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Results for “comparative transcriptomics”

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PubMed2004

Neuroendocrine transcriptome in genetic hypertension: multiple changes in diverse adrenal physiological systems.

The genetic basis of hypertension in the genetically/hereditary hypertensive (BPH/2) mouse strain is incompletely understood, although a recent genome scan uncovered evidence for several susceptibility loci. To probe the neuroendocrine transcriptome in this disease model, 12 488 probe set microarray experiments were performed on mRNA transcripts from adrenal glands of juvenile (prehypertensive) and adult BPH/2 (hypertensive), as well as the genetically/hereditary low-blood pressure (BPL/1), strains at both time points. To determine the impact of strain (BPH/2 versus BPL/1), age (juvenile versus adult), and the interaction of strain and age on gene expression levels, we performed standard 2-factor ANOVA and computed a concordance coefficient to assess the reproducibility of gene expression measurements among replicates. Of genes with significant (P<0.05) differential expression, 2647 showed strain differences, 982 showed age differences, and 757 exhibited strain-by-age interaction. Fold-changes in gene expression assayed by microarray were confirmed in a subset by real-time polymerase chain reaction (R=0.739, P=0.0094). We used a systems biology approach to evaluate alterations in contributing biochemical pathways and we statistically quantified these global pathway disturbances using the Kolmogorov-Smirnov goodness-of-fit test. We found widespread, indeed global, alterations in patterns of gene expression in diverse systems of BPH/2: in sympathochromaffin transcripts suggesting increased sympathetic stimulation; in vasoconstrictor/vasodilator systems; global reductions in carbohydrate intermediary metabolism; and increases in oxidative stress, with changes in oxygen radical forming and disposition enzymes. These analyses highlight widespread derangements in diverse physiological pathways, providing multiple avenues for further investigation into the pathogenesis of genetic hypertension.

Adrenal Glands↗

PubMed2001

Selective down-regulation of high-affinity IgE receptor (FcepsilonRI) alpha-chain messenger RNA among transcriptome in cord blood-derived versus adult peripheral blood-derived cultured human mast cells.

Substantial numbers of human mast cells (MCs) were generated from umbilical cord blood (CB) and from adult peripheral blood (PB). A single CB progenitor produced 15 436 MCs, whereas a single PB progenitor produced 807 MCs on average. However, PB-derived MCs were far more active than CB-derived MCs in terms of high-affinity IgE receptor (FcepsilonRI)-mediated reactions. One million sensitized PB-derived MCs released 3.6 microg histamine, 215 pg IL-5, and 14 ng granulocyte macrophage-colony-stimulating factor (GM-CSF), whereas 10(6) sensitized CB-derived MCs released only 0.8 microg histamine, 31 pg IL-5, and 0.58 ng GM-CSF on anti-IgE challenge. However, ionophore A23 187 released similar levels of histamine from the 2 MC types. PB-derived MCs highly expressed surface FcepsilonRI alpha chain, and CB-derived MCs almost lacked it in the absence of IgE. PB-derived MCs expressed approximately 5 times higher levels of messenger RNA (mRNA) for FcepsilonRI alpha chain than CB-derived MCs, but mRNAs for beta and gamma chains of the receptors were equally expressed. Among the approximately 5600 kinds of full-length human genes examined by using the high-density oligonucleotide probe-array system, FcepsilonRIalpha was ranked the fifth most increased transcript in PB-derived MCs. The 4 other increased transcripts were unrelated to MC function. These results suggest that IgE-mediated reactions may be restricted during early infancy through the selective inhibition of FcepsilonRIalpha transcription, which is probably committed at progenitor stages and is, at least in part, cytokine-insensitive.

Adult↗

PubMed2005

Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack.

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and E occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

Alternaria↗

PubMed2004

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity.

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

Amino Acid Sequence↗

PubMed2005

Transcriptome responses to carbon tetrachloride and pyrene in the kidney and liver of juvenile rainbow trout (Oncorhynchus mykiss).

