Differential Gene Expression Studies Research Articles

Application of genefishing discovery system on differential gene expression study for prokaryotic system

GeneFishing Discovery is a system applied on disease and gene related research for eukaryotes and the starting material for the study could be either total RNA or mRNA. In this study we demonstrate the use of this discovery system for a prokaryotic system by modifying the eukaryotic protocol using the poly (A)-tailing approach. As an example we compared the gene expression pattern of two closely related species of bacteria under the same family (Burkholderia thailandensis andBurkholderia cepacia) with satisfactory results. Key words: Genefishing, differential expression, prokaryotic system.

Molecular mechanisms of viral infection and propagation: An overview of the second Advanced Summer School in Africa

Molecular mechanisms of viral infection and propagation: An overview of the second Advanced Summer School in Africa

Identification of candidate genes important for frost tolerance in Festuca pratensis Huds. by transcriptional profiling

Studies of differential gene expression between cold acclimated (CA) and non-cold acclimated (NA) plants yield insight into how plants prepare for cold stress at the transcriptional level. Furthermore genes involved in the cold acclimation process are good candidate loci for genetic variation in frost tolerance and winter survival. In this study we combine different approaches to try to decode the genetics of cold acclimation and frost tolerance in meadow fescue (Festuca pratensis Huds). An EST library of cold acclimation responsive genes was established by suppression subtractive hybridization (SSH), and a microarray experiment was used to identify gene expression differences between high and low frost tolerance genotypes in response to cold acclimation. Many genes known to be involved in CA in other species were confirmed to be involved in CA in F. pratensis, however, 18% of the ESTs did not show significant homology to any database proteins. Seven genes were found to be differentially expressed (>2-fold) between high and low frost tolerance genotypes. Two of these genes, FpQM and FpTPT, represent interesting candidate genes for frost tolerance in perennial forage grasses.

Examining smoking-induced differential gene expression changes in buccal mucosa

BackgroundGene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood. However, a less invasive method of sample collection is of interest because of the discomfort and specialized personnel necessary for blood sampling especially if multiple samples are being collected. Buccal mucosa cells are easily collected and may be an alternative sample material for biomarker testing. A limited number of studies, primarily in the smoker/oral cancer literature, address this tissue's efficacy as an RNA source for expression analysis. The current study was undertaken to determine if total RNA isolated from buccal mucosa could be used as an alternative tissue source to assay relative gene expression.MethodsTotal RNA was isolated from swabs, reverse transcribed and amplified. The amplified cDNA was used in RT-qPCR and microarray analyses to evaluate gene expression in buccal cells. Initially, RT-qPCR was used to assess relative transcript levels of four genes from whole blood and buccal cells collected from the same seven individuals, concurrently. Second, buccal cell RNA was used for microarray-based differential gene expression studies by comparing gene expression between a group of female smokers and nonsmokers.ResultsAn amplification protocol allowed use of less buccal cell total RNA (50 ng) than had been reported previously with human microarrays. Total RNA isolated from buccal cells was degraded but was of sufficient quality to be used with RT-qPCR to detect expression of specific genes. We report here the finding of a small number of statistically significant differentially expressed genes between smokers and nonsmokers, using buccal cells as starting material. Gene Set Enrichment Analysis confirmed that these genes had a similar expression pattern to results from another study.ConclusionsOur results suggest that despite a high degree of degradation, RNA from buccal cells from cheek mucosa could be used to detect differential gene expression between smokers and nonsmokers. However the RNA degradation, increase in sample variability and microarray failure rate show that buccal samples should be used with caution as source material in expression studies.

A large-scale study of differential gene expression in monocyte-derived macrophages infected with several strains of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is a significant concern to the American and European dairy industries and possibly to human health. MAP possesses the rare ability to survive and replicate in infected macrophages, cells that are typically able to destroy pathogens. Little is known about what changes occur in MAP-infected macrophages that prevent phagosome maturation and lead to intracellular survival of the bacteria. In this study, a bovine immunologically specific cDNA microarray was used to study genes whose expression was altered in monocyte-derived macrophages (MDM) when these cells were infected with 10 different strains of MAP bacteria. Although we used MAP strains isolated from four different host species, cluster analysis of each strains influence in infected MDMs showed no species of origin specific MAP alterations in the host transcriptome. However, MAP strain K10 was observed as a clear outlier in the cluster analysis. Additionally, we observed two SuperShedder MAP strains clustering very closely together compared to the other strains in this study. Overall, microarray analysis yielded 78 annotated genes whose expression was altered by MAP infection, regardless of strain. Few of these genes have been previously studied in the context of Johne's disease or other mycobacterium-caused diseases. Large groups of apoptosis genes, transcription factors and cytokines were found to be differentially expressed in infected monocyte-derived macrophages as well as several genes not previously linked to MAP-host interactions. Identifying novel host genes affected by MAP infection of macrophages may lead to a more complete picture of this complex host-pathogen interaction.

