A Mixture model with random-effects components for clustering correlated gene-expression profiles

Motivation: The clustering of gene profiles across some experimental conditions of interest contributes significantly to the elucidation of unknown gene function, the validation of gene discoveries and the interpretation of biological processes. However, this clustering problem is not straightforward as the profiles of the genes are not all independently distributed and the expression levels may have been obtained from an experimental design involving replicated arrays. Ignoring the dependence between the gene profiles and the structure of the replicated data can result in important sources of variability in the experiments being overlooked in the analysis, with the consequent possibility of misleading inferences being made. We propose a random-effects model that provides a unified approach to the clustering of genes with correlated expression levels measured in a wide variety of experimental situations. Our model is an extension of the normal mixture model to account for the correlations between the gene profiles and to enable covariate information to be incorporated into the clustering process. Hence the model is applicable to longitudinal studies with or without replication, for example, time-course experiments by using time as a covariate, and to cross-sectional experiments by using categorical covariates to represent the different experimental classes. Results: We show that our random-effects model can be fitted by maximum likelihood via the EM algorithm for which the E(expectation) and M(maximization) steps can be implemented in closed form. Hence our model can be fitted deterministically without the need for time-consuming Monte Carlo approximations. The effectiveness of our model-based procedure for the clustering of correlated gene profiles is demonstrated on three real datasets, representing typical microarray experimental designs, covering time-course, repeated-measurement and cross-sectional data. In these examples, relevant clusters of the genes are obtained, which are supported by existing gene-function annotation. A synthetic dataset is considered too.

MediPlEx - a tool to combine in silico & experimental gene expression profiles of the model legume Medicago truncatula

Background: Expressed Sequence Tags (ESTs) are in general used to gain a first insight into gene activities from a species of interest. Subsequently, and typically based on a combination of EST and genome sequences, microarray-based expression analyses are performed for a variety of conditions. In some cases, a multitude of EST and microarray experiments are conducted for one species, covering different tissues, cell states, and cell types. Under these circumstances, the challenge arises to combine results derived from the different expression profiling strategies, with the goal to uncover novel information on the basis of the integrated datasets. Findings: Using our new analysis tool, MediPlEx (MEDIcago truncatula multiPLe EXpression analysis), expression data from EST experiments, oligonucleotide microarrays and Affymetrix GeneChips® can be combined and analyzed, leading to a novel approach to integrated transcriptome analysis. We have validated our tool via the identification of a set of well-characterized AM-specific and AM-induced marker genes, identified by MediPlEx on the basis of in silico and experimental gene expression profiles from roots colonized with AM fungi. Conclusions: MediPlEx offers an integrated analysis pipeline for different sets of expression data generated for the model legume Medicago truncatula. As expected, in silico and experimental gene expression data that cover the same biological condition correlate well. The collection of differentially expressed genes identified via MediPlEx provides a starting point for functional studies in plant mutants.

PR gene families of citrus: their organ specific-biotic and abiotic inducible expression profiles based on ESTs approach

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sub-telomere directed gene expression during initiation of invasive aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the sigma(54) regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma(54)-dependent manner. A more complete picture of the sigma(54) regulon was achieved by combining the transcriptome data with an in silico search for potential sigma(54)-dependent promoters, using a position weight matrix approach. One of these sigma(54)-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma(54)-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma(54) regulon.

A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

Background: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/.S3T source code and datasets can also be downloaded from the aforementioned website.

Identification of protein-coding and non-coding RNA expression profiles in CD34(+) and in stromal cells in refractory anemia with ringed sideroblasts

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods: In this study, gene expression profiles of CD34(+) cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results: In CD34(+) cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value <= 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value <= 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions: These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34(+) cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Gene expression profiling of papillary thyroid carcinoma identifies transcripts correlated with BRAF mutational status and lymph node metastasis

