Scale Gene Expression Analysis Research Articles

Androgen Receptor Activity and Radiotherapeutic Sensitivity in African-American Men with Prostate Cancer: A Large Scale Gene Expression Analysis and Meta-Analysis of RTOG Trials

Population data suggests that African-American (AfA) men have an increased mortality from prostate cancer (PCa) compared to White (C) men. Socioeconomic variables contribute to this disparity, yet intrinsic biological differences remain plausible. Herein, we investigate the interplay of androgen receptor activity (AR-A) and radiotherapeutic sensitivity to provide a molecular rationale to help explain the disparity in outcomes for AfA men with PCa. Transcriptome-wide expression profiles of FFPE tumor samples from 5,831 localized PCa patients were used. Tissue was obtained from a prospective population cohort (n=5,239) and two retrospective cohorts with long-term outcomes (n=592). Predicted radiation sensitivity was measured using the 24-gene post-operative radiation therapy (RT) outcome score (PORTOS). AR-A was defined from the pooled expression of nine canonical AR-target genes. Clinical radiosensitivity was validated using individual patient data from four RTOG trials (92-02, 94-08, 94-13, and 99-10), comprised of 6,011 patients (18.5% AfA). Competing risk adjustments were used for all survival analyses for biochemical recurrence (BCR) and distant metastases (DM). In men treated by surgery from the prospective cohort, low AR-A tumors were significantly more likely to develop DM (10-year rate: 37% vs 17%, p=0.008). On multivariable analysis after adjusting for Gleason grade, T-stage, PSA, margin status, and lymph node invasion, low AR-A remained independently prognostic for DM (p=0.03). After generating a matched cohort of AfA and C patients, AfA tumors were more likely to have low AR-A (p<0.001). However, AfA tumors had decreased double strand break repair pathway expression (p<0.001) and increased predicted RT sensitivity (p<0.001). This suggests that AfA men may have improved outcomes with RT. To clinically test whether AfA tumors are more radiosensitive, we leveraged four large RTOG trials of men treated with RT. On both unadjusted and propensity weighted cohorts (adjusting for age, performance status, PSA, Gleason grade, T-stage, N-stage, and use/duration of hormone therapy), AfA men had significantly improved outcomes compared to C men (BCR: HR 0.82; 95% CI 0.74-0.92; p = 0.0005 and DM: HR 0.70; (95% CI 0.57-0.86; p = 0.0008). Our data suggest that there are population-level differences in AR signaling and DNA repair in AfA and C men’s PCa, which transcriptionally suggest that AfA men may harbor more radiosensitive tumors. To our knowledge, this is the first report demonstrating that AfA men may have improved outcomes compared to C men treated with RT, which is consistent with our hypothesis regarding diversity in AR-A, and warrants further investigation.

Axonal regeneration in zebrafish spinal cord

In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.

BackgroundWhole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).ResultsUsing published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson’s r = 0.92) and Ion Torrent Proton (Pearson’s r = 0.92). We used ROC, Matthew’s correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations.ConclusionsOur study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2270-1) contains supplementary material, which is available to authorized users.

