Expression Of Several Candidate Genes Research Articles

Characterization of signature trends across the spectrum of non-alcoholic fatty liver disease using deep learning method

AimsThe timely diagnosis of different stages in NAFLD is crucial for disease treatment and reversal. We used hepatocellular ballooning to determine different NAFLD stages. Main methodsWe analyzed differentially expressed genes (DEGs) in 78 patients with NAFLD and in healthy controls from previously published RNA-seq data. We identified two expression types in NAFLD progression, calculated the predictive power of candidate genes, and validated them in an independent cohort. We also performed cancer studies with these candidates retrieved from the Cancer Genome Atlas. Key findingsWe identified 103 DEGs in NAFLD patients compared to healthy controls: 75 genes gradually increased or decreased in the NAFLD stage, whereas 28 genes showed differences only in NASH. The former were enriched in negative regulation and binding-related genes; the latter were involved in positive regulation and cell proliferation. Feature selection showed the gradual up- or down-regulation of 21 genes in NASH compared to controls; 18 were highly expressed only in NASH. Using deep-learning method with subset of features from lasso regression, we obtained reliable determination performance in NAFL and NASH (accuracy: 0.857) and validated these genes using an independent cohort (accuracy: 0.805). From cancer studies, we identified significant differential expression of several candidate genes in LIHC; 5 genes were gradually up-regulated and 6 showing high expression only in NASH were influential to patient survival. SignificanceThe identified biomolecular signatures may determine the spectrum of NAFLD and its relationship with HCC, improving clinical diagnosis and prognosis and enabling a therapeutic intervention for NAFLD.

Differential gene expression in chronically irradiated herbaceous species from the Chernobyl exclusion zone

Purpose Transcriptional activity of genes related to ionizing radiation responses in chronically irradiated plant populations at radioactively contaminated territories can be a cost-effective and precise approach for stress response evaluation. However, there are limits to studying non-model plants in field conditions. The work studies the transcriptional activity of candidate genes of adaptation to chronic radiation exposure in plant populations from radioactively contaminated territories of the Chernobyl. Materials and methods In this work, we studied plant species with different sensitivity to acute irradiation: Trifolium repens L., Taraxacum officinale Wigg., and Dactylis glomerata L., sampled in the Chernobyl exclusion zone. The differential expression of several candidate genes of adaptation to chronic radiation exposure in the leaves of these species was analyzed, including homologs of Arabidopsis thaliana genes SLAC1, APX1, GPX2, CAB1, NTRB, PP2-B11, RBOH-F, HY5, SnRK2.4, PDS1, CIPK20, SIP1, PIP1, TIP1. Results and conclusions All studied species were characterized by upregulation of the CAB1 homolog, encoding chlorophyll a/b binding protein, at radioactively contaminated plots. An increase in the expression of genes associated with water and hydrogen peroxide transport, intensity of photosynthesis, and stress responses (homolog of aquaporin TIP1 for T. repens; homologs of aquaporin PIP1 and transcription factor HY5 for D. glomerata; homolog of CBL-interacting serine/threonine protein kinase CIPK20 for T. officinale) was revealed. The methodological approach for studying gene expression in non-model plant species is described, which may allow large-scale screening studies of candidate genes in various plant species abundant in radioactively contaminated areas.

Eggshell decalcification and skeletal mineralization during chicken embryonic development: defining candidate genes in the chorioallantoic membrane

During chicken embryonic development, skeleton calcification mainly relies on the eggshell, whose minerals are progressively solubilized and transported to the embryo via the chorioallantoic membrane (CAM). However, the molecular components involved in this process remain undefined. We assessed eggshell demineralization and calcification of the embryo skeleton after 12 and 16 d of incubation, and analyzed the expression of several candidate genes in the CAM: carbonic anhydrases that are likely involved in secretion of protons for eggshell dissolution (CA2, CA4, CA9), ions transporters and regulators (CALB1, SLC4A1, ATP6V1B2, SGK1, SCGN, PKD2) and vitamin-D binding protein (GC).Our results confirmed that eggshell weight, thickness, and strength decreased during incubation, with a concomitant increase in calcification of embryonic skeletal system. In the CAM, the expression of CA2 increased during incubation while CA4 and CA9 were expressed at similar levels at both stages. SCL4A1 and SCGN were expressed, but not differentially, between the two stages, while the expression of ATP6V1B2 and PKD2 genes decreased. The expression of SGK1 and TRPV6 increased over time, although the expression of the latter gene was barely detectable. In parallel, we analyzed the expression of these candidate genes in the yolk sac (YS), which mediates the transfer of yolk minerals to the embryo during the first half of incubation. In YS, CA2 expression increases during incubation, similar to the CAM, while the expression of the other candidate genes decreases. Moreover, CALB1 and GC genes were found to be expressed during incubation in the YS, in contrast to the CAM where no expression of either was detected.This study demonstrates that the regulation of genes involved in the mobilization of egg minerals during embryonic development is different between the YS and CAM extraembryonic structures. Identification of the full suite of molecular components involved in the transfer of eggshell calcium to the embryo via the CAM should help to better understand the role of this structure in bone mineralization.