We report the effects of the hepatotoxic compound carbon tetrachloride (CCl(4)) and pyrene, a model polycyclic aromatic hydrocarbon, on the transcriptomes of juvenile rainbow trout kidneys and livers. Fish were exposed to sublethal doses for 4 days and expression of 1273 genes was measured using a cDNA microarray. Efforts were focused on differentiating between unspecific responses and those that can be regarded as molecular signatures of CCl(4) and pyrene toxicities. Expression profiles were analyzed in terms of Gene Ontology categories. Universal reactions to chemical toxicity were observed in metallothionein, HSP90 and mitochondrial proteins of oxidative phosphorylation, which were induced in both tissues. Several genes showed similar responses to both compounds in either kidney or liver; most of the effects are implicated in hematopoiesis and immune response. Stimulation of mitochondrial and heat shock proteins was greater in the liver than in the kidney, whereas genes involved in transcription, humoral immune response and apoptosis were suppressed. Pyrene and CCl(4) caused opposite effects on expression of several genes, including HSP-27, macrophage receptor Marco, metalloproteinases (MMP9 and MMP13), and delta-6 fatty acid desaturase. Pyrene affected mainly genes implicated in the maintenance of the genetic apparatus, immune response, glycolysis, and iron homeostasis. CCl(4) affected the structural proteins and genes involved in cellular stress, protein folding, and steroid metabolism. Overall, pyrene suppressed a range of protective or acclimative reactions, many of which were stimulated with CCl(4). Additionally, gene profiling analyses indicated adaptive and potentially maladaptive reactions to toxicity. For instance, stimulation of mitochondrial proteins coincided with suppression of catalase, whereas CCl(4) down-regulated fatty acid metabolism and peroxisomal proteins. A number of candidate biomarkers for ecotoxicological risk assessment were identified as our understanding of mechanisms of pyrene and CCl(4) toxicities in rainbow trout increased.

Animals↗

PubMed2005

Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD), Crohn disease (CD), and ulcerative colitis (UC) are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11), CD patients (n = 10) and UC patients (n = 10). 33P-radiolabeled cDNA from purified poly(A)+ RNA extracted from biopsies (unpooled) was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome) and CDH11 (cadherin 11, type 2). By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

Adolescent↗

PubMed2025

Higher Expression of HPV16 Derived E7_LI Transcript Observed in Men With HIV and Recurrent Anal Cancer.

Squamous cell carcinoma of the anus (SCCA) or anal cancer (AC) is an understudied cancer with a high occurrence rate in people with HIV (PWH), especially men having sex with men (MSM). Furthermore, AC recurs in approximately one-fourth of patients who undergo standard care with chemoradiation therapy (CRT). Using bulk RNA sequencing data of AC obtained from 12 patients with non-recurrent (NR, N = 9) or recurrent (R, N = 3) cancer, we previously showed upregulated expression of key immune genes in the NR compared to the R group. Although the main causative agent of AC is high-risk human papillomavirus (HPV), association of host and viral RNA transcript expression contributing to AC recurrence has not been extensively studied. The objective of the current study was to determine whether enrichment of specific HPV genotypes and/or HPV gene expression patterns differentiate the two groups and if any specific viral (HPV) and host (human) immune mediators correlate with each other. Using bulk RNA sequencing data and VIRTUS 2, we detected viral RNA reads mapping to seven high-risk and six low-risk HPV types, of which the high-risk HPV16 observed in 83% (10/12) AC tumors (7/9 NR and 3/3 R). Rate of all HPV genomes trended toward a decrease in NR AC isolates and correlation between HPV types was more commonly observed in low-risk ones. Analysis of HPV 16 gene expression profile showed a significantly lower positivity rate for a polycistronic transcript encoding for E7^L1 in the NR group (1/9, NR vs. 3/3, R, p < 0.05). An unbiased correlation analysis of HPV-human transcript expression showed a direct correlation between HPV transcripts and human genes involved in cell growth. The data also identified human transcripts showing an inverse correlation with HPV gene expression. These included genes involved in negative regulation of growth, proliferation, and immune response. Taken together, these data indicate that concurrent analyses of viral and host factors in the same tumor can identify potential new therapeutic targets to ameliorate cancer recurrence post-treatment.

Humans↗

PubMed2005

Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2006

Gene expression profiling of human macrophages at late time of infection with Mycobacterium tuberculosis.

Macrophages play an essential role in the immune response to Mycobacterium tuberculosis (Mtb). Previous transcriptome surveys, by means of micro- and macroarrays, investigated the cellular gene expression profile during the early phases of infection (within 48 hr). However, Mtb remains within the host macrophages for a longer period, continuing to influence the macrophage gene expression and, consequently, the environment in which it persists. Therefore, we studied the transcription patterns of human macrophages for up to 7 days after infection with Mtb. We used a macroarray approach to study 858 human genes involved in immunoregulation, and we confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (q-rt RT-PCR) and by enzyme-linked immunosorbent assay the most relevant modulations. We constantly observed the up-regulation in infected macrophages versus uninfected, of the following genes: interleukin-1 beta and interleukin-8, macrophage inflammatory protein-1 alpha, growth-related oncogene-beta, epithelial cell-derived neutrophil-activating peptide-78, macrophage-derived chemokine, and matrix metalloproteinase-7; whereas macrophage colony-stimulating factor-receptor and CD4 were down-regulated in infected macrophages. Mtb is able to withstand this intense cytokine microenvironment and to survive inside the human macrophage. Therefore we simultaneously investigated by q-rt RT-PCR the modulation of five mycobacterial genes: the alternative sigma factors sigA, sigE and sigG, the alpha-crystallin (acr) and the superoxide dismutase C (sodC) involved in survival mechanisms. The identified host and mycobacterial genes that were expressed until 7 days after infection, could have a role in the interplay between the host immune defences and the bacterial escape mechanisms.