Gene expression and functional annotation of human choroid plexus epithelium failure in Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is the most common form of dementia. AD has a multifactorial disease etiology and is currently untreatable. Multiple genes and molecular mechanisms have been implicated in AD, including ß-amyloid deposition in the brain, neurofibrillary tangle accumulation of hyper-phosphorylated Tau, synaptic failure, oxidative stress and inflammation. Relatively little is known about the role of the blood-brain barriers, especially the blood-cerebrospinal fluid barrier (BCSFB), in AD. The BCSFB is involved in cerebrospinal fluid (CSF) production, maintenance of brain homeostasis and neurodegenerative disorders.ResultsUsing an Agilent platform with common reference design, we performed a large scale gene expression analysis and functional annotation of the Choroid Plexus Epithelium (CPE), which forms the BCSFB. We obtained 2 groups of freshly frozen Choroid Plexus (CP) of 7 human donor brains each, with and without AD: Braak stages (0–1) and (5–6). We cut CP cryo-sections and isolated RNA from cresyl-violet stained, laser dissected CPE cells. Gene expression results were analysed with T-tests (R) and the knowledge-database Ingenuity.We found statistically significantly altered gene expression data sets, biological functions, canonical pathways, molecular networks and functionalities in AD-affected CPE. We observed specific cellular changes due to increased oxidative stress, such as the unfolded protein response, E1F2 and NRF2 signalling and the protein ubiquitin pathway. Most likely, the AD-affected BCSFB barrier becomes more permeable due to downregulation of CLDN5. Finally, our data also predicted down regulation of the glutathione mediated detoxification pathway and the urea cycle in the AD CPE, which suggest that the CPE sink action may be impaired. Remarkably, the expression of a number of genes known to be involved in AD, such as APP, PSEN1, PSEN2, TTR and CLU is moderate to high and remains stable in both healthy and affected CPE. Literature labelling of our new functional molecular networks confirmed multiple previous (molecular) observations in the AD literature and revealed many new ones.ConclusionsWe conclude that CPE failure in AD exists. Combining our data with those of the literature, we propose the following chronological and overlapping chain of events: increased Aß burden on CPE; increased oxidative stress in CPE; despite continuous high expression of TTR: decreased capability of CPE to process amyloid; (pro-) inflammatory and growth factor signalling by CPE; intracellular ubiquitin involvement, remodelling of CPE tight junctions and, finally, cellular atrophy. Our data corroborates the hypothesis that increased BCSFB permeability, especially loss of selective CLDN5-mediated paracellular transport, altered CSF production and CPE sink action, as well as loss of CPE mediated macrophage recruitment contribute to the pathogenesis of AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2159-z) contains supplementary material, which is available to authorized users.

Prognostic Relevance of Circulating Tumor Cells in Molecular Subtypes of Breast Cancer.

Circulating tumor cells (CTCs) can be detected in the peripheral blood of breast cancer patients with early and metastatic disease. Recent data suggest that immune pathologic characteristics between the primary tumor, metastatic colonies and CTCs are discordant and that CTCs possess an independent phenotype that is associated with prognosis and treatment efficacy. Large scale gene expression analysis has provided the possibility to stratify breast cancer according to the gene expression fingerprint of primary tumor tissue into five intrinsic molecular subtypes which can be associated with different clinical outcome. As a consequence of the different prognostic power of primary tumors' characteristics and CTCs several groups have started to investigate if CTCs might be disseminated differentially within these breast cancer subtypes. They determined the CTC number in immunohistochemical subtypes to validate if CTCs may provide differential and more specific prognostic information within each subtype. This review provides an overview of the outcome of some recently published data gathered from early and metastatic breast cancer.

FoxM1 as a novel therapeutic target for cancer drug therapy.

Current cancer therapy mainly focuses on identifying novel targets crucial for tumorigenesis. The FoxM1 is of preference as an anticancer target, due to its significance in execution of mitosis, cell cycle progression, as well as other signal pathways leading to tumorigenesis. FoxM1 is partially regulated by oncoproteins or tumor suppressors, which are often mutated, lost, or overexpressed in human cancer. Since sustaining proliferating signaling is an important hallmark of cancer, FoxM1 is overexpressed in a series of human malignancies. Alarge- scale gene expression analysis also identified FoxM1 as a differentially-expressed gene in most solid tumors. Furthermore, overexpressed FoxM1 is correlated with the prognosis of cancer patients, as verified in a series of malignancies by Cox regression analysis. Thus, extensive studies have been conducted to explore the roles of FoxM1 in tumorigenesis, making it an attractive target for anticancer therapy. Several antitumor drugs have been reported to target or inhibit FoxM1 expression in different cancers, and down-regulation of FoxM1 also abrogates drug resistance in some cancer cell lines, highlighting a promising future for FoxM1 application in the clinic.

