DAVID Gene Ontology Research Articles

Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation Analysis Reveal Insights into the Molecular Mechanism of Cordia myxa in the Treatment of Liver Cancer.

Traditional treatments of cancer have faced various challenges, including toxicity, medication resistance, and financial burdens. On the other hand, bioactive phytochemicals employed in complementary alternative medicine have recently gained interest due to their ability to control a wide range of molecular pathways while being less harmful. As a result, we used a network pharmacology approach to study the possible regulatory mechanisms of active constituents of Cordia myxa for the treatment of liver cancer (LC). Active constituents were retrieved from the IMPPAT database and the literature review, and their targets were retrieved from the STITCH and Swiss Target Prediction databases. LC-related targets were retrieved from expression datasets (GSE39791, GSE76427, GSE22058, GSE87630, and GSE112790) through gene expression omnibus (GEO). The DAVID Gene Ontology (GO) database was used to annotate target proteins, while the Kyoto Encyclopedia and Genome Database (KEGG) was used to analyze signaling pathway enrichment. STRING and Cytoscape were used to create protein-protein interaction networks (PPI), while the degree scoring algorithm of CytoHubba was used to identify hub genes. The GEPIA2 server was used for survival analysis, and PyRx was used for molecular docking analysis. Survival and network analysis revealed that five genes named heat shot protein 90 AA1 (HSP90AA1), estrogen receptor 1 (ESR1), cytochrome P450 3A4 (CYP3A4), cyclin-dependent kinase 1 (CDK1), and matrix metalloproteinase-9 (MMP9) are linked with the survival of LC patients. Finally, we conclude that four extremely active ingredients, namely cosmosiin, rosmarinic acid, quercetin, and rubinin influence the expression of HSP90AA1, which may serve as a potential therapeutic target for LC. These results were further validated by molecular dynamics simulation analysis, which predicted the complexes with highly stable dynamics. The residues of the targeted protein showed a highly stable nature except for the N-terminal domain without affecting the drug binding. An integrated network pharmacology and docking study demonstrated that C. myxa had a promising preventative effect on LC by working on cancer-related signaling pathways.

Comparative genome characterization of Leptospira interrogans from mild and severe leptospirosis patients.

Leptospirosis is a zoonotic disease caused by spirochetes from the genus Leptospira. In Thailand, Leptospira interrogans is a major cause of leptospirosis. Leptospirosis patients present with a wide range of clinical manifestations from asymptomatic, mild infections to severe illness involving organ failure. For better understanding the difference between Leptospira isolates causing mild and severe leptospirosis, illumina sequencing was used to sequence genomic DNA in both serotypes. DNA of Leptospira isolated from two patients, one with mild and another with severe symptoms, were included in this study. The paired-end reads were removed adapters and trimmed with Q30 score using Trimmomatic. Trimmed reads were constructed to contigs and scaffolds using SPAdes. Cross-contamination of scaffolds was evaluated by ContEst16s. Prokka tool for bacterial annotation was used to annotate sequences from both Leptospira isolates. Predicted amino acid sequences from Prokka were searched in EggNOG and David gene ontology database to characterize gene ontology. In addition, Leptospira from mild and severe patients, that passed the criteria e-value < 10e-5 from blastP against virulence factor database, were used to analyze with Venn diagram. From this study, we found 13 and 12 genes that were unique in the isolates from mild and severe patients, respectively. The 12 genes in the severe isolate might be virulence factor genes that affect disease severity. However, these genes should be validated in further study.

Bioinformatics Analysis Identifies a Novel Role of GINS1 Gene in Colorectal Cancer.

BackgroundColorectal cancer (CRC) is one of the most lethal malignancies and the incidence of CRC has been on the rise. Herein, we aimed to identify effective biomarkers for early diagnosis and treatment of colorectal cancer via bioinformatic tools.MethodsTo identify differentially expressed genes (DEGs) in CRC, we downloaded CRC gene expression data from GSE24514 and GSE110223 datasets in Gene Expression Omnibus (GEO) and employed R to analyze the data. We further performed functional enrichment analysis of the DEGs on the DAVID gene ontology analysis tool. STRING database and Cytoscape visualization tool were employed to construct a PPI (protein–protein interaction) network and establish intensive intervals in the network. Immunohistochemistry, qRT-PCR and Western blotting were performed to identify the expression level of GINS1 in CRC. In vitro and in vivo experiments were performed to assess the impact of GINS1 in the pathogenesis of CRC in terms of proliferation, migration and metastasis.ResultsAmong the two datasets, 389 DEGs were identified and used to construct a PPI network. These genes were mainly involved in cell proliferation and cell cycle. Among them, 15 genes including GINS1 were found to be strongly associated with the PPI network. We further performed immunohistochemistry, qRT-PCR and Western blotting to identify that GINS1 expression was higher in CRC than in paired normal tissues. Moreover, in vitro and in vivo experiments demonstrated GINS1 could promote the proliferation, invasion and migration of colorectal cancer cells.ConclusionsGINS1 could be considered as a potential biomarker for CRC patients.

