPA) technology in cell growth and cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5165. doi:10.1158/1538-7445.AM2013-5165

Heritability assessment of cartilage metabolism. A twin study on procollagen II N-terminal propeptide (PIIANP)

Heritability assessment of cartilage metabolism. A twin study on procollagen II N-terminal propeptide (PIIANP)

Abstract WP102: Interleukin-21 Promotes Neuronal Autophagy Following Cerebral Ischemia

Currently there is no treatment for the inflammatory responses that develop following cerebral ischemia. We have previously shown that the CD4+ T cell derived inflammatory cytokine IL-21 is highly upregulated in ischemic mouse brain following transient middle cerebral artery occlusion (tMCAO) and promotes neuronal tissue injury. We also found IL-21+ CD4+ cells in perivascular spaces of ischemic human brain tissue; however, the primary effects and cellular targets of IL-21 remain unknown. Thus, we compared genome-wide whole brain gene expression in wild-type (WT) and IL-21 -/- mice following 1 hour tMCAO (n=3-5). RNA from whole brain was analyzed by Mouse Gene 1.0 ST microarray (Affymetrix) revealing that 2019 gene fragments representing 1505 genes had >3 fold higher expression in WT brain compared to IL-21 -/- after 24 hours reperfusion. Gene score resampling of annotated probe scores identified biological process gene ontology (GO) terms with 3-8 member genes most significantly over-represented including: GO:0010507 Negative Regulation of Autophagy, GO:0090174 Organelle Membrane Fusion, GO:0000046 Autophagic vacuole fusion, and GO:0016242 Negative regulation of macroautophagy (p <0.001). These findings suggested a role for IL-21 in neuronal autophagy_consistent with our earlier reports of reduced expression of autophagy related gene 6 (ATG6) in infarcted brain tissue of IL-21 -/- mouse compared to WT mice. Thus, we hypothesized that IL-21 could directly induce autophagy in hypoxic neuronal cells. IL-21 receptor was expressed on 10% of CD45- cells isolated from healthy mouse brain. Furthermore, using RT-PCR we detected Il21r mRNA expression on neuronal (Neura2A), astrocytic, and endothelial cell lines (MB114) with highest expression levels on Neuro2A cells. Surface expression on Neuro2A cells and primary neurons was confirmed by immunofluorescence. In preliminary experiments primary neurons treated with 256 ng/mL rIL-21 for 4 hours following 1-2 hour oxygen glucose deprivation showed increased expression of ATG6 compared to control treated cells (p<.05). In conclusion, these data suggest that IL-21 could directly promote neuronal autophagy following cerebral ischemia.

Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain

In the past decades, reports on bivalves' pathogens and associated mortalities have steadily increased. To face pathogenic micro-organisms, bivalves rely on innate defenses established in hemocytes which are essentially based on phagocytosis and cytotoxic reactions. As a step towards a better understanding of the molecular mechanisms involved in the mussel Mytilus edulis innate immune system, we constructed and sequenced a normalized cDNA library specific to M. edulis hemocytes unchallenged (control) and challenged with Vibrio splendidus LGP32 strain for 2, 4 and 6 h. A total of 1,024,708 nucleotide reads have been generated using 454 pyrosequencing. These reads have been assembled and annotated into 19,622 sequences which we believe cover most of the M. edulis hemocytes transcriptome. These sequences were successfully assigned to biological process, cellular component, and molecular function Gene Ontology (GO) categories. Several transcripts related to immunity and stress such as some fibrinogen related proteins and Toll-like receptors, the complement C1qDC, some antioxidant enzymes and antimicrobial peptides have already been identified. In addition, Toll-like receptors signaling pathways and the lysosome and apoptosis mechanisms were compared to KEGG reference pathways. As an attempt for large scale RNA sequencing, this study focuses on identifying and annotating transcripts from M. edulis hemocytes regulated during an in vitro experimental challenge with V. splendidus. The bioinformatic analysis provided a reference transcriptome, which could be used in studies aiming to quantify the level of transcripts using high-throughput analysis such as RNA-Seq.