ATP-binding Cassette Transporter ABCB5 Gene is Expressed with Variability in Malignant Melanoma

Background Melanoma is a malignant neoplasm with high metastatic disease risk and elevated mortality. Incidence of melanoma varies according to geographic region and genetic background. Epidemiological studies indicate that acral melanoma (AM) is among the most common melanomas in the Mexican population. While extensive studies have identified genes associated with melanoma, little is known about the genes involved in the pathogenesis of AM. Objective To compare the gene expression patterns between primary melanoma and normal skin. Methods We used 10 samples of fresh acral melanomas and normal skin for the study of differential gene expression and 22 samples of melanoma for in situ hybridization. Results We first identified a gene that was present in a sample of AM and absent in normal skin. DNA sequencing of this differentially expressed gene revealed that it corresponded to ABCB5, a gene recently implicated in the regulation of progenitor cell fusion. Furthermore, we detected ABCB5 expression in other melanoma specimens by RT-PCR. We showed that nine out of ten melanomas were positive for ABCB5 while only one melanoma and normal skin samples were negative. All ABCB5 expressing melanomas had variable gene expression according to in situ hybridization studies, suggesting that the ABCB5 gene may be differentially regulated by individual melanomas. Conclusions The ABCB5 gene may be related to the properties of chemoresistance and aggressiveness of melanoma. The high expression found in samples of acral melanoma may provide more insight on the pathogenesis of this common type of melanoma in the Mexican population, frequently associated with poor prognosis.

Lactoferrin as an effector molecule in the skeleton

Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. In recent years, studies have shown that lactoferrin also acts on the skeleton to promote bone growth. Lactoferrin stimulates the proliferation and differentiation of the bone forming cells, the osteoblasts, and acts as a survival factor for these cells. Lactoferrin also inhibits osteoclastogenesis, reducing the number of cells that can actively resorb bone, thus producing a greater overall increase in bone volume. In vivo, local injection of lactoferrin results in substantial increases in bone area, establishing lactoferrin as an effector molecule in the skeleton. Investigations of the mechanism of action of lactoferrin in bone cells showed that the mitogenic effect of lactoferrin in osteoblasts is mediated mainly through LRP1, a member of the low density lipoprotein receptor-related proteins. Lactoferrin induces activation of p42/44 MAPK signaling as well as PI3-kinase-dependent phosphorylation of Akt in osteoblasts. Differential gene expression studies indicated a possible role for the activation of IGF1, Ptgs2 and Nfatc1 in mediating the mitogenic activity of lactoferrin in osteoblasts. Lactoferrin is a positive regulator of bone with a possible physiological role in bone growth and healing. There is a growing interest in the potential use of lactoferrin for the improvement of bone health, and in a number of recent studies dietary lactoferrin supplementation improved bone mineral density and bone strength. Lactoferrin appears to be a promising candidate for the development of an anabolic therapeutic factor for osteoporosis.

ATP-binding Cassette Transporter ABCB5 Gene is Expressed with Variability in Malignant Melanoma

Background Melanoma is a malignant neoplasm with high metastatic disease risk and elevated mortality. Incidence of melanoma varies according to geographic region and genetic background. Epidemiological studies indicate that acral melanoma (AM) is among the most common melanomas in the Mexican population. While extensive studies have identified genes associated with melanoma, little is known about the genes involved in the pathogenesis of AM. Objective To compare the gene expression patterns between primary melanoma and normal skin. Methods We used 10 samples of fresh acral melanomas and normal skin for the study of differential gene expression and 22 samples of melanoma for in situ hybridization. Results We first identified a gene that was present in a sample of AM and absent in normal skin. DNA sequencing of this differentially expressed gene revealed that it corresponded to ABCB5, a gene recently implicated in the regulation of progenitor cell fusion. Furthermore, we detected ABCB5 expression in other melanoma specimens by RT-PCR. We showed that nine out of ten melanomas were positive for ABCB5 while only one melanoma and normal skin samples were negative. All ABCB5 expressing melanomas had variable gene expression according to in situ hybridization studies, suggesting that the ABCB5 gene may be differentially regulated by individual melanomas. Conclusions The ABCB5 gene may be related to the properties of chemoresistance and aggressiveness of melanoma. The high expression found in samples of acral melanoma may provide more insight on the pathogenesis of this common type of melanoma in the Mexican population, frequently associated with poor prognosis.

