Genome Expression Analysis Research Articles

Heterogeneity in global gene expression profiles between biopsy specimens taken peri-surgically from primary ER-positive breast carcinomas.

BackgroundGene expression is widely used for the characterisation of breast cancers. Variability due to tissue heterogeneity or measurement error or systematic change due to peri-surgical procedures can affect measurements but is poorly documented. We studied the variability of global gene expression between core-cuts of primary ER+ breast cancers and the impact of delays to tissue stabilisation due to sample X-ray and of diagnostic core cutting.MethodsTwenty-six paired core-cuts were taken immediately after tumour excision and up to 90 minutes delay due to sample X-ray; 57 paired core-cuts were taken at diagnosis and 2 weeks later at surgical excision. Whole genome expression analysis was conducted on extracted RNA. Correlations and differences were assessed between the expression of individual genes, gene sets/signatures and intrinsic subtypes.ResultsTwenty-three and 56 sample pairs, respectively, were suitable for analysis. The range of correlations for both sample sets were similar with the majority being >0.97 in both. Correlations between pairs for 18 commonly studied genes were also similar between the studies and mainly with Pearson correlation coefficients >0.6 except for a small number of genes, which had a narrow-dynamic range (e.g. MKI67, SNAI2). There was no systematic difference in intrinsic subtyping between the first and second sample of either set but there was c.15 % discordance between the subtype assignments between the pairs, mainly where the subtyping of individual samples was less certain. Increases in the expression of several stress/early-response genes (e.g. FOS, FOSB, JUN) were found in both studies and confirmed findings in earlier smaller studies. Increased expression of IL6, IGFBP2 and MYC (by 17 %, 14 % and 44 %, respectively) occurred between the samples taken 2 weeks apart and again confirmed findings from an earlier study.ConclusionsThere is generally good correlation in gene expression between pairs of core-cuts except where genes have a narrow dynamic range. Similar correlation coefficients to the average gene expression profiles of intrinsic subtype, particularly LumA and LumB, can lead to discordances between assigned subtypes. Substantial changes in expression of early-response genes occur within an hour after surgery and in IL6, IGFB2 and MYC as a result of diagnostic core-cut biopsy.Trial registrationTrial number CRUK/07/015. Study start date September 2008.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0696-2) contains supplementary material, which is available to authorized users.

Development and validation of a custom microarray for global transcriptome profiling of the fungus Aspergillus nidulans.

Transcriptome profiling is a powerful tool for identifying gene networks from whole genome expression analysis in many living species. Here is described the first extensively characterized platform using Agilent microarray technology for transcriptome analysis in the filamentous fungus Aspergillus (Emericella) nidulans. We developed and validated a reliable gene expression microarray in 8×15K format, with predictive and experimental data establishing its specificity and sensitivity. Either one or two 60-mer oligonucleotide probes were selected for each of 10,550 nuclear as well as 20 mitochondrial coding sequences. More than 99% of probes were predicted to hybridize with 100% identity to their aimed specific A. nidulans target only. Probe sensitivity was supported by a highly narrow distribution of melting temperatures together with thermodynamic features, which strongly favored probe-target perfect match hybridization, in comparison with predicted secondary structures. Array quality was evaluated through transcriptome comparison of two A. nidulans strains, differing by the presence or not of Escherichia coli LacZ transgene. High signal-to-noise ratios were measured, and signal reproducibility was established at intra-probe and inter-probe levels. Reproducibility of microarray performances was assessed by high correlation between two-color dye signals and between technical replicates. Results were confirmed by RT-qPCR analysis on five genes. Though it covers 100% of the A. nidulans targeted coding sequences, this low density array allows limited experimental costs and simplified data analysis process, making it suitable for studying gene expression in this model organism through large numbers of experimental conditions, in basic, biomedical or industrial microbiology research fields.

Ten-Eleven Translocation Genes are Downregulated in Endometriosis.