Abstract 2363: Large scale gene expression analysis identifies key oncogenic pathways across hematological malignancies

Abstract Hematological malignancies, consisting of all the subtypes of leukemia and lymphoma together with multiple myeloma, constitute a diverse assembly of cancers manifesting primarily in the circulatory or immune systems. Common to all of them is their developmental origin in the hematopoietic cell lineage, which normally produces mature blood cells. For this reason, numerous transcriptomic aberrations, along with their underlying mutations, are shared by different subtypes of hematological malignancies. To enable concurrent gene expression analysis across a comprehensive range of leukemias, lymphomas and multiple myeloma, we compiled a hematological dataset from gene expression microarray data publicly available in the Gene Expression Omnibus data repository. This manually curated collection includes over six thousand neoplastic and nearly nine hundred healthy samples of multiple lineages descending from the hematopoietic stem cell. The cancer samples were manually re-annotated in a congruent manner into categories with three hierarchical levels so as to allow analysis at various levels of specificity. For instance, an early B cell acute lymphocytic leukemia (ALL) sample is classified first as leukemia, secondly as ALL, and in the third, most specific level, as early B-ALL. The power of the dataset lays not only in its extensiveness in terms of subtypes of hematological malignancies, but also in the sheer number of samples within these categories. This enables the discovery of expression signatures with high statistical significance, as well as identification of key oncogenic pathways and regulatory networks within different subtypes. To demonstrate these analyses, we used our database to search for any specific expression patterns of small nucleolar RNAs (snoRNAs) in acute leukemias. Among thousands of leukemia patients, we identified a specific signature of SNORD114 in acute promyelocytic leukemia, previously revealed in a very restricted cohort of patients. Citation Format: Thomas Liuksiala, Kaisa Teittinen, Kirsi Granberg, Merja Heinäniemi, Matti Annala, Antti Ylipää, Markku Mäki, Matti Nykter, Olli Lohi. Large scale gene expression analysis identifies key oncogenic pathways across hematological malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2014-2363

A conserved BDNF, glutamate- and GABA-enriched gene module related to human depression identified by coexpression meta-analysis and DNA variant genome-wide association studies.

Large scale gene expression (transcriptome) analysis and genome-wide association studies (GWAS) for single nucleotide polymorphisms have generated a considerable amount of gene- and disease-related information, but heterogeneity and various sources of noise have limited the discovery of disease mechanisms. As systematic dataset integration is becoming essential, we developed methods and performed meta-clustering of gene coexpression links in 11 transcriptome studies from postmortem brains of human subjects with major depressive disorder (MDD) and non-psychiatric control subjects. We next sought enrichment in the top 50 meta-analyzed coexpression modules for genes otherwise identified by GWAS for various sets of disorders. One coexpression module of 88 genes was consistently and significantly associated with GWAS for MDD, other neuropsychiatric disorders and brain functions, and for medical illnesses with elevated clinical risk of depression, but not for other diseases. In support of the superior discriminative power of this novel approach, we observed no significant enrichment for GWAS-related genes in coexpression modules extracted from single studies or in meta-modules using gene expression data from non-psychiatric control subjects. Genes in the identified module encode proteins implicated in neuronal signaling and structure, including glutamate metabotropic receptors (GRM1, GRM7), GABA receptors (GABRA2, GABRA4), and neurotrophic and development-related proteins [BDNF, reelin (RELN), Ephrin receptors (EPHA3, EPHA5)]. These results are consistent with the current understanding of molecular mechanisms of MDD and provide a set of putative interacting molecular partners, potentially reflecting components of a functional module across cells and biological pathways that are synchronously recruited in MDD, other brain disorders and MDD-related illnesses. Collectively, this study demonstrates the importance of integrating transcriptome data, gene coexpression modules and GWAS results for providing novel and complementary approaches to investigate the molecular pathology of MDD and other complex brain disorders.

Edman sequencing and amino acid analysis in the proteomic age

In an era when large scale gene expression analysis and MS/proteomics are playing increasingly important roles, our review of 148 samples sequenced last year reveals that Edman degradation has evolved from being used to elucidate complete protein sequences to fulfilling several roles for which it is uniquely well suited. These include determining N‐termini of limited cleavage products and constructs used in structure/function and biophysical studies, and of proteins whose start sequences are not well predicted; and for QC of recombinant proteins (e.g., of antigens used to produce vaccines against tuberculosis). The demand for Edman sequencing may expand as emphasis shifts from completing genome sequences to identifying and understanding protein functions, interactions, and post‐translational modifications. In addition, our review of 1,558 amino acid analysis (AAA) samples indicates that hydrolysis/AAA is used to determine the accurate protein/peptide concentrations needed for quantitative biophysical studies and to match protein amounts for comparative proteome analyses such as iTRAQ, to verify the frequency of incorporation of modifications, and the compositions of synthetic and naturally occurring peptides (e.g., differentiating between isobaric Ile/Leu in peptides sequenced by de novo MS/MS). We also will report on preliminary LC‐MRM AAA studies.