Cigarette smoke and electronic cigarettes differentially activate bronchial epithelial cells

BackgroundThe use of electronic cigarettes (ECIGs) is increasing, but the impact of ECIG-vapor on cellular processes like inflammation or host defense are less understood. The aim of the present study was to compare the acute effects of traditional cigarettes (TCIGs) and ECIG-exposure on host defense, inflammation, and cellular activation of cell lines and primary differentiated human airway epithelial cells (pHBE).MethodsWe exposed pHBEs and several cell lines to TCIG-smoke or ECIG-vapor. Epithelial host defense and barrier integrity were determined. The transcriptome of airway epithelial cells was compared by gene expression array analysis. Gene interaction networks were constructed and differential gene expression over all groups analyzed. The expression of several candidate genes was validated by qRT-PCR.ResultsBacterial killing, barrier integrity and the expression of antimicrobial peptides were not affected by ECIG-vapor compared to control samples. In contrast, TCIGs negatively affected host defense and reduced barrier integrity in a significant way. Furthermore ECIG-exposure significantly induced IL-8 secretion from Calu-3 cells but had no effect on NCI-H292 or primary cells. The gene expression based on array analysis distinguished TCIG-exposed cells from ECIG and room air-exposed samples.ConclusionThe transcriptome patterns of host defense and inflammatory genes are significantly distinct between ECIG-exposed and TCIG-treated cells. The overall effects of ECIGs on epithelial cells are less in comparison to TCIG, and ECIG-vapor does not affect host defense. Nevertheless, although acute exposure to ECIG-vapor induces inflammation, and the expression of S100 proteins, long term in vivo data is needed to evaluate the chronic effects of ECIG use.

Transcriptome profile and association study revealed STAT3 gene as a potential quality marker of bovine gametes.

The present study was aimed to investigate differences in molecular signatures in oocytes derived from Holstein-Friesian heifers with different genetic merit for fertility, euthanized during day 0 or day 12 of the estrous cycle. Moreover, association between single nucleotide polymorphisms (SNPs) of ODC1 and STAT3 genes and bull fertility traits was investigated. The gene expression patterns were analyzed using cDNA array and validated with quantitative real-time polymerase chain reaction (PCR). The result revealed that several genes have shown not only to be regulated by fertility merit but also by the day of oocyte recovery during the estrous cycle. The STAT3 gene was found to be upregulated in oocytes recovered from animals with high fertility merit at both day 0 and day 12. Some other genes like PTTG1, ODC1 and TUBA1C were downregulated at day 0 and upregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In contrast, the transcript abundance of TPM3 was upregulated at day 0 and downregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In addition, ODC1 and STAT3 were found to be associated (P < 0.05) with sperm quality traits as well as flow cytometry parameters. Therefore, the expression of several candidate genes including ODC1 and STAT3 was related to the genetic merit of the cow. In addition polymorphisms in these two genes were found to be associated with bull semen quality.

Integrated analysis of transcriptome-wide m6A methylome of osteosarcoma stem cells enriched by chemotherapy.

Aim: To analyze the m6A methylome of osteosarcoma stem cells (OSCs). Materials & methods: Chemoresistant OSCs were enriched by doxorubicin treatment. Expression of m6A-related enzymes was detected by quantitative real-time-PCR and western blot. MeRIP-seq and RNA-seq were performed to identify differences in m6A methylation and gene expression. Data analysis was conducted to explore the modified genes and their clinical significance. Results: Three m6A-related enzymes were altered in OSCs. Differentially methylated genes were enriched in some pathways regulating pluripotency of stem cells. The expression of several candidate genes were found consistent with that in GSE33458 dataset, and associated with poor prognosis in osteosarcoma patients. Conclusion: m6A may play a role in the emergence and maintaining of OSCs and affect the prognosis.