Our previous whole genome expression analysis of endometriomas suggested dysregulation of the ten-eleven translocation genes (TET1, TET2, and TET3), involved in converting 5- methylcytosine to 5-hydroxymethylcytosine (5-hmC). The objective of this study was to validate the expression of TET genes in ectopic and eutopic endometrium and in primary cultures of human endometrial stromal fibroblasts (HESF) during in vitro decidualization and to quantify 5-hmC levels in patients with endometriosis. Blood, eutopic endometrium, and endometriotic tissues were collected at time of gynecologic surgery. HESF cultures were created from eutopic endometrium of women without (HESF-CONTROL) and with endometriosis (HESF-ENDO) and underwent in vitro decidualization. Genomic DNA from blood and tissues underwent quantification of the absolute amount of 5-hmC using ELISA. The expression of TET1, TET2, and TET3 was decreased in endometriosis compared to non-endometriosis control eutopic endometrium. Surprisingly, the global amount of 5-hmC was higher in ectopic endometrium than control eutopic endometrium, while genomic DNA from blood of women with endometriosis contained statistically significantly less 5-hmC than women without endometriosis. Expression of TET1, TET2, and TET3 was decreased in non-decidualized HESFENDO. Upon in vitro decidualization, control HESF showed decreased expression of TET3, while decidualized HESF-ENDO showed no statistically significant change in expression of TET1, TET2, or TET3. These results indicate that the TET genes are downregulated in ectopic endometrium and in HESF-ENDO, and suggest for the first time that TET genes play a role in endometriosis. High global amounts of 5-hmC in endometriotic tissues suggest unique epigenetic regulation in these tissues.

Abstract P6-07-13: New insights on HER2 amplification from the constitutional and somatic standpoints: Results from the ICGC and SIGNAL/Phare studies

Abstract Background: HER2-positive (HER2+) breast cancers are defined by the amplification and/or overexpression of the human epidermal growth factor receptor (HER2/ERBB2) gene on chromosome region 17q12. Although anti-HER2 targeted therapies have greatly improved treatment of HER2+ breast cancer, the magnitude of benefit varies widely between patients. Deciphering the genomic and genetic heterogeneity of HER2+ breast cancer may provide a basis to better understand their natural history, opening new avenues of treatment. Methods: As part of the ICGC Breast Cancer Working Group effort, we combined whole genome sequencing and transcriptomic analyses of 64 HER2+ primary invasive carcinomas, and a genome wide association study (GWAS) of over 9.836 breast cancer patients in the prospective SIGNAL/PHARE cohort (NCT00381901 – RECF1098). Results: Using WGS data we precisely delineate the ERBB2 amplicon as a 106 kb region involving six genes and show that the amplification mechanism was consistent with breakage-fusion-bridge (BFB) cycles. Four RNA expression-based groups were identified, displaying specific genomic alterations in terms of amplification, rearrangements and mutations. On other hand, GWAS analyses failed to identify any constitutional variants associated with HER2 amplification. Discussion: By combining whole genome sequencing and expression analysis, we provide evidence showing that HER2+ tumours display considerably more molecular heterogeneity than previously reported. These results are reinforced with the lack of association between any genetic variants and HER2 amplification from GWAS analyses. Taken as a whole, these results suggest that HER2+ breast cancers do not represent per se a homogeneous subtype, but are distributed along the whole breast cancer spectrum, from ER-positive luminal to ER-negative basal phenotype. Genome alterations present in HER2+ tumors are in accordance with these phenotypes, and it is likely that the HER2 amplification is a secondary event in the course of tumorigenesis, not favored by any particular constitutional or somatic genetic variants. Citation Format: Vincent-Salomon A, Ferrari A, Pivot X, Macgrogan G, Arnould L, Treilleux I, Romieux G, Sertier A-S, Thomas E, Tonon L, Boyault S, Kielbassa J, Letexier V, Pauporte I, Birbaum D, Saintigny P, Cox D, Viari A. New insights on HER2 amplification from the constitutional and somatic standpoints: Results from the ICGC and SIGNAL/Phare studies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-13.