Identification of genes involved in the floral transition at the shoot apical meristem of Lolium perenne L. by use of suppression subtractive hybridisation

Generally, heading in Lolium perenne L. (perennial ryegrass) is associated with a reduction in the feed quality of the forage and therefore extending the period of vegetative growth during the growing season will contribute to an improvement in quality. The genetic control of floral transition has been well studied in model plant species but less research has been done in economically important crop species such as perennial ryegrass. A differential gene expression study was performed between two full sibling lines of L. perenne with contrasting flowering time. Suppression subtractive hybridization was used to identify 155 transcripts differentially expressed between the two sibling lines in the shoot apical meristem after primary and during secondary induction. Transcripts with a putative role in the floral transition were further characterized, through floral induction stages, by real time RT–PCR. This revealed five genes with greater than tenfold difference in expression between the lines during floral induction. Furthermore, a putative methyl binding domain protein and bHLH transcription factor were identified, which show clear differential expression patterns through floral induction and may act as potential enhancers of flowering in L. perenne.

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy.

Development of a cDNA microarray of zebra mussel ( Dreissena polymorpha) foot and its use in understanding the early stage of underwater adhesion

The underwater adhesion of the zebra mussel ( Dreissena polymorpha) to substrates is a complex process that is controlled by a delicate apparatus, the byssus. As a critical activity of the byssus glands embedded in the zebra mussel feet, byssogenesis is highly active to produce numerous byssal threads from the settled juvenile stage through the adult stage in its life cycle. This lifelong activity helps the zebra mussel to firmly attach to substrata underwater, thereby causing severe economic and ecologic impacts. In an attempt to better understand the zebra mussel's byssus activity, a cDNA microarray (ZMB) including 716 genes, generated from a Suppression Subtractive Hybridization (SSH) cDNA library, was printed and used for the comparison of gene expression during zebra mussel adhesion and non-adhesion. To better understand the byssogenesis mechanism, RNA samples from the zebra mussel feet with byssogenesis and without byssogenesis were used in a two-color hybridization to reveal the gene differential expression in the two states. Based on the P values ( P < 0.05), Fifty-two ESTs were found as differentially expressed genes and were divided into two groups, upregulated and downregulated groups according to there logFC values. With the false discovery rate (FDR) adjustment, seven were identified from the upregulated group and nine from the downregulated group. Phylogenetic analysis indicated that the four excretory gland peptide-like protein (EGP) encoding genes in upregulated group are structurally different than the two in the downregulated list. The amino acid composition analysis on the proteins, which were encoded by the up- or downregulated ESTs without homologues (NH) suggested that seven of the NH proteins are biochemically similar to the novel foot proteins from other mussels. The quantitative reverse transcription PCR (QRT-PCR) proved the uniqueness of the templates in the array, and also confirmed the differentially expressed genes identified by microarray experiment. Our findings demonstrated that the zebra mussel byssus cDNA microarray is an efficient tool for the studies of differential gene expression in different byssogenesis states, thereby revealing important details of the underwater adhesion.

Is cancer a genetic program with an unknown function?

Cancer is a tissue that requires a genetic program, possibly involving hundreds of genes, as indicated by studies of differential gene expression performed with microarrays in various types of cancer. Cancer is thus a biological process of considerable precision as described by Schrödinger's equation, and it is not simply the random expression of hundreds of genes, but rather a program that gives neoplastic tissue characteristics that are independent of the originating tissue: invasion capacity and destruction of neighbouring tissues, metastasis, and capacity to progressively disengage from the genetic program of the original tissue. This program is common to all types of cancer, and does not appear to be related to the genetic program of any known differentiated tissue. In other words, cancer's genetic program appears to be universal in all tissues, since cancer behaves similarly in all tissues; though depending on the tissue in which it originates it will show certain specific characteristics, reflecting the extent to which the neoplastic cells are able to escape from the characteristics of the original tissue.

Leaf senescence and gene expression.

Abstract The long-standing view of leaf senescence as an organized genetically controlled process has received convincing support from recent genome-wide studies of differential gene expression. Many hundreds of senescence-associated genes (SAGs) appear to be up- or down-regulated for the sake of nutrient salvage, including a wide array of SAGs that encode proteases, nucleases, lipid-, carbohydrate- and amino acid-degrading enzymes. Mobilization and export of nutrients from senescing leaves is accompanied by complex expression of a special set of genes for transporters. Another category of genes with altered transcription rates was shown to control protective- or stress-response functions necessary to keep the cells viable till the task of salvage is completed. Coordinated activation or inactivation of various subsets of SAGs is governed by a comprehensive network of regulatory genes, such as genes for transcription factors and hormone pathways. Transcriptome data and mutant screens provide a foundation for further analysis of physiological functions of senescence-expressed genes and subsequent manipulation for the benefits of plant breeding.