Identification of differentially expressed genes in the oviduct of two rabbit lines divergently selected for uterine capacity using suppression subtractive hybridization

Suppressive subtractive hybridization libraries from oviduct at 62 h post-mating of two lines of rabbits divergently selected for uterine capacity were generated to identify differentially expressed genes. A total of 438 singletons and 126 contigs were obtained by cluster assembly and sequence alignment of 704 expressed sequence tags (ESTs), of which 54% showed homology to known proteins of the non-redundant NCBI databases. Differential screening by dot blot validated 71 ESTs, of which 47 showed similarity to known genes. Transcripts of genes were functionally annotated in the molecular function and the biological process gene ontology categories using the BLAST2GO software and were assigned to reproductive developmental process, immune response, amino acid metabolism and degradation, response to stress and apoptosis terms. Finally, three interesting genes, PGR, HSD17B4 and ERO1L, were identified as overexpressed in the low line using RT-qPCR. Our study provides a list of candidate genes that can be useful to understanding the molecular mechanisms underlying the phenotypic differences observed in early embryo survival and development traits.

Global gene expression analysis in skin biopsies of European red deer experimentally infected with bluetongue virus serotypes 1 and 8

Bluetongue virus (BTV) is a double-stranded RNA virus transmitted by blood-feeding biting midges of the genus Culicoides to wild and domestic ruminants, causing high morbidity and variable mortality. The aim of this study was to characterize differential gene expression in skin biopsies of red deer (Cervus elaphus) hinds experimentally infected with BTV serotypes 1 and 8. Skin biopsies were collected from BTV-1 and BTV-8 experimentally infected and control hinds at 14 and 98 days post-infection (dpi). Global gene expression profile in response to BTV infection was characterized at 14dpi using a bovine microarray together with real-time RT-PCR analysis of differentially expressed genes at 14 and 98dpi. Eighteen genes were upregulated and three were downregulated in response to virus infection, with no significant differences between BTV-1 and BTV-8 infected hinds. Seven unique genes, six upregulated (ISG15, PSMB8, PSMB9, BOLA, C1qA, C4) and one downregulated (FOS) were over-represented after conditional test for biological process gene ontology, which affected five molecular pathways (RIG-1, proteasome, MHC-1, complement, TLR) implicated in host immune response. BTV infection had a minor and transient effect on gene expression in hinds, as shown by the very few genes that were differentially expressed in response to infection at 14dpi, most of which had similar expression levels between infected and uninfected animals at 98dpi. These results suggested that red deer could control BTV infection with little effect on host molecular pathways.

Improving biomarker list stability by integration of biological knowledge in the learning process.

BackgroundThe identification of robust lists of molecular biomarkers related to a disease is a fundamental step for early diagnosis and treatment. However, methodologies for biomarker discovery using microarray data often provide results with limited overlap. It has been suggested that one reason for these inconsistencies may be that in complex diseases, such as cancer, multiple genes belonging to one or more physiological pathways are associated with the outcomes. Thus, a possible approach to improve list stability is to integrate biological information from genomic databases in the learning process; however, a comprehensive assessment based on different types of biological information is still lacking in the literature. In this work we have compared the effect of using different biological information in the learning process like functional annotations, protein-protein interactions and expression correlation among genes.ResultsBiological knowledge has been codified by means of gene similarity matrices and expression data linearly transformed in such a way that the more similar two features are, the more closely they are mapped. Two semantic similarity matrices, based on Biological Process and Molecular Function Gene Ontology annotation, and geodesic distance applied on protein-protein interaction networks, are the best performers in improving list stability maintaining almost equal prediction accuracy.ConclusionsThe performed analysis supports the idea that when some features are strongly correlated to each other, for example because are close in the protein-protein interaction network, then they might have similar importance and are equally relevant for the task at hand. Obtained results can be a starting point for additional experiments on combining similarity matrices in order to obtain even more stable lists of biomarkers. The implementation of the classification algorithm is available at the link: http://www.math.unipd.it/~dasan/biomarkers.html.

