Angiogenesis-related gene expression profile with independent prognostic value in advanced ovarian carcinoma.

BACKGROUND Ovarian carcinoma is the most important cause of gynecological cancer-related mortality in Western societies. Despite the improved median overall survival in patients receiving chemotherapy regimens such as paclitaxel and carboplatin combination, relapse still occurs in most advanced diseased patients. Increased angiogenesis is associated with rapid recurrence and decreased survival in ovarian cancer. This study was planned to identify an angiogenesis-related gene expression profile with prognostic value in advanced ovarian carcinoma patients. METHODOLOGY/PRINCIPAL FINDINGS RNAs were collected from formalin-fixed paraffin-embedded samples of 61 patients with III/IV FIGO stage ovarian cancer who underwent surgical cytoreduction and received a carboplatin plus paclitaxel regimen. Expression levels of 82 angiogenesis related genes were measured by quantitative real-time polymerase chain reaction using TaqMan low-density arrays. A 34-gene-profile which was able to predict the overall survival of ovarian carcinoma patients was identified. After a leave-one-out cross validation, the profile distinguished two groups of patients with different outcomes. Median overall survival and progression-free survival for the high risk group was 28.3 and 15.0 months, respectively, and was not reached by patients in the low risk group at the end of follow-up. Moreover, the profile maintained an independent prognostic value in the multivariate analysis. The hazard ratio for death was 2.3 (95% CI, 1.5 to 3.2; p<0.001). CONCLUSIONS/SIGNIFICANCE It is possible to generate a prognostic model for advanced ovarian carcinoma based on angiogenesis-related genes using formalin-fixed paraffin-embedded samples. The present results are consistent with the increasing weight of angiogenesis genes in the prognosis of ovarian carcinoma.

Changes in gene expression profile in human subcutaneous adipose tissue during significant weight loss.

Objective: To analyze the expression of peroxisome proliferator-activated receptor-γ1 and 2 (PPARγ1 and 2), 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), and leptin in adipose tissue (AT) of obese women during weight loss following Roux-en-Y gastric bypass (RYGB) and to compare these levels with those obtained in AT of nonobese subjects. Methods: Gene expression was determined by real-time RT-PCR prior to surgery and at 3, 6, and 12 months after RYGB. Results: All obese patients lost weight, reaching a mean BMI of 29.3 ± 1.0 kg/m(2) at 1 year after surgery (-33.9 ± 1.5% of their initial body weight). In obese subjects leptin and 11βHSD1 were over-expressed, whereas PPARγ1 was expressed at lower levels compared to controls. After surgery, leptin and 11βHSD1 gene expression decreased, whereas PPARγ1 expression increased. At 12 months after RYGB, these 3 genes had reached levels similar to the controls. In contrast, PPARγ2 gene expression was not different between groups and types of tissue and remained unchanged during weight loss. We found a positive correlation between BMI and levels of gene expression of leptin and 11βHSD1. Conclusion: Gene expression of leptin, PPARγ1, and 11βHSD1 in AT is modified in human obesity. This default is completely corrected by RYGB. Copyright © 2012 S. Karger GmbH, Freiburg.

Response to Infliximab in Crohn's Disease: Genetic Analysis Supporting Expression Profile.

Substantial proportion of Crohn's disease (CD) patients shows no response or a limited response to treatment with infliximab (IFX) and to identify biomarkers of response would be of great clinical and economic benefit. The expression profile of five genes (S100A8-S100A9, G0S2, TNFAIP6, and IL11) reportedly predicted response to IFX and we aimed at investigating their etiologic role through genetic association analysis. Patients with active CD (350) who received at least three induction doses of IFX were included and classified according to IFX response. A tagging strategy was used to select genetic polymorphisms that cover the variability present in the chromosomal regions encoding the identified genes with altered expression. Following genotyping, differences between responders and nonresponders to IFX were observed in haplotypes of the studied regions: S100A8-S100A9 (rs11205276* G/rs3014866* C/rs724781* C/rs3006488* A; P = 0.05); G0S2 (rs4844486* A/rs1473683* T; P = 0.15); TNFAIP6 (rs11677200* C/rs2342910* A/rs3755480* G/rs10432475* A; P = 0.10); and IL11 (rs1126760* C/rs1042506* G; P = 0.07). These differences were amplified in patients with colonic and ileocolonic location for all but the TNFAIP6 haplotype, which evidenced significant difference in ileal CD patients. Our results support the role of the reported expression signature as predictive of anti-TNF outcome in CD patients and suggest an etiological role of those top-five genes in the IFX response pathway.