Cells, Cultured↗

PubMed2005

Identification of hypertension-related genes through an integrated genomic-transcriptomic approach.

In search for the genetic basis of hypertension, we applied an integrated genomic-transcriptomic approach to identify genes involved in the pathogenesis of hypertension in the Sabra rat model of salt-susceptibility. In the genomic arm of the project, we previously detected in male rats two salt-susceptibility QTLs on chromosome 1, SS1a (D1Mgh2-D1Mit11; span 43.1 cM) and SS1b (D1Mit11-D1Mit4; span 18 cM). In the transcriptomic arm, we studied differential gene expression in kidneys of SBH/y and SBN/y rats that had been fed regular diet or salt-loaded. We used the Affymetrix Rat Genome RAE230 GeneChip and probed >30,000 transcripts. The research algorithm called for an initial genome-wide screen for differentially expressed transcripts between the study groups. This step was followed by cluster analysis based on 2x2 ANOVA to identify transcripts that were of relevance specifically to salt-sensitivity and hypertension and to salt-resistance. The two arms of the project were integrated by identifying those differentially expressed transcripts that showed an allele-specific hypertensive effect on salt-loading and that mapped within the defined boundaries of the salt-susceptibility QTLs on chromosome 1. The differentially expressed transcripts were confirmed by RT-PCR. Of the 2933 genes annotated to rat chromosome 1, 1102 genes were identified within the boundaries of the two blood pressure QTLs. The microarray identified 2470 transcripts that were differentially expressed between the study groups. Cluster analysis identified genome-wide 192 genes that were relevant to salt-susceptibility and/or hypertension, 19 of which mapped to chromosome 1. Eight of these genes mapped within the boundaries of QTLs SS1a and SS1b. RT-PCR confirmed 7 genes, leaving TcTex1, Myadm, Lisch7, Axl-like, Fah, PRC1-like, and Serpinh1. None of these genes has been implicated in hypertension before. These genes become henceforth targets for our continuing search for the genetic basis of hypertension.

Algorithms↗

PubMed2006

Gender-specific expression of complex-type N-glycans in schistosomes.

Sex-specific gene expression by Schistosoma mansoni worms has been demonstrated at the transcriptome as well as the proteome levels. In view of the important role of glycans in the biology of schistosomes and the interaction with their human host, we have investigated the sex-specific protein glycosylation. Mass spectrometric profiling and structural characterization of PNGase F-released N-glycans revealed the following gender-specific glycosylation patterns: Complex-type N-glycans of females mainly carried Gal(beta1-4)GlcNAc (LacNAc) and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc (Lewis x) antennae structures, whereas GalNAc(beta1-4)GlcNAc- (LacdiNAc; LDN) and GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc (LDN-F) were prevalent in N-glycans from males. LDN(-F) motifs were found to occur as repeats on the antennae of large N-glycans that contained up to seven LDN(-F) units. The female complex-type glycans were mostly di-antennary and tri-antennary, whereas male structures were predominantly of the mono-antennary and di-antennary type. Oligomannosidic N-glycans were expressed at similar levels in females and males. The localization of the sex-biased glycan motifs was studied by immunofluorescence microscopy using defined anti-glycan monoclonal antibodies (mAbs). The Lewis x element was strongly expressed in the gut of both males and females, but with respect to tegument localization, the females expressed this structure, while Lewis x seemed to be almost completely absent from the male tegument. The expression of LDN-F was predominantly detected in the parenchyma of both male and female worms as well as in the tegument of the male ventral cavity facing the female. LDN was detected in the tegument of male and female worms at similar levels. The sex-specific expression and differential localization of these antigenic glycan motifs in schistosomes may play a role in male-female interactions during conjugal biology and may lead to a differential immune reaction of the host to the two sexes.

Animals↗

PubMed2002

Full-length-enriched cDNA libraries from Echinococcus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences.

The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60% of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27% of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were <100 nt, although approximately 8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by >60 nt. Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E. granulosus mRNAs appear to be insensitive to the enzymatic treatments used to 'oligo-cap' unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms.

5' Untranslated Regions↗

PubMed2004

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Alternative Splicing↗