Characterization of basal and estrogen-regulated antisense transcription in breast cancer cells: Role in regulating sense transcription

Estrogen-responsive breast cancer cells exhibit both basal and estrogen-regulated transcriptional programs, which lead to the transcription of many different transcription units (i.e., genes), including those that produce coding and non-coding sense (e.g., mRNA, lncRNA) and antisense (i.e., asRNA) transcripts. We have previously characterized the global basal and estrogen-regulated transcriptomes in estrogen receptor alpha (ERα)-positive MCF-7 breast cancer cells. Herein, we have mined genomic data to define three classes of antisense transcription in MCF-7 cells based on where their antisense transcription termination sites reside relative to their cognate sense mRNA and lncRNA genes. These three classes differ in their response to estrogen treatment, the enrichment of a number of genomic features associated with active promoters (H3K4me3, RNA polymerase II, open chromatin architecture), and the biological functions of their cognate sense genes as analyzed by DAVID gene ontology. We further characterized two estrogen-regulated antisense transcripts arising from the MYC gene in MCF-7 cells, showing that these antisense transcripts are 5′-capped, 3′-polyadenylated, and localized to different compartments of the cell. Together, our analyses have revealed distinct classes of antisense transcription correlated to different biological processes and response to estrogen stimulation, uncovering another layer of hormone-regulated gene regulation.

Platelet RNA Splicing Profiles Can Distinguish IgM MGUS Patients from Healthy Individuals

Introduction Within the general population over 50 years, 3% is affected with a monoclonal gammopathy of undetermined significance (MGUS). Although MGUS generally demands no treatment, interest in treating premalignant stages to prevent progression to overt malignancies has grown. Better insight in processes underlying progression of MGUS is therefore warranted. Platelet RNA profiles have been shown to reflect information on the topical tumor environment in various cancer types, including multiple myeloma (MM). Our aim was to investigate whether IgM MGUS patients can be distinguished from healthy controls (HCs) using platelet RNA splicing profiles, and to relate IgM MGUS splicing profiles to genes associated with Waldenström's Macroglobulinemia (WM). Methods Twenty-four IgM MGUS patients with presence of a paraprotein confirmed through immunofixation consented to participation. The thromboSeq protocol for platelet RNA sequencing (RNA-Seq) based classification (Best et al. Nat Protoc 2019) was used to acquire platelet RNA profiles from all MGUS subjects. RNA was extracted from platelets isolated from whole blood. After reverse transcription and SMARTer amplification, 100 bp single-read sequencing was performed on the Illumina Hiseq 2500 platform. Raw sequencing data were processed with Trimmomatic, aligned to a reference genome with STAR and mapped with HTSeq. Mapping data of 29 age and blood storage time matched HCs were also included in the analysis. Differential expression of spliced RNAs expressed as log2 fold change (logFC) was assessed with a likelihood-ratio ANOVA and visualized in a heatmap constructed with differentially spliced RNAs that passed the particle swarm optimized (PSO) false discovery rate (FDR) threshold. RNA profiles were further explored and related to WM and MM by DAVID gene ontology enrichment analyses. Results Mean age in the MGUS and HC groups was 67.2 (SD 7.7) and 66.9 (SD 6.7) years. Thirteen of 24 MGUS patients suffered from IgM mediated anti-myelin associated glycoprotein (MAG) neuropathy; the other 11 were asymptomatic. Of 3,426 high-abundant spliced RNAs detected, 1,371 demonstrated significant differential expression (FDR &lt;0.05) and allowed perfect distinction of MGUS from HCs after PSO (Fig. 1; P &lt;0.0001). The two distinct MGUS phenotypes (neuropathy vs. asymptomatic) could not be distinguished. Top 50 overexpressed RNA isoforms were mainly involved in apoptotic processes, transcription regulation, cellular defense response and immune response regulation. WM associated genes (logFC in parentheses) BCL7C (1.35), CASP8 (1.75), CD79A (2.65), CD81 (1.58), DUSP1 (2.32), HLA-DRB1 (1.83), JAK3 (0.65), NFKBIA (3.59), NFKB2 (1.63), REL (1.35) and TNFRSF14 (0.75), as well as MM associated JUND (4.61) and CCNL1 (3.11), were overexpressed in MGUS. TNFAIP3, negatively regulating the NF-κB pathway, was highly overexpressed (logFC 4.07). JAK1 and JAK2 mRNAs were mildly though significantly depleted in the MGUS group. We also found overexpression of CCNL2 (1.81), MS4A1 (2.17) and WNK1 (0.79), which have been shown to be enriched in WM relative to IgM MGUS. Top 50 depleted RNAs were mainly involved in protein phosphorylation, intracellular signal transduction and response to tumor necrosis factor. CASP3 (-1.43), CASP4 (-0.62) and TNFSF4 (-0.99) mRNAs, of which overexpression is associated with WM, were depleted in MGUS. Conclusions For the first time, we have demonstrated widespread differential expression of spliced RNAs in platelets of MGUS patients as compared to healthy controls. We found evidence of overexpression of various genes that have been associated with WM. The differences were mainly in pathways related to apoptosis, signaling and immune response regulation. This is in line with findings that balanced elevation of free light chains, possibly due to a chronic inflammatory state, increases risk of developing a monoclonal gammopathy (Kumar et al. Blood Cancer J 2019). Overexpression of NFKBIA, NFKB2 andTNFAIP3 in MGUS possiblysuggests activation of the NF-κB pathway, a key step in MYD88 mediated progression to WM. JAK subtype expression levels may reflect that the JAK/STAT pathway, also important for progression to WM, has not (yet) been aberrantly activated in our MGUS subjects. Future research could be focused on studying the development of MGUS and MGUS progression to WM and MM using differentially expressed splice junctions in platelets. Disclosures Minnema: Amgen: Honoraria; Celgene Corporation: Honoraria, Research Funding; Gilead: Honoraria; Jansen Cilag: Honoraria; Servier: Honoraria.