Correlation between vasoconstrictor roles and mRNA expression of alpha1-adrenoceptor subtypes in blood vessels of genetically engineered mice.

We examined the contribution of each alpha(1)-adrenoceptor (AR) subtype in noradrenaline (NAd)-evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration-response curves (CRCs) for NAd in the thoracic aortas of wild-type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD(2) value in rank order is as follows: WT mice (8.21) > alpha(1B)-adrenoceptor knockout (alpha(1B)-KO) (7.77) > alpha(1D)-AR knockout (alpha(1D)-KO) (6.44) > alpha(1B)- and alpha(1D)-AR double knockout (alpha(1BD)-KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and alpha(1B)-KO mice (7.12) or alpha(1D)-KO (6.19) and alpha(1BD)-KO (6.29) mice. However, the CRC maximum responses to NAd in alpha(1D)- and alpha(1BD)-KO mice were significantly lower than those in WT and alpha(1B)-KO mice. Except in the thoracic aortas of alpha(1BD)-KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the alpha(1D)-AR gene. In the thoracic aorta, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.25 and 8.46, respectively, and in alpha(1B)-KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.34 and 7.47, respectively, and in alpha(1B)-KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and alpha(1B)-KO mice, which showed low affinities to BMY7378. We performed a quantitative analysis of the mRNA expression of each alpha(1)-AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each alpha(1)-AR subtype in mediating vascular contraction. The rank order of each alpha(1)-AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, alpha(1D)-AR > alpha(1B)-AR > alpha(1A)-AR in the thoracic aorta and alpha(1D)-AR > alpha(1A)-AR > alpha(1B)-AR in the mesenteric artery. No dramatic compensatory change of alpha(1)-AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.

A TARBP2-dependent miRNA expression profile underlies cancer stem cell properties and provides candidate therapeutic reagents in Ewing sarcoma.

We have recently demonstrated that human pediatric mesenchymal stem cells can be reprogrammed toward a Ewing sarcoma family tumor (ESFT) cancer stem cell (CSC) phenotype by mechanisms that implicate microRNAs (miRNAs). Here, we show that the miRNA profile of ESFT CSCs is shared by embryonic stem cells and CSCs from divergent tumor types. We also provide evidence that the miRNA profile of ESFT CSCs is the result of reversible disruption of TARBP2-dependent miRNA maturation. Restoration of TARBP2 activity and systemic delivery of synthetic forms of either of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC clonogenicity and tumor growth in vivo. Our observations suggest that CSC self-renewal and tumor maintenance may depend on deregulation of TARBP2-dependent miRNA expression.

Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis.

PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-beta-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. beta-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.

Temporal changes in mRNA expression of the brain nutrient transporters in the lithium-pilocarpine model of epilepsy in the immature and adult rat.

The lithium-pilocarpine model mimics most features of human temporal lobe epilepsy. Following our prior studies of cerebral metabolic changes, here we explored the expression of transporters for glucose (GLUT1 and GLUT3) and monocarboxylates (MCT1 and MCT2) during and after status epilepticus (SE) induced by lithium-pilocarpine in PN10, PN21, and adult rats. In situ hybridization was used to study the expression of transporter mRNAs during the acute phase (1, 4, 12 and 24h of SE), the latent phase, and the early and late chronic phases. During SE, GLUT1 expression was increased throughout the brain between 1 and 12h of SE, more strongly in adult rats; GLUT3 increased only transiently, at 1 and 4h of SE and mainly in PN10 rats; MCT1 was increased at all ages but 5-10-fold more in adult than in immature rats; MCT2 expression increased mainly in adult rats. At all ages, MCT1 and MCT2 up-regulation was limited to the circuit of seizures while GLUT1 and GLUT3 changes were more widespread. During the latent and chronic phases, the expression of nutrient transporters was normal in PN10 rats. In PN21 rats, GLUT1 was up-regulated in all brain regions. In contrast, in adult rats GLUT1 expression was down-regulated in the piriform cortex, hilus and CA1 as a result of extensive neuronal death. The changes in nutrient transporter expression reported here further support previous findings in other experimental models demonstrating rapid transcriptional responses to marked changes in cerebral energetic/glucose demand.