Analysis of TLR-9, CD90 and VEGF Expressions in the tumor-Osteosarcoma Mesenchymal Stem Cell

For further investigation on the effect of Mesenchymal Stem Cell (MSC) on tumor / cancer growth, it is necessary to start with tumor MSC profile from tumor / cancer tissue. In this study, we use MSC from osteosarcoma tissue as a representative. The MSC profile analysis method used were the comparing the phenotype and characteristics of MSC fromosteosarcoma tissue with MSC from normal adiposa tissue. The characteristics of MSC were represented by protein expression of several candidate genes namely TLR-9, CD-90 and VEGF. From the results of immunofluorescence staining on adiposa MSC compared withosteosarcoma MSC, it has obviously seen that although the phenotypes were similar, there was no significant difference in CD-90 expression in osteosarcoma MSC and adiposa MSC. Although VEGF expression appears to be slightly prominent in osteosarcoma MSCs, the difference between the two was also not significant. Whereas TLR9 expression is very prominent in osteosarcoma MSC and significantly different from adiposa MSC. Thus TLR9 may be of concern in subsequent studies to investigate the mechanism of MSC triggers tumor growth.

Factors involved in early polarization of the anterior-posterior axis in the milkweed bug Oncopeltus fasciatus.

The axes of insect embryos are defined early in the blastoderm stage. Genes involved in this polarization are well known in Drosophila, but less so in other insects, such as the milkweed bug Oncopeltus fasciatus. Using quantitative PCR, we looked at differential expression of several candidate genes for early anterior-posterior patterning and found that none of them are expressed asymmetrically in the early blastoderm. We then used an RNA-Seq approach to identify novel candidate genes that might be involved in early polarization in Oncopeltus. We focused on transcription factors (TFs) as these are likely to be central players in developmental processes. Using both homology and domain based identification approaches, we were unable to find any TF encoding transcripts that are expressed asymmetrically along the anterior-posterior axis at early stages. Using a GO-term analysis of all asymmetrically expressed mRNAs, we found an enrichment of genes relating to mitochondrial function in the posterior at the earliest studied time-point. We also found a gradual enrichment of transcription related activities, giving us a putative time frame for the maternal to zygotic transition. Our dataset provides us with a list of new candidate genes in early development, which can be followed up experimentally.

Mutation analysis of the NKX2.5 gene in Iranian pediatric patients with congenital hypothyroidism.

The embryonic development of the thyroid gland is regulated by the expression of several candidate genes which are related to congenital hypothyroidism. These genes include the numerous critical thyroid transcription factors such as NKX2.1, NKX2.5, FOXE1, and PAX8. The molecular analysis of these loci will be essential to the explanation of the participation of these transcription activators in the etiology of hypothyroidism. Among them, the role of NKX2.5 is important during the early thyroid morphogenesis and in controlling thyroidal cell differentiation and migration. Importantly, NKX2.5 change nucleotides are recognized to be central to the genesis of congenital hypothyroidism. A case-control study was conducted in 65 unrelated patients, diagnosed with primary congenital hypothyroidism and all of them were diagnosed according to the clinical presentations of thyroid hypoplasia and without cardiovascular defects. Mutational screening of the entire NKX2-5 coding sequence was performed in a cohort of pediatric patients by PCR-SSCP and direct sequencing. We identified two known variations 73C>T (R25C) and 63A>G (E21E) in patients with thyroid hypothyroidism. Both of them are located in conserved region of the gene and previously reported in cases with thyroid dysgenesis and congenital heart defects. There was a significance association between 63A>G variation with primary hypothyroidism (p=0.003). These SNPs are probably related to thyroid hypoplasia because the allele frequency of the 63A>G polymorphism was significantly different in patients and controls and also R25C variation not observed in healthy cases.