Characterization of resistance to pine wood nematode infection in Pinus thunbergiiusing suppression subtractive hybridization

BackgroundPine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. In the 40 years since the pathogen was identified, the physiological changes occurring as the disease progresses have been characterized using anatomical and biochemical methods, and resistant trees have been selected via breeding programs. However, no studies have assessed the molecular genetics, e.g. transcriptional changes, associated with infection-induced physiological changes in resistant or susceptible trees.ResultsWe constructed seven subtractive suppression hybridization (SSH) cDNA libraries using time-course sampling of trees inoculated with pine wood nematode at 1, 3, or 7 days post-inoculation (dpi) in susceptible trees and at 1, 3, 7, or 14 dpi in resistant trees. A total of 3,299 sequences was obtained from these cDNA libraries, including from 138 to 315 non-redundant sequences in susceptible SSH libraries and from 351 to 435 in resistant SSH libraries. Using Gene Ontology hierarchy, those non-redundant sequences were classified into 15 subcategories of the biological process Gene Ontology category and 17 subcategories of the molecular function category. The transcriptional components revealed by the Gene Ontology classification clearly differed between resistant and susceptible libraries. Some transcripts were discriminative: expression of antimicrobial peptide and putative pathogenesis-related genes (e.g., PR-1b, 2, 3, 4, 5, 6) was much higher in susceptible trees than in resistant trees at every time point, whereas expression of PR-9, PR-10, and cell wall-related genes (e.g., for hydroxyproline-rich glycoprotein precursor and extensin) was higher in resistant trees than in susceptible trees at 7 and 14 dpi.ConclusionsFollowing inoculation with pine wood nematode, there were marked differences between resistant and susceptible trees in transcript diversity and the timing and level of transcripts expressed in common; in particular, expression of stress response and defense genes differed. This study provided new insight into the differences in the physiological changes between resistant and susceptible trees that have been observed in anatomical and biochemical studies.

A network-based maximum link approach towards MS identifies potentially important roles for undetected ARRB1/2 and ACTB in liver cancer progression

Hepatocellular Carcinoma (HCC) ranks among the deadliest of cancers and has a complex etiology. Proteomics analysis using iTRAQ provides a direct way to analyse perturbations in protein expression during HCC progression from early- to late-stage but suffers from consistency and coverage issues. Appropriate use of network-based analytical methods can help to overcome these issues. We built an integrated and comprehensive Protein-Protein Interaction Network (PPIN) by merging several major databases. Additionally, the network was filtered for GO coherent edges. Significantly differential genes (seeds) were selected from iTRAQ data and mapped onto this network. Undetected proteins linked to seeds (linked proteins) were identified and functionally characterised. The process of network cleaning provides a list of higher quality linked proteins, which are highly enriched for similar biological process gene ontology terms. Linked proteins are also enriched for known cancer genes and are linked to many well-established cancer processes such as apoptosis and immune response. We found that there is an increased propensity for known cancer genes to be found in highly linked proteins. Three highly-linked proteins were identified that may play an important role in driving HCC progression - the G-protein coupled receptor signalling proteins, ARRB1/2 and the structural protein beta-actin, ACTB. Interestingly, both ARRB proteins evaded detection in the iTRAQ screen. ACTB was not detected in the original dataset derived from Mascot but was found to be strongly supported when we re-ran analysis using another protein detection database (Paragon). Identification of linked proteins helps to partially overcome the coverage issue in shotgun proteomics analysis. The set of linked proteins are found to be enriched for cancer-specific processes, and more likely so if they are more highly linked. Additionally, a higher quality linked set is derived if network-cleaning is performed prior. This form of network-based analysis complements the cluster-based approach, and can provide a larger list of proteins on which to perform functional analysis, as well as for biomarker identification.

Genome-wide analysis reveals the active roles of keratinocytes in oral mucosal adaptive immune response

To elucidate the roles of oral keratinocytes in the adaptive immune response of oral mucosa, global gene expression analysis was performed by microarray technique and integrating computational methods, including hierarchical clustering, biological process Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, self-organizing maps (SOMs) and biological association network analysis (BAN). Raw data from microarray experiments were uploaded to the Gene Expression Omnibus Database, http://www.ncbi.nlm.nih.gov/geo/ (GEO accession GSE28035). We identified 666 differentially expressed genes in the early stage (48 h) and 993 in the late stage (96 h) of the oral mucosal adaptive immune response. The analysis revealed that oral keratinocytes exerted diverse biological functions in different stages of immune response. Specifically, in 48 h the differentially expressed genes encompassed an array of biological ontology associated with immune response, such as antigen processing and presentation, and positive regulation of T-cell-mediated cytotoxicity. Several pathways which have been reported to be critical in inflammation, including mitogen-activated protein kinase pathway, were activated. Furthermore, after BAN construction, some putative hub genes and networks such as interleukin-1α and its subnetwork were recognized. Taken together, these results give substantial evidence to support the active roles of keratinocytes in the oral mucosal adaptive immune response.

