rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Trypanosoma cruzi Gene Expression in Response to Gamma Radiation

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.

Gene expression of peripheral blood lymphocytes may discriminate patients with schizophrenia from controls

To identify a classifier in schizophrenia, blood gene expression profiling was applied to patients with schizophrenia under different treatments and to controls. Expression of six genes discriminated patients with sensitivity of 89.3% and specificity of 90%, supporting the use of peripheral blood as biological material for diagnosis in schizophrenia. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray

Abstract Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process.

Gene expression responses to acute and chronic heat stress in the common reef-building coral Pocillopora verrucosa

Global climate change is impacting coral reefs worldwide, with approximately 19% of reefs being permanently degraded, 15% showing symptoms of imminent collapse, and 20% at risk of becoming critically affected in the next few decades. This alarming level of reef degradation is mainly due to an increase in frequency and intensity of natural and anthropogenic disturbances. Recent evidence has called into question whether corals have the capacity to acclimatize or adapt to climate changes and some groups of corals showed inherent physiological tolerance to environmental stressors. The aim of the present study was to evaluate mRNA expression patterns underlying differences in thermal tolerance in specimen of the common reef-building coral Pocillopora verrucosa collected at different locations in Bangka Island waters (North Sulawesi, Indonesia). Part of the experimental work was carried out at the CoralEye Reef Research Outpost (Bangka Island). This includes sampling of corals at selected sites and at different depths (3 and 12 m) as well as their experimental exposure to an increased water temperature under controlled conditions for 3 and 7 days. Levels of mRNAs encoding ATP synthase (ATPs) NADH dehydrogenase (NDH) and a 70kDa Heat Shock Protein (HSP70) were evaluated by quantitative real time PCR. Transcriptional profiles evaluated under field conditions suggested an adaptation to peculiar local environmental conditions in corals collected at different sites and at the low depth. Nevertheless, high–depth collected corals showed a less pronounced site-to-site separation suggesting more homogenous environmental conditions. Exposure to an elevated temperature under controlled conditions pointed out that corals adapted to the high depth are more sensitive to the effects of thermal stress, so that reacted to thermal challenge by significantly over-expressing the selected gene products. Being continuously exposed to fluctuating environmental conditions, low-depth adapted corals are more resilient to the stress stimulus, and indeed showed unaffected or down-regulated mRNA expression profiles. Overall these results highlight that transcriptional profiles of selected genes involved in cellular stress response are modulated by natural seasonal temperature changes in P. verrucosa. Moreover, specimens living in more variable habitats (low-depth) exhibit higher basal HSP70 mRNA levels, possibly enhancing physiological tolerance to environmental stressors.

Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma

Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use.

14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

Renal allografts with IF/TA display distinct expression profiles of metzincins and related genes

Chronic renal allograft injury is often reflected by interstitial fibrosis (IF) and tubular atrophy (TA) without evidence of specific etiology. In most instances, IF/TA remains an irreversible disorder, representing a major cause of long-term allograft loss. As members of the protease family metzincins and functionally related genes are involved in fibrotic and sclerotic processes of the extracellular matrix (ECM), we hypothesized their deregulation in IF/TA. Gene expression and protein level analyses using allograft biopsies with and without Banff'05 classified IF/TA illustrated their deregulation. Expression profiles of these genes differentiated IF/TA from Banff'05 classified Normal biopsies in three independent microarray studies and demonstrated histological progression of IF/TA I to III. Significant upregulation of matrix metalloprotease-7 (MMP-7) and thrombospondin-2 (THBS-2) in IF/TA biopsies and sera was revealed in two independent patient sets. Furthermore, elevated THBS-2, osteopontin (SPP1) and beta-catenin may play regulatory roles on MMP. Our findings further suggest that deregulated ECM remodeling and possibly epithelial to mesenchymal transition (EMT) are implicated in IF/TA of kidney transplants, and that metzincins and related genes play an important role in these processes. Profiling of these genes may be used to complement IF/TA diagnosis and to disclose IF/TA progression in kidney transplant recipients.