Purpose: To identify papillary thyroid carcinoma (PTC)-associated transcripts, we compared the gene expression profiles of three Serial Analysis of Gene Expression libraries generated from thyroid tumors and a normal thyroid tissue. Experimental Design: Selected transcripts were validated in a panel of 57 thyroid tumors using quantitative PCR (qPCR). An independent set of 71 paraffin-embedded sections was used for validation using immunohistochemical analysis. To determine if PTC-associated gene expression could predict lymph node involvement, a separate cohort of 130 primary PTC (54 metastatic and 76 nonmetastatic) was investigated. The BRAF(V600E) mutational status was compared with qPCR data to identify genes that might be regulated by abnormal BRAF/MEK/extracellular signal-regulated kinase signaling. Results: We identified and validated new PTC-associated transcripts. Three genes (CST6, CXCL14, and DHRS3) are strongly associated with PTC. Immunohistochemical analysis of CXCL14 confirmed the qPCR data and showed protein expression in PTC epithelial cells. We also observed that CST6, CXCL14, DHRS3, and SPP1 were associated with PTC lymph node metastasis, with CST6, CXCL14, and SPP1 being positively correlated with metastasis and DHRS3 being negatively correlated. Finally, we found a strong correlation between CST6 and CXCL14 expression and BRAF(V600E) mutational status, suggesting that these genes may be induced subsequently to BRAF activation and therefore may be downstream in the BRAF/MEK/extracellular signal-regulated kinase signaling pathway. Conclusion: CST6, CXCL14, DHRS3, and SPP1 may play a role in PTC pathogenesis and progression and are possible molecular targets for FTC therapy.

Deregulation of LOXL1 and HTRA1 Gene Expression in Endometriosis

Endometriosis is a gynecologic disease characterized by the presence of endometrial tissue outside the uterine cavity. Although 15% of the female population in reproductive age is affected by endometriosis, its pathogenesis remains unclear. According to the most accepted pathogenesis hypothesis, endometrial fragments from the menstrual phase are transported through the uterine tubes to the peritoneal cavity, where they undergo implantation and growth, invading adjacent tissues. However, the establishment of the disease requires that endometrial cells present molecular characteristics favoring the onset and progression of ectopic implantation. In this investigation, we analyzed the differential gene expression profiles of peritoneal and ovarian endometriotic lesions compared to the endometrial tissue of nonaffected women using rapid subtraction hybridization (RaSH). In our study, this method was applied to samples of endometriotic lesions from affected women and to biopsies of endometrium of healthy women without endometriosis, where we could identify 126 deregulated genes. To evaluate the expression of genes found by RaSH method, we measured LOXL1, HTRA1, and SPARC genes by real-time polymerase chain reaction. Significant different expression was obtained for HTRA1 and LOXL1, upregulated in the ectopic endometrium, suggesting that these genes are involved in the physiopathology of endometriosis and may favor the viability of endometrial cells at ectopic sites.

Alterations in gene expression and activity during squamous cell carcinoma development

This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLD-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P less than or equal to 0.05; 1.9% of genes screened) altered in neoplastic cells compared with normal cells. Of these genes, 10 genes were up-regulated and 27 genes were down-regulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the preneoplastic IEC-1 cells. Immunohistochemical staining of patient tumors was used to verify the cDNA array analysis. Our analysis indicated that alterations in genes associated with extracellular matrix production and apoptosis are disrupted in preneoplastic cells, whereas later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or epidermal growth factor (EGF) receptor/mitogen-activated protein kinase kinase (MAPKK)/MAPK/activator protein-1 (AP-1) signaling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they did not contribute to the neoplastic phenotype.

Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes

Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 It recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Gene Expression Profiling and Association Studies Implicate the Neuregulin Signaling Pathway in Behçet's Disease Susceptibility

Behçet's disease (BD) is a complex disease with genetic and environmental risk factors implicated in its etiology; however, its pathophysiology is poorly understood. To decipher BD's genetic underpinnings, we combined gene expression profiling with pathway analysis and association studies. We compared the gene expression profiles in peripheral blood mononuclear cells (PBMCs) of 15 patients and 14 matched controls using Affymetrix microarrays and found that the neuregulin signaling pathway was over-represented among the differentially expressed genes. The Epiregulin (EREG), Amphiregulin (AREG), and Neuregulin-1 (NRG1) genes of this pathway stand out as they are also among the top differentially expressed genes. Twelve haplotype tagging SNPs at the EREG-AREG locus and 15 SNPs in NRG1 found associated in at least one published BD genome-wide association study were tested for association with BD in a dataset of 976 Iranian patients and 839 controls. We found a novel association with BD for the rs6845297 SNP located downstream of EREG, and replicated three associations at NRG1 (rs4489285, rs383632, and rs1462891). Multifactor dimensionality reduction analysis indicated the existence of epistatic interactions between EREG and NRG1 variants. EREG-AREG and NRG1, which are members of the epidermal growth factor (EGF) family, seem to modulate BD susceptibility through main effects and gene–gene interactions. These association findings support a role for the EGF/ErbB signaling pathway inBD pathogenesis that warrants further investigation and highlight the importance of combining genetic and genomic approaches to dissect the genetic architecture of complex diseases.

Reconstruction of the regulatory network for Bacillus subtilis and reconciliation with gene expression data

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00275

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.