Regulation of host cellular gene transcription by Bovine Leukemia Virus (BLV) Tax, wild type and mutants

BLV is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle, and is closely related to HTLV-1. The Tax protein of BLV is a transcriptional activator of viral replication, a key contributor to the oncogenic potential and a positive and negative regulator of apoptosis. In our previous study, we identified interesting mutant BLV Tax with elevated (TaxD247G) or reduced (TaxS240P) transactivation activity on the replication and propagation of BLV. However, the effects of mutation on other functions besides its function as a transcriptional activator are unknown. To address this, by microarray analysis, we here identified different cellular genes in response to Tax wild type and mutants. We constructed expression vectors encoding Tax wild type (TaxWT), TaxD247G or TaxS240P; introduced into HeLa cells and performed a microarray analysis. Up- and down-regulation of 122, 118 and 139 genes were detected for TaxWT, TaxS240P and TaxD247G, respectively, but TaxS240P down regulated more genes than TaxWT or TaxD247G. For comparative purposes, we distributed the genes according to Gene Ontology processes and identified expressed genes involved in transcription, signal transduction, immune response, cell proliferation, cell growth and apoptosis, which were affected by Tax. Interestingly, there was a notable fold change difference between up-regulated genes involved in transcription, signal transduction and cell proliferation induced by TaxS240P or TaxD247G. In addition, genes down regulated related to immune/viral response belonged to the interferon family. Thus, our result may be useful for understanding Tax functions and its regulation which cellular factors.

Characterization of Microrna Expression Profile In Platelets In Sickle Cell Disease

AbstractAbstract 2030Sickle cell disease (SCD) is associated with a high rate of thromboembolic complications, particularly in patients with advanced vasculopathy. Recent functional studies have revealed pathological platelet activation associated with intravascular hemolysis, the severity of pulmonary hypertension (PAH), and impaired NO-signaling in platelets mediated by NO scavenging by plasma hemoglobin (Villagra J et al, Blood 2007; Hu W et al, Blood 2010, prepublished online). In addition, our previous amplified platelet transcriptome studies in SCD patients have demonstrated dysregulated arginine and polyamine metabolism, potentially contributing to this dysfunction at the transcriptional level (Raghavachari N et al, Circulation 2007). New RNA technologies for purification of total RNA including small RNAs now allow for more refined study of transcriptional events in platelets from individual patients. We have undertaken the present study to demonstrate and validate the presence and differential expression of microRNAs (miRNAs) in platelets from patients with SCD compared with African American control subjects. We hypothesize that posttranscriptional regulation of the platelet function by miRNAs is an important contributor to SCD pathogenesis associated with pathological platelet activation. To date, there have been no studies undertaken to study the role of platelet miRNAs in SCD. Platelet rich plasma samples from 43 subjects were obtained from University of Pittsburgh Medical Center (UPMC) and National Heart, Lung, Blood Institute (NHLBI). Out of these there were 27 SCD patients and 17 were sex-, race- matched healthy controls. Out of the SCD patients, 18 were known to have PAH and 14 were on hydroxyurea (HU) at the time of study enrollment. Total platelet RNA including miRNAs were extracted using the miRNeasy Minikit (Qiagen, Valencia, CA) with yield in the range of 110–1875 ng from 16 ml of whole blood per donor. We hybridized these samples on a miRNA microarray platform (Agilent version 3) containing 866 human miRNAs. The array data was normalized using quantile normalization and were analyzed with GeneSpring GX 11 software. Differential expression profiles between sickle cell disease patients and controls were determined separately for the NHLBI and UPMC cohort to detect a greater than 2 fold difference change in miRNA expression at p-value of 0.05. Our final expression list included only common miRNAs identified in both cohorts, allowing for an additional level of validation. Final validation was performed by RT-PCR of a subset of the differentially expressed miRNAs. Our analysis identified 42 significantly differentially expressed miRNAs in platelets when comparing the SCD with the control groups, at a fold-change > 2 and p-value <0.05. Out of these there were 18 up- and 24 down-regulated miRNAs. Interestingly, within the SCD group, there were 14 differentially expressed miRNAs between the subjects on HU and those off HU and 23 differentially expressed miRNAs between subjects with and without PAH. Further confirmation of relative expression of downregulated miR-376a and miR409-3p along with upregulated 1225-3p was conducted by real-time PCR. Computational analysis revealed that predicted targets of these 3 miRNAs belong to the regulation of transcription, transcription factor activity and regulation of RNA metabolic process Gene Ontology (GO) annotations. These results are currently being directly tested by evaluating the effect of perturbing mir-376, mir 409-3pand mir-1225-3p on platelet activation, differentiation and budding. The results will be updated at the meeting. The present study confirms the existence of rplatelet miRNAs, their differential expression in SCD and lays down the foundations to study their role in the molecular pathogenesis of SCD. Future evaluation of miRNA-target mRNA interactions will provide an experimental framework for the study of disease-specific platelet biology and potentially identify biomarkers for predicting patients at high risk of developing vasculopathy. Disclosures:No relevant conflicts of interest to declare.