Reduction of avian influenza virus shedding by administration of Toll-like receptor ligands to chickens

Avian influenza viruses (AIV) are of concern to the poultry industry. Outbreaks of AIV highlight the urgent need for effective control measures. Prophylactic strategies should be explored that rapidly elicit immunity against the virus. Toll-like receptors (TLRs) are innate immune molecules that can induce anti-viral responses, therefore the application of TLR ligands as prophylactic agents in chickens is gaining more attention. We hypothesized that treatment of chickens with TLR ligands reduces the shedding of AIV from infected birds. In addition, the effects of TLR ligand dose and route of administration on the efficiency of TLR ligands to reduce AIV shedding were examined. Chickens were treated with TLR2, 4, 7 and 21 ligands using different doses and routes of administration, 18h before AIV infection. Moreover, the expression of several candidate genes, such as type I interferons, PKR, OAS, viperin and IFITM3 was quantified at 3, 8 and 18h post-treatment with TLR ligands. The results revealed that route of administration and dosage affect the efficacy of TLR ligands to reduce virus shedding. Furthermore, varying effects were observed when different ligands were applied. Our results demonstrated that all TLR ligand treatments reduced AIV shedding, with the CpG-ODN 1826 being the most efficacious to reduce oral virus shedding, whereas LPS from Escherichia coli 026:B6 resulted in the largest reduction in cloacal virus shedding. Moreover, TLR ligands induced the expression of genes involved in antiviral responses such as type I interferons and interferon-stimulated genes in chicken trachea and cecal tonsils. These results raise the possibility of treatment of chickens with TLR ligands as anti-viral agents.

Different Molecular Mechanisms Account for Drought Tolerance in Coffea canephora var. Conilon

The effects of water deficit on photochemical parameters and expression of several candidate genes were investigated in drought-tolerant clone 73 of Coffea canephora submitted to slowly imposed water limitation. Under irrigation, this clone showed low values of stomatal conductance (g s ) and of CO2 assimilation rates (A) suggesting that it had a great efficiency in controlling stomatal closure and transpiration. After water withdrawal, this clone reached a −3.0 MPa after 15 days without irrigation and showed a slow decrease in the pre-dawn leaf water potential. Under drought, the suppression of A was accompanied by maintenance of photochemical quenching (q P) and internal to ambient CO2 concentration (Ci/Ca) ratios as well as by a decrease of non-photochemical quenching (q N). This is confirmed by the transport rate/CO2 assimilation (ETR/A) rates that suggested the participation of an alternative electron sink protecting the photosynthetic apparatus against photoinhibition. At the transcriptomic level, high up-regulation of genes encoding for a dehydrin (CcDH3), an ascorbate peroxidase (CcAPX1), a prephenate-dehydrogenase like protein (CcPDH1) and a non-symbiotic haemoglobin (CcNSH1) was also observed upon drought suggesting a strong induction of antioxidant and osmoprotection systems in this clone. High expression levels of gene-encoding ABA receptors (CcPYL3 and CcPYL7) under water limitation were also observed suggesting the involvement of the ABA signaling pathway in response to drought. All these results where compared to those previously obtained for drought-tolerant clones 14 and 120. Our results demonstrated the existence of different mechanisms amongst the drought-tolerant coffee clones regarding water deficit.

Lead tolerance and accumulation in Hirschfeldia incana, a Mediterranean Brassicaceae from metalliferous mine spoils.

Lead is a heavy metal of particular concern with respect to environmental quality and health. The lack of plant species that accumulate and tolerate Pb is a limiting factor to understand the molecular mechanisms involved in Pb tolerance. In this study we identified Hirschfeldia incana, a Brassicaceae collected from metalliferous mine spoils in Morocco, as a Pb accumulator plant. H. incana exhibited high Pb accumulation in mine soils and in hydroponic cultures. Major Pb accumulation occurred in the roots and a part of Pb translocated from the roots to the shoots, even to the siliques. These findings demonstrated that H. incana is a Pb accumulator species. The expression of several candidate genes after Pb-exposure was measured by quantitative PCR and two of them, HiHMA4 and HiMT2a, coding respectively for a P1B-type ATPase and a metallothionein, were particularly induced by Pb-exposure in both roots and leaves. The functional characterization of HiHMA4 and HiMT2a was achieved using Arabidopsis T-DNA insertional mutants. Pb content and primary root growth analysis confirmed the role of these two genes in Pb tolerance and accumulation. H. incana could be considered as a good experimental model to identify genes involved in lead tolerance and accumulation in plants.