Proteomic Profiling of Extracellular Vesicles Isolated From Cerebrospinal Fluid of Former National Football League Players at Risk for Chronic Traumatic Encephalopathy.

Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of repetitive mild traumatic brain injury, such as American football players. Initial neuropathologic changes in CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau) neurofibrillary tangles and other aggregates in neurons, astrocytes and cell processes in an irregular pattern often at the depths of the cortical sulci. In later stages, the p-tau depositions become widespread and is associated with neurodegeneration. Extracellular vesicles (EVs) are known to carry neuropathogenic molecules, most notably p-tau. We therefore examined the protein composition of EVs isolated from the cerebrospinal fluid (CSF) of former National Football League (NFL) players with cognitive and neuropsychiatric dysfunction, and an age-matched control group (CTRL) with no history of contact sports or traumatic brain injury. EVs were isolated from the CSF samples using an affinity purification kit. Total tau (t-tau) and tau phosphorylated on threonine181 (p-tau181) in CSF-derived EVs from former NFL players and CTRL participants were measured by ultrasensitive immunoassay. The t-tau and p-tau181 levels of CSF-derived EV were positively correlated with the t-tau and p-tau181 levels of total CSF in former NFL players, respectively, but not in the CTRL group. 429 unique proteins were identified from CSF-derived EVs and quantified by TMT-10 plex method. The identified protein molecules were significantly enriched for the extracellular exosome molecules, Alzheimer’s disease pathway and Age/Telomere Length ontology as determined by DAVID Gene Ontology analysis. Ingenuity pathway analysis of the differentially expressed EV proteins revealed enrichment of canonical liver/retinoid X receptor activation pathway. Upstream effect analysis predicted MAPT (tau) as an upstream regulator in former NFL players. These data will be useful for understanding the EV-mediated disease spread and development of novel EV biomarkers for CTE and related disorders.