Gene expression changes in spleens of the wildlife reservoir species, Eurasian wild boar (Sus scrofa), naturally infected with Brucella suis biovar 2

Brucella suis is responsible for swine brucellosis worldwide. Of the five different B. suis biovars (bv.), bv. 2 appears restricted to Europe where it is frequently isolated from wild boar and hares, can infect pigs and can cause human brucellosis. In this study, the differential gene expression profile was characterized in spleens of Eurasian wild boar naturally infected with B. suis bv. 2. Of the 20,201 genes analyzed in the microarray, 633 and 1,373 were significantly (fold change > 1.8; P < 0.01) upregulated and downregulated, respectively, in infected wild boar. The analysis was focused on genes that were over represented after conditional test for biological process gene ontology. Upregulated genes suggested that B. suis bv. 2 infection induced cell maturation, migration and/or proliferation in infected animals. The genes downregulated in infected wild boar impaired the activity of several important cellular metabolic pathways such as metabolism, cytoskeleton organization and biogenesis, immune response and lysosomal function and vesicle-mediated transport. In addition, the response to stress, sperm fertility, muscle development and apoptosis seemed to be also impaired in infected animals. These results suggested that B. suis bv. 2 may use strategies similar to other smooth brucellae to facilitate intracellular multiplication and the development of chronic infections. To our knowledge, this is the first report of the analysis of gene expression profile in hosts infected with B. suis bv. 2, which is important to understand the molecular mechanisms at the host-pathogen interface in the main reservoir species with possible implications in the zoonotic cycle of the pathogen.

Examination of the relationship between essential genes in PPI network and hub proteins in reverse nearest neighbor topology

BackgroundIn many protein-protein interaction (PPI) networks, densely connected hub proteins are more likely to be essential proteins. This is referred to as the "centrality-lethality rule", which indicates that the topological placement of a protein in PPI network is connected with its biological essentiality. Though such connections are observed in many PPI networks, the underlying topological properties for these connections are not yet clearly understood. Some suggested putative connections are the involvement of essential proteins in the maintenance of overall network connections, or that they play a role in essential protein clusters. In this work, we have attempted to examine the placement of essential proteins and the network topology from a different perspective by determining the correlation of protein essentiality and reverse nearest neighbor topology (RNN).ResultsThe RNN topology is a weighted directed graph derived from PPI network, and it is a natural representation of the topological dependences between proteins within the PPI network. Similar to the original PPI network, we have observed that essential proteins tend to be hub proteins in RNN topology. Additionally, essential genes are enriched in clusters containing many hub proteins in RNN topology (RNN protein clusters). Based on these two properties of essential genes in RNN topology, we have proposed a new measure; the RNN cluster centrality. Results from a variety of PPI networks demonstrate that RNN cluster centrality outperforms other centrality measures with regard to the proportion of selected proteins that are essential proteins. We also investigated the biological importance of RNN clusters.ConclusionsThis study reveals that RNN cluster centrality provides the best correlation of protein essentiality and placement of proteins in PPI network. Additionally, merged RNN clusters were found to be topologically important in that essential proteins are significantly enriched in RNN clusters, and biologically important because they play an important role in many Gene Ontology (GO) processes.

Valproic Acid–Induced Deregulation In Vitro of Genes Associated In Vivo with Neural Tube Defects

The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.

Comparison of gene expression responses to hypoxia in viviparous ( Xiphophorus) and oviparous ( Oryzias) fishes using a medaka microarray

Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka ( Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species ( Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant ( p < 0.05) ± 3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 × 10 − 5 ) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).