OP054: p16-Directed transcriptome profiling in oral squamous cell carcinoma

Purpose Immunohistochemical staining of p16 is routinely used as a surrogate for human papilloma virus infection in oropharyngeal squamous cell carcinoma; however, its relevance and association with oral squamous cell carcinoma (OSCC) outcomes require further investigation. We used fluorescence immunohistochemistry (F-IHC) and automated quantitative analysis (AQUA) to evaluate p16 expression in OSCC samples. We compared the transcriptomes of patients with high p16 expression (p16+) and patients with low p16 expression (p16−). Materials and methods 56 OSCC patients were included in the study. Clinical data were obtained from the Alberta Cancer Registry and chart review. p16 protein expression was quantified using F-IHC and AQUA on tissue microarrays containing triplicate cores of formalin-fixed paraffin-embedded (FFPE) tissue. Kaplan–Meier curves were used to analyze the association between p16 expression and 5-year disease-specific survival (DSS). The Illumina Whole Genome cDNA-mediated annealing, selection, extension and ligation (WG-DASL) assay was employed to assess gene expression. Results High p16 expression was associated with significantly improved DSS ( p = 0.01). 68 genes were differentially regulated in p16+ versus p16− samples. Pathway enrichment analysis revealed that the p16+ group overexpressed genes involved in the immune response, cell adhesion, mesoderm and ectoderm development and blood vessel morphogenesis. The improved prognosis observed in p16+ patients may be explained by the increased expression of genes encoding tumor suppressors such as SPINT2 and NEFH, T cell glycoprotein CD2, transcription factor ELF3, ERAP1/2 aminopeptidases and downregulation of oncogenes such as a Ras homolog, NRIP3 and a gene implicated in metastasis (FABP4). Conclusions We identified distinct gene expression patterns in OSCC patients based on their p16 expression status that may explain the improved prognosis of p16+ patients. The differential expression of several candidate genes is being confirmed by real-time PCR. The genes identified using this high-throughput technique provide mechanistic insights and potential therapeutic targets in OSCC.

Abstract 1809: Analysis of molecular networks that drive astrocytoma initiation and progression.

Abstract High grade astrocytomas, anaplastic astrocytoma and glioblastoma multiforme (GBM) remain incurable in spite of advanced aggressive treatments including surgery, radiation and chemotherapy. To study pathways and mechanisms involved in the development of high grade astrocytomas, we used mouse models wherein key molecular pathways perturbed in human GBMs were inactivated or induced via Cre-driven adult astrocyte-specific system. Inhibition of Rb pathway via expression of T121, a N-terminal fragment of SV40 large T antigen (T: TgGZT121, GFAP-CreERTM) initiated diffuse grade II astrocytoma formation by 2 months after tamoxifen treatment which in some cases developed to grade III pathology 1.5 year after induction. Activation of KRas pathway (TR: TgGZT121, Kras+/lsl-G12D, GFAP-CreERTM) facilitated progression to grade III anaplastic astrocytoma tumor masses 4-5 months post induction which in a few cases developed to grade IV glioblastoma with some features of human disease. Additional PTEN loss or haploinsufficiency (TRPhet: TgGZT121, Kras+/lsl-G12D, PTEN+/fl, GFAP-CreERTM; TRPnull: TgGZT121, Kras+/lsl-G12D, PTENfl/fl, GFAP-CreERTM) led to rapid development of glioblastoma with characteristic features of angiogenesis and necrosis observed in human disease. Transcriptome analyses of GBM in our mouse models showed concordance with highly aggressive mesenchymal and proneural subclasses. To identify disease-specific gene networks involved in astrocytoma initiation and progression, we analyzed more than 300 brain samples from tamoxifen-induced T, TR, TRPhet and TRPnull mice and corresponding controls at different time points after induction for gene and miRNA expression by microarray and Nanostring technology. The genes that were induced early and gradually increased in expression with tumor grade belonged to several key molecular networks: DNA replication and repair, cell division and chromosome transmission fidelity, cell cycle progression, metabolism, and pathways important for embryonic stem cell biology. Pathways significantly induced at later stages of disease (grades III-IV) included Notch and Wnt signaling, inflammatory response genes, p53 signaling, and RNA processing. Significantly downregulated pathways were related to neuronal development and function. We have confirmed expression of several candidate genes in mouse tumor samples and cell lines derived from low and high grade tumors. Currently we are investigating potential role of the selected candidates in astrocytoma development by in vitro and in vivo functional analysis. In summary, we utilized mouse models to determine global molecular changes during astrocytoma initiation and progression to high grade. These studies are important for understanding the mechanisms of the disease and may facilitate the development of new therapies. Citation Format: Yaroslava Ruzankina, Burak Kutlu, Sophie Wang, Yurong Song, Deborah Householder, Philip Martin, Maureen Baran, Simone Difilippantonio, Leroy Hood, Terry Van Dyke. Analysis of molecular networks that drive astrocytoma initiation and progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2013-1809