Abstract B17: Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model

Abstract Sphingosine kinase 1 (SphK1) phosphorylates pro-apoptotic sphingosine to form pro-survival sphingosine-1-phosphate (S1P), leading to cancer progression. In breast cancer, high levels of SphK1 mRNA are found in 80% of the patients, with average increase of 2-fold in breast tumors compared with normal tissue from the same patient. Recent studies suggest that SphK1 expression is associated with disease-free survival in ER-negative and HER2-positive breast tumors. In addition, inhibition of SphK1 in HER2-positive breast cancer cell line reduced S1P-induced HER2 and ERK1/2 activation. While these evidences suggest that SphK1 may play an important role in HER2-positive breast cancer, the exact role of SphK1 in breast tumorigenesis remains unclear. The aim of this project is to define the role of SphK1 in HER2-positive breast tumorigenesis and to investigate whether the pathway components are potential target for breast cancer prevention and therapies. Here, we report that SphK1 is required for HER2/neu-induced breast tumorigenesis and SphK1 regulates claudin-2 (CLDN2) to promote tumor development perhaps by mediating HER2. In MMTV-neu transgenic mouse model, genetic deletion of SphK1, which was accompanied by reduced S1P and increased C16-ceramide levels, significantly inhibited breast tumor development, with reduced incidence (SphK1-/-, 19%; SphK1+/-, 26%; and SphK1+/+, 57%; p < 0.05) and multiplicity (SphK1-/-, 0.27 ± 0.13; SphK1+/-, 0.32 ± 0.07; and SphK1+/+, 0.67 ± 0.14 tumors per animal; p < 0.05). Unexpectedly, SphK1 deletion did not affect tumor growth as results showed no differences in tumor volume. However, this is not surprising when considering the fact that the tumors were allowed to grow until tumor reached 20 mm in longer diameter, at which they were sacrificed regardless of age. Interestingly, whole genome expression analysis by Illumina MouseWG-6 v2.0 Expression BeadChip Array revealed significant reduction of CLDN2 expression in tumors from SphK1 deficient mice (p < 0.0001). In support to these findings, overexpression of SphK1 in MDA-MB-453 human breast cancer cells, which HER2 is amplified but lacks hormone receptors, lead to increase in CLDN2 expression (p < 0.05). Furthermore, overexpression of SphK1 in MDA-MB-453 cells also enhanced cell proliferation, colony formation, and migration/invasion, and drug resistance to cisplatin. The relevance of this finings in humans was confirmed by immunostaining of SphK1 and CLDN2 in HER2-positive breast cancer tumors and normal tissues (n = 93 and 35 subjects, respectively). The results showed that the expression of SphK1 was higher in tumors compared to adjacent normal tissue (p < 0.0001). Furthermore, higher SphK1 expression was observed in high grade tumors (vs low grade; p < 0.001) and ER-PR- tumors (vs ER+PR+; p < 0.0001). Interestingly, we observed similar staining pattern of CLDN2 proteins, where CLDN2 expression was high in tumors (p < 0.0001), particularly in high grade tumors (p < 0.01) and ER-PR- tumors (p < 0.0001). Moreover, we found a strong correlation between SphK1 and CLDN2 expressions in tumors but not in normal tissues, confirming the results obtained in animal study. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis, and targeting SphK1 and/or CLDN2 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment. Citation Format: Yoshiko Shimizu, Hideki Furuya, Paulette M. Tamashiro, Steve Goodison, Owen Chan, Ian Pagano, Kayoko Iino, Rafael Peres, Kanani Hokutan, Lenora W.M. Loo, Brenda Hernandez, Aung Naing, Charles J. Rosser, Toshihiko Kawamori. Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B17.

Correlating behaviour and gene expression endpoints in the dopaminergic system after modafinil administration in mouse

The mechanisms of action of modafinil continue to be poorly characterised and its potential for abuse in preclinical models remains controverted. The aim of this study was to further elucidate the mechanism of action of modafinil, through a potential behavioural and molecular association in the mouse. A conditioned place preference (CPP) paradigm was implemented to investigate the rewarding properties of modafinil. Whole genome expression and qRT-PCR analysis were performed on the ventral tegmental area (VTA), nucleus accumbens (NAC) and prefrontal cortex (PFC) of modafinil-treated and control animals. Modafinil administration (65mg/kg) induced an increase in locomotor activity, an increase in the change of preference for the drug paired side after a conditioning period as well as changes to gene expression profiles in the VTA (120 genes), NAC (23 genes) and PFC (19 genes). A molecular signature consisting of twelve up-regulated genes was identified as common to the three brain regions. Multiple linear correlation analysis showed a strong correlation (R2>0.70) between the behavioural and molecular endpoints in the three brain regions. We show that modafinil had a concomitant effect on CPP, locomotor activity, and up-regulation of interferon-γ (IFN-γ) regulated genes (Gbp2, Gbp3, Gbp10, Cd274, Igtp), while correlating the latter set of genes with behaviour changes evaluated through the CPP. A potential association can be proposed based on the dysregulation of p47 family genes and Gbp family of IFN-γ induced GTPases. In conclusion, these findings suggest a link between the behavioural and molecular events in the context of modafinil administration.