SURVIVAL PREDICTION FOR BRAIN TUMOR PATIENTS USING GENE EXPRESSION DATA

Brain tumor is one of the most aggressive types of cancer in humans, with an estimated median survival time of 12 months and only 4% of the patients surviving more than 5 years after disease diagnosis. Until recently, brain tumor prognosis has been based only on clinical information such as tumor grade and patient age, but there are reports indicating that molecular profiling of gliomas can reveal subgroups of patients with distinct survival rates. We hypothesize that coupling molecular profiling of brain tumors with clinical information might improve predictions of patient survival time and, consequently, better guide future treatment decisions. In order to evaluate this hypothesis, the general goal of this research is to build models for survival prediction of glioma patients using DNA molecular profiles (U133 Affymetrix gene expression microarrays) along with clinical information. First, a predictive Random Forest model is built for binary outcomes (i.e. short vs. long-term survival) and a small subset of genes whose expression values can be used to predict survival time is selected. Following, a new statistical methodology is developed for predicting time-to-death outcomes using Bayesian ensemble trees. Due to a large heterogeneity observed within prognostic classes obtained by the Random Forest model, prediction can be improved by relating time-to-death with gene expression profile directly. We propose a Bayesian ensemble model for survival prediction which is appropriate for high-dimensional data such as gene expression data. Our approach is based on the ensemble "sum-of-trees" model which is flexible to incorporate additive and interaction effects between genes. We specify a fully Bayesian hierarchical approach and illustrate our methodology for the CPH, Weibull, and AFT survival models. We overcome the lack of conjugacy using a latent variable formulation to model the covariate effects which decreases computation time for model fitting. Also, our proposed models provides a model-free way to select important predictive prognostic markers based on controlling false discovery rates. We compare the performance of our methods with baseline reference survival methods and apply our methodology to an unpublished data set of brain tumor survival times and gene expression data, selecting genes potentially related to the development of the disease under study. A closing discussion compares results obtained by Random Forest and Bayesian ensemble methods under the biological/clinical perspectives and highlights the statistical advantages and disadvantages of the new methodology in the context of DNA microarray data analysis.

Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^

Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells

A hierarchical order of gene expression has been proposed to control developmental events in hematopoiesis, but direct demonstration of the temporal relationships between regulatory gene expression and differentiation has been difficult to achieve. We modified a single-cell PCR method to detect 2-fold changes in mRNA copies per cell (dynamic range, 250–250,000 copies/cell) and used it to sequentially quantitate gene expression levels as single primitive (CD34+,CD38−) progenitor cells underwent differentiation to become erythrocytes, granulocytes, or monocyte/macrophages. Markers of differentiation such as CD34 or cytokine receptor mRNAs and transcription factors associated with their regulation were assessed. All transcription factors tested were expressed in multipotent progenitors. During lineage-specific differentiation, however, distinct patterns of expression emerged. SCL, GATA-2, and GATA-1 expression sequentially extinguished during erythroid differentiation. PU.1, AML1B, and C/EBPα expression profiles and their relationship to cytokine receptor expression in maturing granulocytes could be distinguished from similar profiles in monocytic cells. These data characterize the dynamics of gene expression accompanying blood cell development and define a signature gene expression pattern for specific stages of hematopoietic differentiation.

Lipophosphoglycan from Leishmania suppresses agonist-induced interleukin 1β gene expression in human monocytes via a unique promoter sequence

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1β (IL-1β) and tumor necrosis factor α are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1β steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1β mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor α and Staphylococcus induction of IL-1β gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1β mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the −310 to −57 nucleotide region of the IL-1β promoter was found to mediate LPG’s inhibitory activity. The requirement for the −310 to −57 promoter gene sequence for LPG’s effect is demonstrated by the abrogation of LPG’s inhibitory activity by truncation or deletion of the −310 to −57 promoter gene sequence. Furthermore, the minimal IL-1β promoter (positions −310 to +15) mediated LPG’s inhibitory activity with dose and kinetic profiles that were similar to LPG’s suppression of steady-state IL-1β mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a “gene silencer,” a function, to our knowledge, not previously described. LPG’s inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

The TRANSFAC system on gene expression regulation

The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

The Mouse Gene Expression Database (GXD)

The Gene Expression Database (GXD) is a community resource of gene expression information for the laboratory mouse. By combining the different types of expression data, GXD aims to provide increasingly complete information about the expression profiles of genes in different mouse strains and mutants, thus enabling valuable insights into the molecular networks that underlie normal development and disease. GXD is integrated with the Mouse Genome Database (MGD). Extensive interconnections with sequence databases and with databases from other species, and the development and use of shared controlled vocabularies extend GXD’s utility for the analysis of gene expression information. GXD is accessible through the Mouse Genome Informatics web site at http://www.informatic s.jax.org/ or directly at http://www.informatics.jax.org/me nus/expression_menu.shtml.