Abstract PD7-01: Bromodomain protein 3 is a novel therapeutic target in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is a subtype of breast cancer comprising 10% of breast tumours. ILC is characterised by a loss of E-cadherin, and is generally estrogen receptor (ER) positive. The majority of ILC breast cancers are treated with endocrine therapy, although approximately one in three women are de novo resistant to therapy. To identify novel therapeutic targets for the treatment of ILC, we carried out RNA sequencing on 61 primary ILC samples. We found that high expression of the epigenetic reader, bromodomain protein 3 (BRD3) was associated with poor recurrence free survival. We also validated this finding in a separate cohort of 99 ILC patient samples using the METABRIC cohort. Next, we assessed ILC cell lines for sensitivity to JQ1, an inhibitor of BET family proteins. We found that JQ1 inhibited cell growth in all the cell lines tested. Interesting, two of the ILC cell lines were sensitive to JQ1-induced apoptosis, whereas two of the cell lines were inherently resistant to JQ1-induced apoptosis. Using dynamic BH3 profiling we showed that the JQ1 resistant cell lines were dependent on anti-apoptotic protein BCL-XL following JQ1 treatment. Interestingly, we show both in 2D and 3D cultures that JQ1 is synergistic when combined with the BH3 mimetic, ABT-263. Highlighting that combination treatment with JQ1 and ABT-263 may be a novel potential therapeutic option for ILC. To unveil the mechanism underlying resistance to JQ1-induced apoptosis, we performed paired-end RNA sequencing and compared differentially expressed genes in JQ1 sensitive and JQ1 resistant ILC cell lines. DAVID gene ontology analysis identified 6 pathways differentially upregulated in the JQ1 resistant ILC cell line including MAPK, Wnt, and insulin resistance signaling. Interestingly, we found that ILC cell lines, which were resistant to BET inhibition with JQ1, demonstrated high levels of FGFR1-4 both at the mRNA level and the protein level. Combination treatment with JQ1 and the FGFR1 inhibitor PD173074 or following knockdown of FGFR with siRNA, resulted in increased cell death in JQ1 resistant cells. Currently, we are assessing how FGFR signaling enables survival of ILC cells following JQ1 treatment and determining the exact function of BRD3 in ILC. In conclusion, we have identified a novel therapeutic target, BRD3, which may be inhibited using JQ1 in combination with BH3 mimetic ABT-263 or FGFR1 inhibitor for a more effective treatment strategy for ILC. Citation Format: Haley K, Walsh L, Moran B, Das S, Tarrant F, Caldas C, Bernards R, Gallagher W, O'Connor D, Ni Chonghaile T. Bromodomain protein 3 is a novel therapeutic target in invasive lobular carcinoma [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 PD7-01.

Whole Blood RNA-Seq Differentiates Hematopoietic Cell Transplant Recipients with Upper Versus Lower Respiratory Tract Rhinovirus Infection

Background Human rhinovirus (HRV) is the most common virus detected in the respiratory tract following hematopoietic cell transplantation (HCT). Mortality following virologically proven lower respiratory tract infection (LRTI) may be up to 40%. Specific immune processes activated in upper respiratory tract infection (URTI) vs LRTI are not well understood. Methods Whole blood was collected in PAXgene tubes at the time of URTI or proven (HRV detected in the lower tract with radiographic changes) or possible (HRV detected in the upper tract with radiographic changes) LRTI. RNA was extracted and globin-reduced (PAXgene RNA kit; GlobinClear). Libraries were prepared with the TruSeq RNA Access kit (Illumina), and were pooled and sequenced (2 × 50bp) on an Illumina HiSeq 2500. Low quality reads were filtered prior to alignment (TopHat), and associated with genes (HTSeq). Normalization and significance testing were done using the Bioconductor package, edgeR. Differential expression was defined as |log2 (ratio)| ≥ 1 (± 2-fold) with the FDR set to 5%. Gene ontology (GO) enrichment analysis of a concise set of biological process terms (DAVID GO Direct) was performed using the Bioconductor package, goseq. Results Seventeen subjects with HRV infection following HCT were included (URTI, N = 7; proven LRTI without copathogens, N = 4; possible LRTI, N = 6). RNA was of sufficient quantity and quality to perform RNA-seq. We demonstrated differential gene expression between HRV URTI and proven LRTI (Figure 1A). Differential gene expression between HRV URTI and possible LRTI was not as robust (Figure 1B). Principle component analysis of the top 500 most variable genes demonstrated separation between subjects with HRV URTI and HRV proven LRTI (Figure 2). GO enrichment analyses demonstrated enrichment of terms associated with adaptive immune responses in the URTI group (Table). Conclusions HRV URTI and proven LRTI have highly differential gene expression patterns, whereas possible LRTI gene expression appears to more closely resemble that of URTI. Gene enrichment analysis demonstrates potential increased immune activation in the URTI subjects, which may lead to better control of infection. Larger studies are needed to verify these findings. Whole blood RNA-seq is feasible and may be a useful tool for demonstrating unique transcriptomic signatures for clinical phenotypes in HCT recipients and for identifying specific pathways in pathogenesis.