Exploring Realm of Proteases of Leishmania donovani Genome and Gene Expression Analysis of Proteases under Apoptotic Condition.

Genomic analysis of Leishmania donovani has shed light on various proteases in the parasite genome, their classification and sub-cellular localization. Under apoptotic condition of the parasite, gene expression analyses of representative protease(s) from each clan shows altered expression levels of various proteases. The data indicates possible role of various proteases in apoptotic process, directly or indirectly. Over-expression of autophagy related protease genes under apparent apoptotic conditions show some crosstalk between autophagy and apoptosis like cell death of Leishmania parasite. The counter-regulation of these two processes in trypanosomatids in general and Leishmania in specific, needs further investigation.

Genomic identification, phylogeny, and expression analysis of MLO genes involved in susceptibility to powdery mildew in Fragaria vesca.

The MLO (powdery mildew locus O) gene family is important in resistance to powdery mildew (PM). In this study, all of the members of the MLO family were identified and analyzed in the strawberry (Fragaria vesca) genome. The strawberry contains at least 20 members of the MLO family, and the protein sequence contained between 171 and 1485 amino acids, with 0-34 introns. Chromosomal localization showed that the MLOs were unevenly distributed on each of the chromosomes, except for chromosome 4. The greatest number of MLOs (seven) was found on chromosome 3. A phylogenetic tree showed that the MLOs were divided into seven groups (I-VII), four of which consisted of MLOs from strawberry, Arabidopsis thaliana, rice, and maize, suggesting that these genes may have evolved after the divergence of monocots and dicots. Multiple sequence alignment showed that strawberry MLO candidates related to powdery mildew resistance possessed seven highly conserved transmembrane domains, a calmodulin-binding domain, and two conserved regions, all of which are important domains for powdery mildew resistance genes. Expressed sequence tag analysis revealed that the MLOs were induced by multiple abiotic stressors, including low and high temperature, drought, and high salinity. These findings will contribute to the functional characterization of MLOs related to PM susceptibility, and will assist in the development of disease resistance in strawberries.

Deleterious assembly of the lamin A/C mutant p.S143P causes ER stress in familial dilated cardiomyopathy.

Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.

Expression patterns of inflorescence- and sex-specific transcripts in male and female inflorescences of African oil palm (Elaeis guineensis)

Oil palm is a monoecious plant and the sex ratio of the female to male inflorescences on each palm is important for breeding and commercial production.We hypothesise that the sex differences of oil palm inflorescences are due, at least in part, to variations in gene expression so this study aimed to establish the sexual differences by using whole genome expression analysis. We sequenced the transcriptomes of oil palm male and female inflorescences at the earliest stage at which the male and female tissues can be reliably distinguished. From the transcriptome data, we identified 97 potential sex-specific transcripts. Among the validated transcripts, oil palm orthologs of acid phosphatase and DEFICIENS showed male-specific expression patterns whereas orthologs of bZIP transcription factor, late embryogenesis abundant protein and TASSELSEED1 showed female-specific expression patterns. Transcripts for orthologs of acid phosphatase and late embryogenesis abundant protein were also strongly inflorescence-specific. Furthermore, we assembled a broad and dense consensus transcriptome from male and female inflorescences, shoot apical meristem, mesocarp, leaf and root of oil palm, which provides a valuable reference for identification of unique and common transcripts between these tissues. We suggest that the combined expression of inflorescence- and sex-specific transcripts may account for sexual differences of oil palm male and female inflorescences.

Low-power photodynamic therapy induces survival signaling in perihilar cholangiocarcinoma cells.