Profiling of infection specific mRNA transcripts of the European seabass Dicentrarchus labrax

BackgroundThe European seabass (Dicentrarchus labrax), one of the most extensively cultured species in European aquaculture productions, is, along with the gilthead sea bream (Sparus aurata), a prospective model species for the Perciformes which includes several other commercially important species. Massive mortalities may be caused by bacterial or viral infections in intensive aquaculture production. Revealing transcripts involved in immune response and studying their relative expression enhances the understanding of the immune response mechanism and consequently also the creation of vaccines. The analysis of expressed sequence tags (EST) is an efficient and easy approach for gene discovery, comparative genomics and for examining gene expression in specific tissues in a qualitative and quantitative way.ResultsHere we describe the construction, analysis and comparison of a total of ten cDNA libraries, six from different tissues infected with V. anguillarum (liver, spleen, head kidney, gill, peritoneal exudates and intestine) and four cDNA libraries from different tissues infected with Nodavirus (liver, spleen, head kidney and brain). In total 9605 sequences representing 3075 (32%) unique sequences (set of sequences obtained after clustering) were obtained and analysed. Among the sequences several immune-related proteins were identified for the first time in the order of Perciformes as well as in Teleostei.ConclusionThe present study provides new information to the Gene Index of seabass. It gives a unigene set that will make a significant contribution to functional genomic studies and to studies of differential gene expression in relation to the immune system. In addition some of the potentially interesting genes identified by in silico analysis and confirmed by real-time PCR are putative biomarkers for bacterial and viral infections in fish.

Genetic Susceptibility to Autoimmune Disorders: Clues from Gene Association and Gene Expression Studies

Susceptibility to autoimmune disorders results from the interaction of multiple genetic factors that regulate the threshold of autoreactivity. Genome-wide microsatellite screens and large-scale single nucleotide polymorphism (SNP) association studies have identified chromosomal loci that are associated with specific disorders including systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, multiple sclerosis, and diabetes. Numerous candidate gene association studies have in turn investigated the association of specific genes within these chromosomal regions, with susceptibility to autoimmune diseases (e.g. FcgammaReceptors, TYK2 and systemic lupus). More recently, large-scale differential gene expression studies performed on selected tissues from patients with autoimmune disorders, have led to the identification of gene signatures associated with the activation of specific pathways in these diseases (e.g. interferon signature in lupus). In the future, integrated analyses of gene (and protein) expression together with SNP data will allow us to sketch an intelligible picture of the genesis of autoimmunity in humans. This review sets out to illustrate how the most recent advances in the field of systemic lupus erythematosus, rheumatoid arthritis and juvenile arthritis have led to a better understanding of these disorders.

Metastatic Osteosarcoma Gene Expression Differs In Vitro and In Vivo

An understanding of differential gene expression in highly metastatic osteosarcoma could provide gene targets for treatment of metastases. We compared gene expression profiles of high- (LM7) and low- (LM2) metastatic SaOS2-derived cell lines in an in vitro tissue culture model and examined several differentially regulated genes in vivo in a murine orthotopic xenograft model. We hypothesized an orthotopic inoculation of LM2 and LM7 cells would establish a primary lesion and the gene expression profile of cells grafted in this fashion would resemble the gene expression profile observed in an in vitro model. Thirty-five days after inoculation, animals were euthanized and both tibiae were harvested and rapidly frozen in liquid nitrogen. Human-specific GAPDH mRNA was present in two of four tibias inoculated with LM2 cells and three of four tibias inoculated with LM7 cells. Tibiae displaying the presence of human cells were assayed by semiquantitative reverse transcriptase polymerase chain reaction. We observed poor correspondence of in vitro to in vivo gene expression for either cell line. Accordingly, in vitro osteosarcoma gene expression data must be interpreted with caution until confirmed in vivo. Our orthotopic injection model allowed in vivo study of differential gene expression between these two cell lines but did not show radiographic evidence of an established primary lesion.