Molecular networks of hypoxia and neuronal apoptosis in the cochlea

In organotypic cultures, the modiolus (MOD) region of newborn rats shows afourfold higher rate of cell death than the organ of Corti (OC). The differing vulnerability of OC and MOD cells is related to differential expression of numerous genes (DEG). Organotypic cultures of OC and MOD of 3-5-day-old rats were exposed to anormoxic or a hypoxic (pO2 10-20 mmHg; 5 h) atmosphere. Cell death rate and gene expression as detected by c‑DNA microarray analysis were determined 24 h after the culture was created. Genes with modified expression (n = 60) were analyzed for biological processes according to the DAVID Gene Ontology Database (GO). Molecular networks were created using the STRING and ConsensusPathDB databases. The network of the GO annotations "hypoxia", "inflammation", and "mechanical stimulus" indicates the existence of two gene clusters: acluster with pro-inflammatory genes (Ccl3, Cxcl2, Cxcr4, Ccl20) and acluster with hypoxia-associated genes (e.g., c-Jun, Hif1a, and Vegfa). The network of the GO annotations "positive and negative regulation of neuron apoptotic process" suggests that the differential expression of c-Jun, Ngfr, and Casp3 is important for regulation of programmed cell death in neuronal cells of the OC and MOD. While c‑JUN acts as an important modulator of the balance between cell death and survival, the associations of NGFR and CASP3 seem to be significant for the initiation of cell death. The evaluation and application of findings from biostatistical databases is important for understanding the function of individual genes and gene clusters in medically relevant biological processes.

New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling.

Cell membrane-associated epidermal growth factor receptor (EGFR) translocates into a perinuclear/nuclear location upon stimulation, where it complexes with mRNAs. Treatment with radiation and cisplatin decreases the amounts of mRNAs present within this complex. Gene array analyses of mRNAs in complex with immunoprecipitated nEGFR revealed significant enrichment of different mRNA species compared to the control immunoprecipitation. Functional annotation with help of DAVID Gene Ontology Analysis identified under other terms the HIF-1A/VEGF signaling pathway as one of the top scoring KEGG pathways. RT-PCR and western blots revealed the radiation-induced expression of mRNAs and proteins involved in HIF-1A/VEGF signaling. Simultaneously, the levels of the corresponding validated miRNAs within the complex containing nEGFR and mRNAs were decreased. This finding argues that an mRNA/miRNA/nEGFR complex regulates protein expression. Indeed, we detected the GW182, AGO2, PABPC1 and cNOT1 proteins, which belong to the deadenylase complex, in a complex with nuclear EGFR. Erlotinib-mediated inhibition of EGFR kinase reduced the radiation-induced increase in mRNA expression. In this context, erlotinib reduced AGO2 phosphorylation by the EGFR kinase at residue Y393, which was associated with increased cNOT1 deadenylase activity and reduced mRNA stability. To prove the roles of miRNAs in this context, we transfected cells with an inhibitor of Hsa-mir-1180p5, which targets the NFATC4 mRNA, an mRNA associated with VEGF signaling, or pretreated cells with erlotinib. Indeed, Hsa-mir-1180p5 knockdown increased and the erlotinib treatment decreased the expression of the NFATC4 protein. The expression of the NFATC4 protein controlled the cloning efficiency and radiosensitivity of A549 and FaDu tumor cells. Thus, this study is the first to show that a membrane-located tyrosine kinase receptor, such as EGFR, is internalized to a nuclear/perinuclear location upon exposure to stress and modulates the stability and translation of miRNA-selected mRNAs. This mechanism enables cells to directly express proteins in response to EGFR activation and may contribute to treatment resistance in EGFR-overexpressing tumors.

Time Sequential Transcriptome Analysis Identifies Mir-126 As an Early Biomarker for Inv(16) Acute Myeloid Leukemia (AML) Disease Progression