BackgroundPhotodynamic therapy (PDT) of solid cancers comprises the administration of a photosensitizer followed by illumination of the photosensitizer-replete tumor with laser light. This induces a state of local oxidative stress, culminating in the destruction of tumor tissue and microvasculature and induction of an anti-tumor immune response. However, some tumor types, including perihilar cholangiocarcinoma, are relatively refractory to PDT, which may be attributable to the activation of survival pathways in tumor cells following PDT (i.e., activator protein 1 (AP-1)-, nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB)-, hypoxia-inducible factor 1-alpha (HIF-1α)-, nuclear factor (erythroid-derived 2)-like 2 (NFE2L2)-, and unfolded protein response-mediated pathways).MethodsTo assess the activation of survival pathways after PDT, human perihilar cholangiocarcinoma (SK-ChA-1) cells were subjected to PDT with zinc phthalocyanine (ZnPC)-encapsulating liposomes. Following 30-minute incubation with liposomes, the cells were either left untreated or treated at low (50 mW) or high (500 mW) laser power (cumulative light dose of 15 J/cm2). Cells were harvested 90 min post-PDT and whole genome expression analysis was performed using Illumina HumanHT-12 v4 expression beadchips. The data were interpreted in the context of the survival pathways. In addition, the safety of ZnPC-encapsulating liposomes was tested both in vitro and in vivo.ResultsPDT-treated SK-ChA-1 cells exhibited activation of the hypoxia-induced stress response via HIF-1α and initiation of the pro-inflammatory response via NF-кB. PDT at low laser power in particular caused extensive survival signaling, as evidenced by the significant upregulation of HIF-1- (P < 0.001) and NF-кB-related (P < 0.001) genes. Low-power PDT was less lethal to SK-ChA-1 cells 90 min post-PDT, confirmed by annexin V/propidium iodide staining. In vitro toxicogenomics and toxicological testing in chicken embryos and mice revealed that the ZnPC-encapsulating liposomes are non-toxic.ConclusionsPDT-treated perihilar cholangiocarcinoma cells exhibit extensive survival signaling that may translate to a suboptimal therapeutic response and possibly tumor recurrence. These findings encourage the development of photosensitizer delivery systems with co-encapsulated inhibitors of survival pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1994-2) contains supplementary material, which is available to authorized users.

The whole-genome expression analysis of peripheral blood mononuclear cells from aspirin sensitive asthmatics versus aspirin tolerant patients and healthy donors after in vitro aspirin challenge.

BackgroundUp to 30 % of adults with severe asthma are hypersensitive to aspirin and no unambiguous theory exists which provides a satisfactory explanation for the occurrence of aspirin-induced asthma (AIA) in some asthmatic patients. Therefore, the aim of this study was to compare the AIA expression profile against aspirin tolerant asthma (ATA) and healthy volunteers (HV) profile in peripheral blood mononuclear cells (PBMCs) after in vitro aspirin challenge in Caucasian population.MethodsPBMCs were separated from blood of three groups of subjects - 11 AIA, 7 ATA and 15 HV and then stimulated by either 2 μM lysine aspirin or 20 μM lysine as a control. Subsequently, RNA was isolated, transcribed into cDNA and subjected to microarray and qPCR studies. Simultaneously, protein was extracted from PBMCs and used in further immunoblotting analysis.ResultsThe validation of results at mRNA level has shown only three genes, whose expression was significantly altered between comprising groups. mRNA expression of CNPY3 in PBMCs in AIA was significantly lower (-0.41 ± 2.67) than in HV (1.04 ± 2.69), (p = 0.02); mRNA expression of FOSL1 in PBMCs in AIA was also significantly decreased (-0.66 ± 2.97) as opposed to HV (0.31 ± 4.83), (p = 0.02). While mRNA expression of ERAS in PBMCs was increased (1.15 ± 0.23) in AIA in comparison to HV (-1.32 ± 0.41), (p = 0.03). At protein level the changed expression of one protein was confirmed. Protein expression of FOSL1 in PBMCs in AIA was both significantly lower (-0.86 ± 0.08) than in ATA (0.39 ± 0.42), (p = 0.046) and in HV (0.9 ± 0.27), (p = 0.007).ConclusionsThis pilot study implies a positive association between CNPY3, ERAS, FOSL1 and aspirin-intolerant asthma, suggesting that these findings would be useful for further investigations of NSAIDs mechanism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0305-4) contains supplementary material, which is available to authorized users.

Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast.

Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases having matched primary and metastatic tumors) were profiled using gene sequencing, gene copy number analysis, whole genome expression, and protein expression. Whole genome expression analysis demonstrated consistent over-expression of genes involved in angiogenesis including VEGFA, Angiopoietin-2, VCAM1, PDGFRA, and PTTG1. EGFR protein overexpression was observed in 26/27 (96%) of cases with amplification of the EGFR gene in 8/24 (33%) cases. Two EGFR mutations were identified including EGFRvIII and a presumed pathogenic V774M mutation, respectively. The most common pathogenic mutations included TP53 (50%) and PIK3CA (15%). Cases with matched primary and metastatic tumors harbored identical mutations in both sites (PIK3CA/KRAS and RB1 gene mutations, respectively). Tumor expression of PD-L1 immunoregulatory protein was observed in 3/22 (14%) of cases. Overexpression of molecular biomarkers of increased angiogenesis, EGFR and immune checkpoints provides novel targeted therapy options in malignant phyllodes tumors of the breast.