A structural mixed model to shrink covariance matrices for time-course differential gene expression studies

Time-course microarray studies require a particular modelling of covariance matrices when measures are repeated on the same individuals. Taking into account the within-subject correlation in the test statistics for differential gene expression, however, requires a large number of parameters when a gene-specific approach is used, which often results in a lack of power due to the small number of individuals usually considered in microarray experiments. Shrinkage approaches can improve this detection power in differential gene expression studies by reducing the number of parameters, while offering a good flexibility and a small rate of false positives. A natural extension of the shrinkage approach based on a structural mixed model to variance–covariance matrices is proposed. The structural model was used in three configurations to shrink (i) the eigenvalues in an eigenvalue/eigenvector decomposition, (ii) the innovation variances in a Cholesky decomposition, (iii) both the variances and correlation parameters of a gene-by-gene covariance matrix using a Fisher transformation. The proposed methods were applied both to a publicly available data set and to simulated data. They were found to perform well, compared to previously proposed empirical Bayesian approaches, and outperformed the gene-specific or common-covariance methods in many cases.

The molecular pathophysiology of pseudoexfoliation glaucoma

This review summarizes recent observations and discoveries important for a current understanding of the clinical, molecular and genetic aspects of pseudoexfoliation glaucoma. Significant advances in our understanding of the molecular nature of the pseudoexfoliation material and pseudoexfoliation glaucoma have recently been made in the transcriptome, proteome, and genome levels. Differential gene expression studies have identified gene transcripts that are significantly downregulated and upregulated in the eyes of patients with pseudoexfoliation glaucoma relative to nonglaucomatous controls. Many of these differentially regulated genes are involved with extracellular matrix structure and metabolism and responses to stress and inflammation. Proteomic analysis of the pseudoexfoliation material similarly suggests that extracellular matrix and stress response proteins are associated with pseudoexfoliation glaucoma. Most recently, LOXL1 (which is involved with extracellular matrix formation and stability) has been discovered to be the first gene associated with a risk of developing pseudoexfoliation glaucoma. A proposed protein sink model parsimoniously accounts for the multitude of proteins known to be associated with pseudoexfoliation material and describes the orderly molecular formation of this disease causing material in the eye. Our understanding of pseudoexfoliation glaucoma has recently been significantly advanced by cutting edge molecular and genetic approaches to studying this sight threatening disease. An increased understanding of the molecular pathophysiology of pseudoexfoliation glaucoma will lead to improved management and diagnosis and new treatments for pseudoexfoliation glaucoma.

Resveratrol as an Inhibitor of Carcinogenesis

Given the high probability of developing cancer over the period of a normal life span, cancer chemoprevention provides an attractive therapeutic strategy for the delay or reversal of this process. A variety of phytochemicals, such as sulfides, isothiocyanates, glucosinolates, flavonoids, carotenoids, phenols, and diarylhepanoids, are known to mediate chemopreventive responses. Resveratrol, a ubiquitous stilbene found in the diet of human beings (e.g., as a component of grapes and wine), was uncovered by bioassay-guided fractionation and found to mediate cancer chemopreventive activity in a murine model with mechanisms involving various stages of the carcinogenic process. This work spurred a myriad of studies that are summarized in this article. As demonstrated with in vitro. and cell culture models, resveratrol functions through a plethora of mechanisms, which can vary from model to model. Results from differential gene expression studies are daunting. Irrespective of the precise mechanism, however, efficacy has been demonstrated in some animal models, and a critical evaluation of resveratrol data relative to the characteristics of a promising cancer chemopreventive agent leads to favorable consideration. Animal studies have shown cancer inhibitory activity in a number of models, including adenoma, skin, breast, colon, esophagus, glioma, intestinal, liver, and neuroblastoma. Biomarkers are known, and ample quantities of compound can be produced. Dietary administration is feasible. Several small-scale human trials are under way, and human intervention trials may follow. As learned by past experience, data from these trials are necessary prior to drawing any conclusions, but the current cancer chemopreventive profile of resveratrol provides promise for widespread use in the future.

Universal False Discovery Rate Estimation Methodology for Genome-Wide Association Studies

Genome-wide case-control association studies aim at identifying significant differential markers between sick and healthy populations. With the development of large-scale technologies allowing the genotyping of thousands of single nucleotide polymorphisms (SNPs) comes the multiple testing problem and the practical issue of selecting the most probable set of associated markers. Several False Discovery Rate (FDR) estimation methods have been developed and tuned mainly for differential gene expression studies. However they are based on hypotheses and designs that are not necessarily relevant in genetic association studies. In this article we present a universal methodology to estimate the FDR of genome-wide association results. It uses a single global probability value per SNP and is applicable in practice for any study design, using any statistic. We have benchmarked this algorithm on simulated data and shown that it outperforms previous methods in cases requiring non-parametric estimation. We exemplified the usefulness of the method by applying it to the analysis of experimental genotyping data of three Multiple Sclerosis case-control association studies.