Large scale expressed sequence tag (EST) analysis of Metarhizium acridum infecting Locusta migratoria reveals multiple strategies for fungal adaptation to the host cuticle

To infect its insect host, the entomopathogenic fungus Metarhizium acridum has to breach the physical barrier of the host cuticle to gain entry into insect tissue. To identify virulence-associated genes to improve the biocontrol efficacy of M. acridum, it is necessary to understand the genes expressed by M. acridum during its infection of the insect host cuticle. In this study, we performed a large scale gene expression analysis of M. acridum during locust cuticle infection. We report the identification of 4,739 unique expressed sequence tags (ESTs), of which 80 % have not been previously identified. Analysis of the ESTs provided evidence that M. acridum may employ several different adaptation strategies to infect locust cuticle, such as metabolism reprogramming, protein secretion, endocytosis, cell-cycle-regulated morphogenesis and effector-dependent suppression of host immune responses. By searching against the pathogen-host interaction database, several genes associated with different adaptation processes were found to potentially encode important virulence genes. Gene expression pattern analysis by semi-quantitative RT-PCR revealed that M. acridum differentially regulates subsets of genes in response to different infection niches between the host cuticle and hemolymph. The present study provides novel candidate genes to investigate the biology of insect infection by M. acridum.

Structural and inflammatory heterogeneity in subcutaneous adipose tissue: Relation with liver histopathology in morbid obesity

In addition to total body fat, the regional distribution and inflammatory status of enlarged adipose tissue are strongly associated with metabolic co-morbidities of obesity. We recently showed that the severity of histological liver lesions related to obesity increases with the amount of macrophage accumulation in visceral adipose tissue (VAT), while no association was found with the subcutaneous adipose tissue (SAT). In the abdominal region, SAT is anatomically divided into two layers, i.e. superficial (sSAT) and deep (dSAT). The aim of the present study was to test the hypothesis that these distinct compartments differentially contribute to hepatic alterations in obesity. Biopsies of the liver, sSAT, dSAT, and VAT were collected in 45 subjects with morbid obesity (age 43.7±1.6 years; BMI 48.5±1.2kg/m(2)) during bariatric surgery. Large scale gene expression analysis was performed to identify the pathways that discriminate sSAT from dSAT. Adipose tissue macrophages were quantified by immunohistochemistry using HAM56 antibody in subjects scored for liver histopathology. An inflammatory gene pattern discriminates between sSAT and dSAT. dSAT displayed an intermediate level of macrophage accumulation between sSAT and VAT. The abundance of macrophages in dSAT, but not in sSAT, was significantly increased in patients with non-alcoholic steatohepatitis (NASH) and/or fibroinflammatory hepatic lesions. These data show distinct gene signature and macrophage abundance in the two compartments of SAT, with dSAT more closely related to VAT than to sSAT in terms of inflammation and relation with the severity of liver diseases in morbid obesity.

Fine scale analysis of gene expression in Drosophila melanogaster gonads reveals Programmed cell death 4 promotes the differentiation of female germline stem cells

BackgroundGermline stem cells (GSCs) are present in the gonads of Drosophila females and males, and their proper maintenance, as well as their correct differentiation, is essential for fertility and fecundity. The molecular characterization of factors involved in maintenance and differentiation is a major goal both in Drosophila and stem cell research. While genetic studies have identified many of these key factors, the use of genome-wide expression studies holds the potential to greatly increase our knowledge of these pathways.ResultsHere we report a genome-wide expression study that uses laser cutting microdissection to isolate germline stem cells, somatic niche cells, and early differentiating germ cells from female and male gonads. Analysis of this data, in association with two previously published genome-wide GSC data sets, revealed sets of candidate genes as putatively expressed in specific cell populations. Investigation of one of these genes, CG10990 the Drosophila ortholog of mammalian Programmed cell death 4 (Pdcd4), reveals expression in female and male germline stem cells and early differentiating daughter cells. Functional analysis demonstrates that while it is not essential for oogenesis or spermatogenesis, it does function to promote the differentiation of GSCs in females. Furthermore, in females, Pdcd4 genetically interacts with the key differentiation gene bag of marbles (bam) and the stem cell renewal factor eIF4A, suggesting a possible pathway for its function in differentiation.ConclusionsWe propose that Pdcd4 promotes the differentiation of GSC daughter cells by relieving the eIF4A-mediated inhibition of Bam.