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera,Apidae)

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera, Apidae). Metamorphosis in holometabolous insects occurs through two subsequent molting cycles: pupation (metamorphic molt) and adult differentiation (imaginal molt). The imaginal molt in Apis mellifera L. was recently investigated in both histological and physiological-molecular approaches. Although the metamorphic molt in this model bee is extremely important to development, it is not well-known yet. In the current study we used this stage as an ontogenetic scenario to investigate the transcriptional profile of the gene Amlac2, which encodes a laccase with an essential role in cuticle differentiation. Amlac2 expression in epidermis was contrasted with the hemolymph titer of ecdysteroid hormones and with the most evident morphological events occurring during cuticle renewal. RT-PCR semiquantitative analyses using integument samples revealed increased levels of Amlac2 transcripts right after apolysis and during the subsequent pharate period, and declining levels near pupal ecdysis. Compared with the expression of a cuticle protein gene, AmelCPR14, these results highlighted the importance of the ecdysteroid-induced apolysis as an ontogenetic marker of gene reactivation in epidermis for cuticle renewal. The obtained results strengthen the comprehension of metamorphosis in Apis mellifera. In addition, we reviewed the literature about the development of A. mellifera, and emphasize the importance of revising the terminology used to describe honey bee molting cycles.

Immunodensity and mRNA expression of A2A adenosine, D2 dopamine, and CB1 cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment.

RATIONALE: Dopamine D2 receptors are the main target of antipsychotic drugs. In the brain, D2 receptors coexpress with adenosine A2A and CB1 cannabinoid receptors, leading to functional interactions. OBJECTIVES: The protein and messenger RNA (mRNA) contents of A2A, D2, and CB1 receptors were quantified in postmortem prefrontal cortex of subjects with schizophrenia. MATERIALS AND METHODS: The study was performed in subjects suffering schizophrenia (n=31) who mainly died by suicide, matched with non-schizophrenia suicide victims (n=13) and non-suicide controls (n=33). The density of receptor proteins was evaluated by immunodetection techniques, and their relative mRNA expression was quantified by quantitative real-time polymerase chain reaction. RESULTS: In schizophrenia, the densities of A2A (90+/-6%, n=24) and D2-like receptors (95+/-5%, n=22) did not differ from those in controls (100%). Antipsychotic treatment did not induce changes in the protein expression. In contrast, the immunodensity of CB1 receptors was significantly decreased (71+/-7%, n=11; p<0.05) in antipsychotic-treated subjects with schizophrenia but not in drug-free subjects (104+/-13%, n=11). The relative mRNA amounts encoding for A2A, D2, and CB1 receptors were similar in brains of drug-free, antipsychotic-treated subjects with schizophrenia and controls. CONCLUSIONS: The findings suggest that antipsychotics induce down-regulation of CB1 receptors in brain. Since A2A, D2, and CB1 receptors coexpress on brain GABAergic neurons and reductions in markers of GABA neurotransmission have been identified in schizophrenia, a lower density of CB1 receptor induced by antipsychotics could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release, contributing to the normalization of cognitive functions in the disorder.

Sorting live stem cells based on sox2 mRNA expression.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

Evolution of mRNA expression levels of autosomal and sex-linked genes in amniotes