Inv(16)(p13q22)/t(16;16)(p13.1;q22) is one of the most common chromosomal mutations found in human acute myeloid leukemia (AML). This inversion on chromosome 16 creates a fusion gene CBFb-MYH11 and leads to the expression of a fusion protein CBFβ-SMMHC. Expression of Cbfβ-SMMHC (CM) in a conditional Cbfb-MYH11 knock-in mouse model (Cbfb+/56M/Mx1-Cre) leads to development of spontaneous AML with the acquisition of additional genetic and epigenetic alterations. In order to further understand the underlying mechanism(s) driving leukemogenesis and identify predictive biomarkers during disease progression, we performed RNA-seq to track changes of transcriptiome in time series peripheral blood samples using illumina HiSeq sequencer. A cohort of 16 mice were included in the experiment, 9 out of which are CM conditional knock-in mice and the remaining 7 are controls. Peripheral blood samples were collected before the induction of CM expression as the "0" time point samples. After induction, peripheral blood samples were collected at 1, 2, 3.5, 4.5, 5.5 and 6.5 month (experimental end point) after induction, and leukemic samples were collected when mice were moribund as the "end" time point samples. Between 3.5 and 6.5 months, 6 out of 9 CM knock-in mice in this cohort had succumbed to lethal leukemia. Total RNA was isolated for mRNA and microRNA (miR) sequencing library preparation. The mapped sequencing read counts were annotated to genes and differential expression was compared using edgeR. False discovery rate (FDR) were used to adjust for multiple comparisons. Genes with adjusted P value (FDR) <0.05 and fold-change >2 were considered differentially expressed genes. Unsupervised clustering analysis revealed that all diseased mice showed unique disease-related mRNA and miRNA signatures at the end point. The disease-related signatures were absent in all control mice and the three CM conditional knock-in mice that did not develop leukemia. We found that 2,032 mRNA genes and 106 miRs were significantly up-regulated whereas 2,926 mRNA genes and 121 miRs were significantly down-regulated at the leukemia end point. Principal component analysis revealed that the disease-related changes occur quite early, at 1-2 month after induction. Among the earliest changes, miR-126-3p and miR-126-5p, previously reported to be associated with human inv(16) AML, were significantly up-regulated (log fold change =2.09 and 2.36 respectively; p< 0.0001) at 1 month and increased progressively throughout leukemia progression (log fold change = 4.11 and 4.45; p< 0.0001). DAVID gene ontology (GO) analysis of genes negatively correlated with miR-126 expression showed significant enrichment for mitotic cell cycle and cell division GO pathways. In addition, dynamic changes in a number of predicted miR-126 targets were seen during disease progression. We reasoned that high miR-126 expression might contribute to AML progression. Lentiviral miR-126 knock-down in AML LSK (Lin-/Sca1+/cKit+) cells from CM induced mice led to increased apoptosis and reduced quiescence in vitro. To assess the role of miR-126 in leukemia progression in vivo, we then designed a novel CpG-miR-126 inhibitor (Zhang et al, submitted). Mice transplanted with CM+ AML and treated with with anti-miR126 (5 mg/kg, daily, 30 days) had a significant delay in leukemia progression and enhanced survival (control 42% survival v.s. anti-miR-126 80% survival at 70 days; n=7-10), supporting the notion that high miR-126 contributes to disease growth. Further mechanistic investigation of miR-126 function during inv(16) AML progression are ongoing. In conclusion, transcriptome analysis of time series samples during spontaneous leukemia progression allows identification of disease-related changes and early increase of miR-126 as a potential biomarker. Further functional validation is expected to provide mechanistic insights and rational for targeting miR-126 in inv(16) AML. DisclosuresStein:Seattle Genetics: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Stemline Therapeutics: Consultancy, Research Funding; Argios: Research Funding; Celgene: Research Funding.

Characterization of gene expression profiling of mouse tissues obtained during the postmortem interval

Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval. We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3h after death, and 3) mice that were stored at 4°C for 18h after death, as this storage resembles the human autopsy process in Japan. The RNA quality of the brain and the liver was maintained up to 18h during the postmortem interval. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained due to high RNA integrity number (RIN) values, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. Taken together, our findings suggest that under typical autopsy conditions, gene expression profiles that reflect disease pathology can be examined by understanding comprehensive recognition of postmortem fluctuation of gene expression.

Type I interferon related genes are common genes on the early stage after vaccination by meta-analysis of microarray data

The objective of this study was to find common immune mechanism across different kinds of vaccines. A meta-analysis of microarray datasets was performed using publicly available microarray Gene Expression Omnibus (GEO) and Array Express data sets of vaccination records. Seven studies (out of 35) were selected for this meta-analysis. A total of 447 chips (145 pre-vaccination and 302 post-vaccination) were included. Significance analysis of microarrays (SAM) program was used for screening differentially expressed genes (DEGs). Functional pathway enrichment for the DEGs was conducted in DAVID Gene Ontology (GO) database. Twenty DEGs were identified, of which 10 up-regulated genes involved immune response. Six of which were type I interferon (IFN) related genes, including LY6E, MX1, OAS3, IFI44L, IFI6 and IFITM3. Ten down-regulated genes mainly mediated negative regulation of cell proliferation and cell motion. Results of a subgroup analysis showed that although the kinds of genes varied widely between days 3 and 7 post vaccination, the pathways between them are basically the same, such as immune response and response to viruses, etc. For an independent verification of these 6 type I IFN related genes, peripheral blood mononuclear cells (PBMCs) were collected at baseline and day 3 after the vaccination from 8 Enterovirus 71(EV71) vaccinees and were assayed by RT-PCR. Results showed that the 6 DEGs were also upregulated in EV71 vaccinees. In summary, meta-analysis methods were used to explore the immune mechanism of vaccines and results indicated that the type I IFN related genes and corresponding pathways were common in early immune responses for different kinds of vaccines.