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms.In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model.Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration.In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.

Disease- and age-related changes in histone acetylation at gene promoters in psychiatric disorders

Increasing evidence suggests that epigenetic factors have critical roles in gene regulation in neuropsychiatric disorders and in aging, both of which are typically associated with a wide range of gene expression abnormalities. Here, we have used chromatin immunoprecipitation-qPCR to measure levels of acetylated histone H3 at lysines 9/14 (ac-H3K9K14), two epigenetic marks associated with transcriptionally active chromatin, at the promoter regions of eight schizophrenia-related genes in n=82 postmortem prefrontal cortical samples from normal subjects and those with schizophrenia and bipolar disorder. We find that promoter-associated ac-H3K9K14 levels are correlated with gene expression levels, as measured by real-time qPCR for several genes, including, glutamic acid decarboxylase 1 (GAD1), 5-hydroxytryptamine receptor 2C (HTR2C), translocase of outer mitochondrial membrane 70 homolog A (TOMM70A) and protein phosphatase 1E (PPM1E). Ac-H3K9K14 levels of several of the genes tested were significantly negatively associated with age in normal subjects and those with bipolar disorder, but not in subjects with schizophrenia, whereby low levels of histone acetylation were observed in early age and throughout aging. Consistent with this observation, significant hypoacetylation of H3K9K14 was detected in young subjects with schizophrenia when compared with age-matched controls. Our results demonstrate that gene expression changes associated with psychiatric disease and aging result from epigenetic mechanisms involving histone acetylation. We further find that treatment with a histone deacetylase (HDAC) inhibitor alters the expression of several candidate genes for schizophrenia in mouse brain. These findings may have therapeutic implications for the clinical use of HDAC inhibitors in psychiatric disorders.

Kinetics of gene expression and signaling in bovine cumulus cells throughout IVM in different mediums in relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion

In vitro maturation of oocytes is a crucial step in assisted reproductive technologies in cattle; however, the molecular mechanisms of cumulus contribution to oocyte developmental potential require more investigation. Based on transcriptomic data, we studied by using real-time RT-PCR and western blot in bovine cumulus cells, the kinetics of expression of several candidate genes involved in oxidative stress response, apoptosis, steroid metabolism and signal transmission throughout IVM. Phosphorylations of the components of the main signaling pathways were also analyzed. In addition, IVM was performed in different maturation mediums which influenced the cumulus apoptosis, progesterone secretion and oocyte developmental competence. Glutathione-S-transferase A1 (GSTA1) transcript and protein abundance significantly decreased throughout IVM progression. Similarly, transcript levels of FSH receptor and aromatase (CYP19A1) and protein levels of three steroidogenic enzymes (steroidogenic acute regulatory protein, cytochrome P450scc and 3-beta-hydroxysteroid dehydrogenase) decreased along with progression of maturation and especially since 10 hours of IVM. Expression of progesterone receptor (PGR) and clusterin (CLU) mRNA and phosphorylations of protein kinases AKT, MAPK P38 and SMAD2 were particularly increased at 10 hours of IVM. This expression pattern supposed the role of these factors during oocyte metaphase-I check point of meiosis. Levels of CLU, GSTA1 and FSHR transcripts were higher in 199 basic hormone-free medium as compared to the medium 199EM, enriched in gonadotropins and growth factors, in which we recorded the higher developmental rate and progesterone secretion. Higher phosphorylation levels of SMAD2, AKT and MAP kinase JNK1, but not of MAP kinases ERK1/ERK2 or P38, was positively correlated with oocyte developmental competence and progesterone secretion and negatively correlated with cumulus apoptosis rate. These factors and signaling pathways in cumulus cells are potentially involved in controlling different stages of oocyte nuclear maturation and acquirement of its developmental potential.