Comprehensive genomic identification and expression analysis of the nucleobase-ascorbate transporter (NAT) gene family in apple

Nucleobase ascorbate transporters (NATs), also known as nucleobase:cation-symporter 2 (NCS2) proteins, belong to an evolutionarily widespread family of transport proteins. Because little is known about NAT genes in apple (Malus domestica), this papper screened GDR genome databases and identified 14 NATs in that species. Here, presented an overview of this gene family in apple, including structures, chromosome distribution and localization, phylogenies, and motifs. Phylogenetic and structure analyses showed that these genes could be assigned to three clusters. The 14 genes selected here displayed differential expression patterns in various tissues. Expression was very high for MdNAT12, MdNAT4, and MdNAT6 in the shoot tips, mature leaves, and young and mature fruits. MdNAT1 and MdNAT7 tended to show increased expression while the fruits were maturing. MdNAT6 proved responsive to both drought and salinity. Their transcript levels were generally up-regulated significantly during the fruit maturation process as well as in response to the imposition of drought or salt stress. Results suggest that members of this family function in tissue development, fruit maturation, and plant adaptations to adverse growing environments.

Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

Genomic characterization and expression analysis of four apolipoprotein A-IV paralogs in Senegalese sole (Solea senegalensis Kaup).

The apolipoprotein A-IV (ApoA-IV) plays a key role in lipid transport and feed intake regulation. In this work, four cDNA sequences encoding ApoA-IV paralogs were identified. Sequence analysis revealed conserved structural features including the common 33-codon block and nine repeated motifs. Gene structure analysis identified four exons and three introns except for apoA-IVAa1 (with only 3 exons). Synteny analysis showed that the four paralogs were structured into two clusters (cluster A containing apoA-IVAa1 and apoA-IVAa2 and cluster B with apoA-IVBa3 and apoA-IVBa4) linked to an apolipoprotein E. Phylogenetic analysis clearly separated the paralogs according to their cluster organization as well as revealed four subclades highly conserved in Acanthopterygii. Whole-mount analyses (WISH) in early larvae (0 and 1day post-hatch (dph)) showed that the four paralogs were mainly expressed in yolk syncytial layer surrounding the oil globules. Later, at 3 and 5dph, the four paralogs were mainly expressed in liver and intestine although with differences in their relative abundance and temporal expression patterns. Diet supply triggered the intensity of WISH signals in the intestine of the four paralogs. Quantification of mRNA abundance by qPCR using whole larvae only detected the induction by diet at 5dph. Moreover, transcript levels increased progressively with age except for apoA-IVAa2, which appeared as a low-expressed isoform. Expression analysis in juvenile tissues confirmed that the four paralogs were mainly expressed in liver and intestine and secondary in other tissues. The role of these ApoA-IV genes in lipid transport and the possible role of apoA-IVAa2 as a regulatory form are discussed.

Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway.

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6-specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies.

Complete genome and gene expression analyses of Asaia bogorensis reveal unique responses to culture with mammalian cells as a potential opportunistic human pathogen.

Asaia bogorensis, a member of acetic acid bacteria (AAB), is an aerobic bacterium isolated from flowers and fruits, as well as an opportunistic pathogen that causes human peritonitis and bacteraemia. Here, we determined the complete genomic sequence of the As. bogorensis type strain NBRC 16594, and conducted comparative analyses of gene expression under different conditions of co-culture with mammalian cells and standard AAB culture. The genome of As. bogorensis contained 2,758 protein-coding genes within a circular chromosome of 3,198,265 bp. There were two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases: cyoABCD-1 and cyoABCD-2. The cyoABCD-1 operon was phylogenetically common to AAB genomes, whereas the cyoABCD-2 operon belonged to a lineage distinctive from the cyoABCD-1 operon. Interestingly, cyoABCD-1 was less expressed under co-culture conditions than under the AAB culture conditions, whereas the converse was true for cyoABCD-2. Asaia bogorensis shared pathogenesis-related genes with another pathogenic AAB, Granulibacter bethesdensis, including a gene coding pathogen-specific large bacterial adhesin and additional genes for the inhibition of oxidation and antibiotic resistance. Expression alteration of the respiratory chain and unique hypothetical genes may be key traits that enable the bacterium to survive under the co-culture conditions.