Evaluation of Reference Genes for Quantitative Polymerase Chain Reaction across Life Cycle Stages and Tissue Types of Tribolium castaneum

The genome of the genetic model for coleopteran insects, Tribolium castaneum, is now available for downstream applications. To facilitate gene expression studies in T. castaneum, genes were evaluated for suitability as normalizers in comparisons across tissues and/or developmental stages. In less diverse samples, such as comparisons within developmental stages or tissue only, normalizers for mRNA were more stable and consistent. Overall, the genes for ribosomal proteins rps6, rpl13a, rps3, and rps18 were the most stable normalizers for broad scale gene expression analysis in T. castaneum. However, their stability ranking was dependent upon the instrument as well as the analysis program. These data emphasize the need to optimize normalizers used in all real-time polymerase chain reaction experiments specifically for the experimental conditions and thermocycler and to carefully evaluate data generated by computational algorithms.

Large scale analysis of gene expression in the murine embryonic lung

Large scale analysis of gene expression in the murine embryonic lung

871 Eosinophilic Esophagitis (EoE)-Associated Eosinophil Products and Cytokines Reduce Neurogenic Esophageal Smooth Muscle Contractility

In the intestinal epithelium, the Cdx, GATA and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx-2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx-2, GATA-4 and HNF-1α using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx-2 or HNF-1α, alone and in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1α and GATA-4 together induced morphological modifications of the cells towards polarization resulting in the appearance of functional features such as microvilli. HNF-1α was also sufficient to induce the expression of cadherins and dipeptidylpeptidase while in combination with Cdx-2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large scale analysis of gene expression confirmed the cooperative effects of these factors. Finally, while DCAMKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi-1 and Lgr5, were unaffected. These observations show that, cooperatively with Cdx2, HNF-1α acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 initiates the establishment of cell polarity.

Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse

BackgroundSexual dimorphism in brain gene expression has been recognized in several animal species. However, the relevant regulatory mechanisms remain poorly understood. To investigate whether sex-biased gene expression in mammalian brain is globally regulated or locally regulated in diverse brain structures, and to study the genomic organisation of brain-expressed sex-biased genes, we performed a large scale gene expression analysis of distinct brain regions in adult male and female mice.ResultsThis study revealed spatial specificity in sex-biased transcription in the mouse brain, and identified 173 sex-biased genes in the striatum; 19 in the neocortex; 12 in the hippocampus and 31 in the eye. Genes located on sex chromosomes were consistently over-represented in all brain regions. Analysis on a subset of genes with sex-bias in more than one tissue revealed Y-encoded male-biased transcripts and X-encoded female-biased transcripts known to escape X-inactivation. In addition, we identified novel coding and non-coding X-linked genes with female-biased expression in multiple tissues. Interestingly, the chromosomal positions of all of the female-biased non-coding genes are in close proximity to protein-coding genes that escape X-inactivation. This defines X-chromosome domains each of which contains a coding and a non-coding female-biased gene. Lack of repressive chromatin marks in non-coding transcribed loci supports the possibility that they escape X-inactivation. Moreover, RNA-DNA combined FISH experiments confirmed the biallelic expression of one such novel domain.ConclusionThis study demonstrated that the amount of genes with sex-biased expression varies between individual brain regions in mouse. The sex-biased genes identified are localized on many chromosomes. At the same time, sexually dimorphic gene expression that is common to several parts of the brain is mostly restricted to the sex chromosomes. Moreover, the study uncovered multiple female-biased non-coding genes that are non-randomly co-localized on the X-chromosome with protein-coding genes that escape X-inactivation. This raises the possibility that expression of long non-coding RNAs may play a role in modulating gene expression in domains that escape X-inactivation in mouse.