Unraveling Inflammatory Responses using Systems Genetics and Gene-Environment Interactions in Macrophages

Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli. We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide (LPS), or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions, and eQTL "hot spots" that specifically control LPS responses. We used siRNA knockdown of candidate genes to validate an eQTL hot spot in chromosome 8 and identified the gene 2310061C15Rik as a regulator of inflammatory responses in macrophages. We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits that are modeled in the mouse and for the dissection of regulatory relationships between genes.

Changes in whole blood gene expression in obese subjects with type 2 diabetes following bariatric surgery: a pilot study.

BackgroundA pilot study was performed in order to investigate the effects of bariatric surgery on whole blood gene expression profiles in obese subjects with type 2 diabetes.Methodology/Principal FindingsWhole blood from eleven obese subjects with type 2 diabetes was collected in PAXgene tubes prior to and 6–12 months after bariatric surgery. Total RNA was isolated, amplified, labeled and hybridized to Illumina gene expression microarrays. Clinical and expression data were analyzed using a paired t-test, and correlations between changes in clinical trait and transcript levels were calculated. Pathways were identified using Ingenuity Pathway Analysis and DAVID gene ontology software. Overall, bariatric surgery resulted in significant reduction of body mass index, fasting plasma glucose, fasting plasma insulin, and normalization of glycosylated hemoglobin levels. The expression levels of 204 transcripts, representing 200 unique genes, were significantly altered after bariatric surgery. Among the significantly regulated genes were GGT1, CAMP, DEFA1, LCN2, TP53, PDSS1, OLR1, CNTNAP5, DHCR24, HHAT and SARDH, which have been previously implicated in lipid metabolism, obesity and/or type 2 diabetes. Selected findings were replicated by quantitative real-time-PCR. The changes in expression of seven transcripts, WDR35, FLF45244, DHCR24, TIGD7, TOPBP1, TSHZ1, and FAM8A1 were strongly correlated with the changes in body weight, fasting plasma glucose and glycosylated hemoglobin content. The top pathways associated with gene expression changes after bariatric surgery was lipid metabolism, small molecule biochemistry and gene expression. Two antimicrobial peptides were among the transcripts with the largest changes in gene expression after bariatric surgery.Conclusions/SignificanceData from this pilot study suggest that whole blood expression levels of specific transcripts may be useful as biomarkers associated with susceptibility for type 2 diabetes and/or therapeutic response.

Cell Proliferation and Interleukin-6–Type Cytokine Signaling Are Implicated by Gene Expression Responses in Early Optic Nerve Head Injury in Rat Glaucoma

In glaucoma, the optic nerve head (ONH) is the principal site of initial axonal injury, and elevated intraocular pressure (IOP) is the predominant risk factor. However, the initial responses of the ONH to elevated IOP are unknown. Here the authors use a rat glaucoma model to characterize ONH gene expression changes associated with early optic nerve injury. Unilateral IOP elevation was produced in rats by episcleral vein injection of hypertonic saline. ONH mRNA was extracted, and retrobulbar optic nerve cross-sections were graded for axonal degeneration. Gene expression was determined by microarray and quantitative PCR (QPCR) analysis. Significantly altered gene expression was determined by multiclass analysis and ANOVA. DAVID gene ontology determined the functional categories of significantly affected genes. The Early Injury group consisted of ONH from eyes with <15% axon degeneration. By array analysis, 877 genes were significantly regulated in this group. The most significant upregulated gene categories were cell cycle, cytoskeleton, and immune system process, whereas the downregulated categories included glucose and lipid metabolism. QPCR confirmed the upregulation of cell cycle-associated genes and leukemia inhibitory factor (Lif) and revealed alterations in expression of other IL-6-type cytokines and Jak-Stat signaling pathway components, including increased expression of IL-6 (1553%). In contrast, astrocytic glial fibrillary acidic protein (Gfap) message levels were unaltered, and other astrocytic markers were significantly downregulated. Microglial activation and vascular-associated gene responses were identified. Cell proliferation and IL-6-type cytokine gene expression, rather than astrocyte hypertrophy, characterize early pressure-induced ONH injury.