RNA microarray analysis in dried blood spots

Objectives Despite the promises and ever-decreasing costs, the genomic revolution has so far had only a marginal role in epidemiological research. This is at least partly due to lack of minimally-invasive methods of specimen collection that can be used for genomic analysis. We aimed to address this gap by testing whether a minimally-invasive method of collection through dried blood spots provides a good proxy for microarray (RNA expression) analysis in whole blood. Methods We collected venipuncture and dried blood spot samples from 19 individuals and performed whole genome expression analysis on extracted RNA using the Illumina platform. Results We found a correlation of rho = .76 and an agreement of kappa = .45 for detected genes in the two datasets (validity). We found a correlation of rho = .96 and an agreement of kappa = .72 for detected genes in the independent replicates from dried blood spots (reliability). Conclusions Genome-wise RNA expression in dried blood spots shows a moderate-to-high correlation and fair-to-good agreement with RNA expression in whole blood. If these findings are replicated in larger samples, microarray analysis in dried blood spots could be used as a minimally-invasive method to assess genomic underpinning of illness in population studies.

Abstract 3826: A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development

Abstract Background and aims: New generation sequencing and array technologies now allow the systematic investigation and comparison of genetic or epigenetic alterations in AD-CRC. The colorectal adenoma-dysplasia-cancer (AD-CRC) development is characterised by sporadic (&amp;lt;45% frequency) mutations in a limited number of genes. Recent epigenetic investigations showed that methylation of selected genes occurs in high frequency (&amp;gt;95%) in early stages of cancers already. Aims: Evaluation of the role of epigenetic and genetic alterations in the AD-CRC development using new generation sequencing and DNA methylation arrays. Materials and methods: DNA was isolated from fresh frozen biopsy specimens (20 normal; 33 adenomas; 17 CRCs). First, a multiplex PCR panel was designed to amplify mutation hot spots of 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, MSH6, NRAS, PIK3CA, SMAD2, SMAD4, TP53). Further DNA methylation analysis of 94 genes was performed on Human Colon Cancer EpiTect Methyl II Signature PCR Array (Qiagen) from the same DNA specimen. After RNA isolation whole genome expression analysis was performed with HGU133plus2 microarrays (Affymetrix) from 49 normal, 49 adenoma and 49 CRC specimens and on HT-29 cells with and without 5-aza-deoxycytidin treatment. Immunohistochemistry confirmation of expression changes on protein level was performed using tissue microarrays. Results: Mutations were found in 76% of adenomas and 78% of the cancer cases. The average number of mutations found in mutated samples was 1; 1,8; 1,9 and 2,3 in low grade adenomas, high grade adenomas, carcinomas and serrated adenomas, respectively. The APC suppressor gene was mutated in adenomas more frequently than in carcinomas (36% vs. 24%). The most frequently mutated genes were APC, TP53 and KRAS with 36%, 18% and 26% frequencies in adenomas and 24%, 47% and 45% frequencies in carcinomas. DNA methylation was found in 100% of the investigated colorectal adenoma and cancer specimens. Eight genes were found to be methylated in all of the cases. This gen set included SFRP1, MAL, SLIT2, SST, VIM; another set of genes (DKK1, SLI3, TMEFF2) was found to be hypermethylated in adenomas and cancers in &amp;gt;75% of the cases. In adenomas 56 genes, in dysplasias 40 in cancer 37 genes were methylated (&amp;gt;50% of the cases). The effect of methylation could be confirmed by decreased mRNA and protein expression. Demethylation treatment successfully restored the expression profile of the top methylated genes. Conclusion: Methylation of gene sets occurred in early premalignant stages followed by somatic mutations in increasing number through the AD-CRC development. Demethylation treatment could reverse the systematic, robust methylation alterations. Epigenetic alterations precede the somatic mutations of the AD-CRC and show higher significance in CRC development than genetic. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Barnabas Wichmann, Arpad V. Patai, Zsolt Tulassay. A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3826. doi:10.1158/1538-7445.AM2015-3826