Tinkering with the C-function: a molecular frame for the selection of double flowers in cultivated roses.

BackgroundRoses have been cultivated for centuries and a number of varieties have been selected based on flower traits such as petal form, color, and number. Wild-type roses have five petals (simple flowers), whereas high numbers of petals (double flowers) are typical attributes of most of the cultivated roses. Here, we investigated the molecular mechanisms that could have been selected to control petal number in roses.Methodology/Principal FindingsWe have analyzed the expression of several candidate genes known to be involved in floral organ identity determination in roses from similar genetic backgrounds but exhibiting contrasting petal numbers per flower. We show that the rose ortholog of AGAMOUS (RhAG) is differentially expressed in double flowers as compared to simple flowers. In situ hybridization experiments confirm the differential expression of RhAG and demonstrate that in the double-flower roses, the expression domain of RhAG is restricted toward the center of the flower. Conversely, in simple-flower roses, RhAG expression domain is wider. We further show that the border of RhAG expression domain is labile, which allows the selection of rose flowers with increased petal number. Double-flower roses were selected independently in the two major regions for domestication, China and the peri-Mediterranean areas. Comparison of RhAG expression in the wild-type ancestors of cultivated roses and their descendants both in the European and Chinese lineages corroborates the correlation between the degree of restriction of RhAG expression domain and the number of petals. Our data suggests that a restriction of RhAG expression domain is the basis for selection of double flowers in both the Chinese and peri-Mediterranean centers of domestication.Conclusions/SignificanceWe demonstrate that a shift in RhAG expression domain boundary occurred in rose hybrids, causing double-flower phenotype. This molecular event was selected independently during rose domestication in Europe/Middle East and in China.

Circadian clock in Ciona intestinalis revealed by microarray analysis and oxygen consumption

The molecular mechanisms of the endogenous circadian clocks that allow most animals to adapt to environmental cycles have recently been uncovered. The draft genome of the ascidian, Ciona intestinalis, a model animal that is close to vertebrates, has been described. However, the C. intestinalis genome lacks the canonical clock genes such as Per, Bmal and Clock that are shared by vertebrates and insects. Here, we found the circadian rhythms at the physiological and molecular levels. The oxygen consumption rate was lower during the light phase and higher during the dark phase during a day, and the rhythm highly damped and continued under constant darkness. From the microarray analysis, the 396 spots (1.8% of the total; corresponding to 388 clones) were extracted as candidates for circadian expression. We confirmed the circadian expression of several candidate genes by northern blotting. Furthermore, three of four rhythmic expressed genes showed phase-shifts to prolonged light period. However, most of known clock genes did not oscillate. These data suggest that C. intestinalis have a unique molecular circadian clock and the daily environmental change is not such a strong effect for sea squirt in its evolution when compared to vertebrates and insects.

Sensitivity to high salinity in tetraploid citrus seedlings increases with water availability and correlates with expression of candidate genes

We investigated tolerance to high salinity in well-irrigated diploid and tetraploid citrus. Comparisons were made between two diploids (2×) of trifoliate orange (Poncirus trifoliata (L.) Raf.) and willow leaf mandarin (Citrus deliciosa Ten), their respective doubled diploids (4×) and the allotetraploid (FLHORAG1) obtained from the protoplast fusion of trifoliate orange and Willow leaf mandarin. Salinity stress was applied by progressively increasing the concentration of NaCl from 50 mM to 400 mM for 8 weeks. Two-year-old plants were watered daily. Maximum quantum yield of PSII, and leaf and root chloride and sodium content were monitored. We previously reported that under moderate saline stress, citrus 4× genotypes were more tolerant that the 2×, but under these experimental conditions, 4× seedlings were certainly more sensitive to salt stress than 2×, as they accumulated more toxic ions and were more affected than 2×. Chloride accumulation in 4× leaves was greater and the maximum quantum yield of PSII was more reduced in 4× than in 2×. The expression of several candidate genes involved in signal transduction, sodium and chloride transport, osmotic adjustment, regulation of the stomata opening and detoxification processes were also investigated by quantitative real-time reverse transcription-PCR. A high correlation was observed between phenotype of sensitivity to stress and gene expression changes.