Expression and chromosomal organization of mouse meiotic genes

Microarray technology which enables large scale analysis of gene expression and thus comparison between transcriptomes of different cell types, cells undergoing different treatments or cells at different developmental stages has also been used to study the transcriptome involved with spermatogenesis. Many new germ cell-specific genes were determined, and the resulting genes were classified according to different criteria. However, the biological significance of these classifications and their clustering according to developmental transcriptional patterns during spermatogenesis have not yet been addressed. In this study we utilized mouse testicular transcriptome analysis at five distinct post-natal ages (Days 7, 10, 12, 14, and 17), representing distinct meiotic stages, in an attempt to better understand the biological significance of genes clustered into similar expression patterns during this process. Among 790 sequences that showed an expression level change of twofold or more in any of the five key stages that were monitored, relative to the geometric average of all stages, about 40% peaked and about 30% were specifically suppressed at post-natal day 14 (representing the early pachytene stage of spermatocytes), reflecting tight transcriptional regulation at this stage. We also found that each of the six main transcription clusters that were determined was characterized by statistically significant representation of genes related to specific biological processes. Finally, our results indicated that genes important for meiosis are not randomly distributed along the mouse genome but rather preferentially located on specific chromosomes, suggesting for the first time that chromosomal location might be a regulating factor of meiotic gene expression.

Combining next-generation pyrosequencing with microarray for large scale expression analysis in non-model species

BackgroundThe next generation sequencing technologies provide new options to characterize the transcriptome and to develop affordable tools for functional genomics. We describe here an innovative approach for this purpose and demonstrate its potential also for non-model species.ResultsThe method we developed is based on 454 sequencing of 3' cDNA fragments from a normalized library constructed from pooled RNAs to generate, through de novo reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This "virtual transcriptome" provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in Vitis vinifera as if no other sequence information was available for this species. The microarray designed on the berries' transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes in the same samples.ConclusionThis approach provides a powerful method to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.

A role for “omics” technologies in food safety assessment

Risk assessment frameworks, such as those used for GM crops, have detailed comparative analysis with appropriate non-GM counterparts as their cornerstone. Opinions have been voiced that current analytical approaches are too specific and need to be complemented by more unbiased, larger scale analysis of gene expression and protein expression using transcriptomics and proteomics, respectively. In parallel, the use of metabolomics has been advocated as an approach to expand significantly the range of metabolites that can be measured to assess more stringently the potential for any unintended effects. Transcriptomics, proteomics and metabolomics have been termed collectively “omics” technologies. This review assesses the potential for using “omics” techniques in risk assessment. Importantly, the review provides information on sources of natural variation which can result from crop management practices, from interactions between genotype and growing environment and from non-GM breeding systems. This provides an important benchmark for risk assessors and risk managers.

The role of mtDNA mutations in the pathogenesis of age-related hearing loss in mice carrying a mutator DNA polymerase γ

Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related diseases. Previously, we reported that accumulation of mtDNA mutations is associated with age-related hearing loss in mice carrying a mutator allele of the mitochondrial Polg DNA polymerase. To elucidate the role of mtDNA mutations in the pathogenesis of age-related hearing loss or presbycusis, we performed large scale gene expression analysis to identify mtDNA mutation-responsive genes and biological process categories associated with mtDNA mutations by comparing the gene expression patterns of cochlear tissues from 9-month-old mitochondrial mutator and control mice. mtDNA mutations were associated with transcriptional alterations consistent with impairment of energy metabolism, induction of apoptosis, cytoskeletal dysfunction, and hearing dysfunction in the cochlea of aged mitochondrial mutator mice. TUNEL staining and caspase-3 immunostaining analysis demonstrated that the levels of apoptotic markers were significantly increased in the cochleae of mitochondrial mutator mice compared to age-matched controls. These observations support a new model of how mtDNA mutations impact cochlear function whereby accumulation of mtDNA mutations lead to mitochondrial dysfunction, an associated impairment of energy metabolism, and the induction of an apoptotic program. The data presented here provide the first global assessment at the molecular level of the pathogenesis of age-related disease in mitochondrial mutator mice and reveal previously unrecognized biological pathways associated with mtDNA mutations.