Gene Expression Changes In Patients with 5q- Syndrome Treated with Lenalidomide

AbstractAbstract 4641 Introduction:The 5q- syndrome constitutes a World Health Organization (WHO)-defined category of myelodysplastic syndromes (MDS) with the following hematological features: macrocytic anemia, erythroid hypoplasia, normal or elevated platelet count, hypolobulated megakaryocytes and isolated del(5q). Its pathogenesis remains uncertain. Lenalidomide has shown impressive rate of erythroid and in some cases even cytogenetic response. Lenalidomide is anti-angiogenic, anti-adhesive, affects important cytokine circuits and can directly induce growth arrest and apoptosis in certain types of malignant cells. There is also a potent immunomodulatory effect including costimulation of CD4+ and CD8+ T-cells that are partially activated via the T-cell receptor and enhancement of NK cell-mediated lysis. The mechanism of lenalidomide on 5q- syndromes patients is not known, but appears to be suppression of the 5q- clone, resulting in transfusion-independence and a rise in hemoglobin levels. The aim of this study is to investigate the direct effects of lenalidomide on gene expression in isolated CD3+ T-lymphocytes and CD14+ monocytes from 5q- syndromes. Patients and Methods:Six lenalidomide responding patients (three females and three males) with 5q- syndromes (aged 55 to 75 years) and six healthy controls (aged 32 to 74 years) were included in the study. Samples of peripheral blood were collected before the treatment and then at the time of first erytroid response (2-5 months). HumanRef-8 v2 Expression Bead Chips (Illumina) were used to generate expression profiles. The raw data were normalized with the R software, lumi package. Normalized data were filtered by detection p-value <0.01. Differentially expressed genes were identified by Significance Analysis of Microarrays (SAM). Results:558/228 significantly differentially expressed genes between CD14+ monocytes/CD3+ T-lymfocytes patients and control samples were identified (fold change >1.5; p<0.05) before the treatment. This gene list was imported into the DAVID gene ontology and identified 4 significantly affected pathways (p<0.05): Systemic lupus erythematosus (11 genes), NOD-like receptor signaling pathway (8 genes), Cell cycle (9 genes), Pathogenic coli infection (7 genes). Among the most up-regulated genes in monocytes were: globin genes (HBA, HBB, HBG and HBG2), IL8, CDKN1A, TNF, TNFAIP3, BHLHB2, CXCL2, and CD83 genes. In T lymphocytes we have found in addition up-regulation of JUN, FOS and ALAS2. Genes significantly differentially expressed between lenalidomide-treated patient and baseline treatment in CD14+ monocytes was found in 267 genes. Out of these, 90 genes showed down-regulation, while the rest of the genes were up-regulated in patient samples. Using this set of genes into the DAVID gene ontology application, the gene annotation found the following biological processes: Regulation of protein kinase cascade (12 genes), regulation of I-kappaB kinase/NF-kappaB cascade (8 genes), regulation of lymphocyte mediated immunity (4 genes) and negative regulation of apoptosis (12 genes). The following pathways (p<0.05) that contained genes significantly deregulated in patients responding to lenalidomide were: Fc gamma R-mediated phagocytosis, Leukocyte transendothelial migration and Lysosome pathway. The most down-regulated genes by lenalidomide in monocytes were TNF, STAB1, IL1B, MYB, FYN and NFKB2. The most significantly up-regulated genes by lenalidomide in monocytes were NCF1, EEF1G, RPS28, RN28S1, RN28S1 and ARPC1B. No significantly differentially expressed genes were founded in T-lymfocytes before treatment and at the time of response. We observed large variability among patients. Conclusion:Our results indicate that lenalidomide with a still unknown exact effect in 5q- causes significant changes in expression profile in many genes that are probably important in the pathogenesis of the disease and the treatment response. Significant decrease in relation to lenalidomide treatment was observed of tumor necrosis factor, interleukin 1 and transcription factor MYB. We plan next to monitor changes in gene expression during the course of therapy and comparing the gene expression in CD34+ cells before and after lenalidomide therapy. Supported by Celgene Corporation and grants NS/9634 MZCR, MZ0 UHKT2005 and MSM0021620808. Disclosures:No relevant conflicts of interest to declare.