In addition to differences in protein-coding gene sequences, changes in expression resulting from mutations in regulatory sequences have long been hypothesized to be responsible for phenotypic differences between species. However, unlike comparison of genome sequences, few studies, generally restricted to pairwise comparisons of closely related mammalian species, have assessed between-species differences at the transcriptome level. They reported that gene expression evolves at different rates in various organs and in a pattern that is overall consistent with neutral models of evolution. In the first part of my thesis, I investigated the evolution of gene expression in therian mammals (i.e.7 placental and marsupials), based on microarray data from human, mouse and the gray short-tailed opossum (Monodelphis domestica). In addition to autosomal genes, a special focus was given to the evolution of X-linked genes. The therian X chromosome was recently shown to be younger than previously thought and to harbor a specific gene content (e.g., genes involved in brain or reproductive functions) that is thought to have been shaped by specific sex-related evolutionary forces. Sex chromosomes derive from ordinary autosomes and their differentiation led to the degeneration of the Y chromosome (in mammals) or W chromosome (in birds). Consequently, X- or Z-linked genes differ in gene dose between males and females such that the heterogametic sex has half the X/Z gene dose compared to the ancestral state. To cope with this dosage imbalance, mammals have been reported to have evolved mechanisms of dosage compensation.¦In the first project, I could first show that transcriptomes evolve at different rates in different organs. Out of the five tissues I investigated, the testis is the most rapidly evolving organ at the gene expression level while the brain has the most conserved transcriptome. Second, my analyses revealed that mammalian gene expression evolution is compatible with a neutral model, where the rates of change in gene expression levels is linked to the efficiency of purifying selection in a given lineage, which, in turn, is determined by the long-term effective population size in that lineage. Thus, the rate of DNA sequence evolution, which could be expected to determine the rate of regulatory sequence change, does not seem to be a major determinant of the rate of gene expression evolution. Thus, most gene expression changes seem to be (slightly) deleterious. Finally, X-linked genes seem to have experienced elevated rates of gene expression change during the early stage of X evolution. To further investigate the evolution of mammalian gene expression, we generated an extensive RNA-Seq gene expression dataset for nine mammalian species and a bird. The analyses of this dataset confirmed the patterns previously observed with microarrays and helped to significantly deepen our view on gene expression evolution.¦In a specific project based on these data, I sought to assess in detail patterns of evolution of dosage compensation in amniotes. My analyses revealed the absence of male to female dosage compensation in monotremes and its presence in marsupials and, in addition, confirmed patterns previously described for placental mammals and birds. I then assessed the global level of expression of X/Z chromosomes and contrasted this with its ancestral gene expression levels estimated from orthologous autosomal genes in species with non-homologous sex chromosomes. This analysis revealed a lack of up-regulation for placental mammals, the level of expression of X-linked genes being proportional to gene dose. Interestingly, the ancestral gene expression level was at least partially restored in marsupials as well as in the heterogametic sex of monotremes and birds. Finally, I investigated alternative mechanisms of dosage compensation and found that gene duplication did not seem to be a widespread mechanism to restore the ancestral gene dose. However, I could show that placental mammals have preferentially down-regulated autosomal genes interacting with X-linked genes which underwent gene expression decrease, and thus identified a novel alternative mechanism of dosage compensation.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

The reelin gene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that express reelin transcripts. reelinexpression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis,reelin was detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages, reelin was also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages, reelin transcripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions,reelin expression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.

Mineralocorticoid effects in the kidney: correlation between alphaENaC, GILZ, and Sgk-1 mRNA expression and urinary excretion of Na+ and K+.

Aldosterone exerts its effects through interactions with two types of binding sites, the mineralocorticoid (MR) and the glucocorticoid (GR) receptors. Although both receptors are known to be involved in the anti-natriuretic response to aldosterone, the mechanisms of signal transduction leading to modulation of electrolyte transport are not yet fully understood. This study measured the Na(+) and K(+) urinary excretion and the mRNA levels of three known aldosterone-induced transcripts, the serum and glucocorticoid-induced kinase (Sgk-1), the alpha subunit of the epithelial Na(+) channel (alphaENaC), and the glucocorticoid-induced-leucine-zipper protein (GILZ) in the whole kidney and in isolated cortical collecting tubules of adrenalectomized rats treated with low doses of aldosterone and/or dexamethasone. The resulting plasma concentrations of both steroids were close to 1 nmol/L. Aldosterone, given with or without dexamethasone, induced anti-natriuresis and kaliuresis, whereas dexamethasone alone did not. GILZ and alphaENaC transcripts were higher after treatment with either or both hormones, whereas the mRNA abundance of Sgk-1 was increased in the cortical collecting tubule by aldosterone but not by dexamethasone. We conclude the increased expression of Sgk-1 in the cortical collecting tubules is a primary event in the early antinatriuretic and kaliuretic responses to physiologic concentrations of aldosterone. Induction of alphaENaC and/or GILZ mRNAs may play a permissive role in the enhancement of the early and/or late responses; these effects may be necessary for a full response but do not by themselves promote early changes in urinary Na(+) and K(+) excretion.

In leishmaniasis due to Leishmania guyanensis infection, distinct intralesional interleukin-10 and Foxp3 mRNA expression are associated with unresponsiveness to treatment.

The presence of intralesional natural regulatory T cells, characterized by the expression of Foxp3 mRNA, was analyzed in patients with localized leishmaniasis due to Leishmania guyanensis infection that was unresponsive to treatment with pentamidine isethionate. Foxp3 mRNA levels were associated with unresponsiveness to treatment among patients with a lesion duration of 1 month, but this association was not observed among patients with a lesion duration of <1 month. In conclusion, high intralesional expression of Foxp3 might be an indicator of poor response to treatment, depending on the duration of lesions.

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.