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Sample records for quantitative differential expression

  1. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  2. Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method

    PubMed Central

    Yang, Ganglong; Xu, Zhipeng; Lu, Wei; Li, Xiang; Sun, Chengwen; Guo, Jia; Xue, Peng; Guan, Feng

    2015-01-01

    The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia), KK47 (low grade nonmuscle invasive bladder cancer, NMIBC), and YTS1 (metastatic bladder cancer) have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC) progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO) term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer. PMID:26230496

  3. Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

    PubMed

    Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

    2013-04-01

    To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species. PMID:23464874

  4. Quantitative gene expression profiling of mouse brain regions reveals differential transcripts conserved in human and affected in disease models.

    PubMed

    Brochier, Camille; Gaillard, Marie-Claude; Diguet, Elsa; Caudy, Nicolas; Dossat, Carole; Ségurens, Béatrice; Wincker, Patrick; Roze, Emmanuel; Caboche, Jocelyne; Hantraye, Philippe; Brouillet, Emmanuel; Elalouf, Jean-Marc; de Chaldée, Michel

    2008-04-22

    Using serial analysis of gene expression, we collected quantitative transcriptome data in 11 regions of the adult wild-type mouse brain: the orbital, prelimbic, cingulate, motor, somatosensory, and entorhinal cortices, the caudate-putamen, the nucleus accumbens, the thalamus, the substantia nigra, and the ventral tegmental area. With >1.2 million cDNA tags sequenced, this database is a powerful resource to explore brain functions and disorders. As an illustration, we performed interregional comparisons and found 315 differential transcripts. Most of them are poorly characterized and 20% lack functional annotation. For 78 differential transcripts, we provide independent expression level measurements in mouse brain regions by real-time quantitative RT-PCR. We also show examples where we used in situ hybridization to achieve infrastructural resolution. For 30 transcripts, we next demonstrated that regional enrichment is conserved in the human brain. We then quantified the expression levels of region-enriched transcripts in the R6/2 mouse model of Huntington disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease and observed significant alterations in the striatum, cerebral cortex, thalamus and substantia nigra of R6/2 mice and in the striatum of MPTP-treated mice. These results show that the gene expression data provided here for the mouse brain can be used to explore pathophysiological models and disclose transcripts differentially expressed in human brain regions. PMID:18252803

  5. TeratoScore: Assessing the Differentiation Potential of Human Pluripotent Stem Cells by Quantitative Expression Analysis of Teratomas.

    PubMed

    Avior, Yishai; Biancotti, Juan Carlos; Benvenisty, Nissim

    2015-06-01

    Teratoma formation is the gold standard assay for testing the capacity of human pluripotent stem cells to differentiate into all embryonic germ layers. Although widely used, little effort has been made to transform this qualitative assay into a quantitative one. Using gene expression data from a wide variety of cells, we created a scorecard representing tissues from all germ layers and extraembryonic tissues. TeratoScore, an online, open-source platform based on this scorecard, distinguishes pluripotent stem cell-derived teratomas from malignant tumors, translating cell potency into a quantitative measure (http://benvenisty.huji.ac.il/teratoscore.php). The teratomas used for the algorithm also allowed us to examine gene expression differences between tumors with a diploid karyotype and those initiated by aneuploid cells. Chromosomally aberrant teratomas show a significantly different gene expression signature from that of teratomas originating from diploid cells, particularly in central nervous system-specific genes, congruent with human chromosomal syndromes. PMID:26070610

  6. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  7. Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones

    SciTech Connect

    Nguyen, C.; Rocha, D.; Granjeaud, S.

    1995-09-01

    High-throughput measurement of hybridization signatures obtained using complex probes prepared from poly(A){sup +} RNA and high-density cDNA colony filters is described. The performance of the system, elimination of artifacts, and verification of the validity of the data are discussed. cDNAs corresponding to sequences present at levels of approximately 0.01% in the complex probe can be detected. Good correlation is observed between expression profiles determined by this method and by Northern blotting. The method is applied to a preliminary investigation of differential expression in three cell types present in the murine thymus. 41 refs., 3 figs., 1 tab.

  8. Differentiation of five body fluids from forensic samples by expression analysis of four microRNAs using quantitative PCR.

    PubMed

    Sauer, Eva; Reinke, Ann-Kathrin; Courts, Cornelius

    2016-05-01

    Applying molecular genetic approaches for the identification of forensically relevant body fluids, which often yield crucial information for the reconstruction of a potential crime, is a current topic of forensic research. Due to their body fluid specific expression patterns and stability against degradation, microRNAs (miRNA) emerged as a promising molecular species, with a range of candidate markers published. The analysis of miRNA via quantitative Real-Time PCR, however, should be based on a relevant strategy of normalization of non-biological variances to deliver reliable and biologically meaningful results. The herein presented work is the as yet most comprehensive study of forensic body fluid identification via miRNA expression analysis based on a thoroughly validated qPCR procedure and unbiased statistical decision making to identify single source samples. PMID:26878708

  9. Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

    PubMed Central

    Ginaldi, L; Farahat, N; Matutes, E; De Martinis, M; Morilla, R; Catovsky, D

    1996-01-01

    AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts. Images PMID:8813949

  10. Proteomic analysis from haploid and diploid embryos of Quercus suber L. identifies qualitative and quantitative differential expression patterns.

    PubMed

    Gómez, Aranzazu; López, Juan Antonio; Pintos, Beatriz; Camafeita, Emilio; Bueno, Ma Angeles

    2009-09-01

    Quercus suber L. is a Mediterranean forest species with ecological, social and economic value. Clonal propagation of Q. suber elite trees has been successfully obtained from in vitro-derived somatic and gametic embryos. These clonal lines play a main role in breeding and genetic studies of Q. suber. To aid in unravelling diverse genetic and biological unknowns, a proteomic approach is proposed. The proteomic analysis of Q. suber somatic and gametic in vitro culture-derived embryos, based on DIGE and MALDI-MS, has produced for the first time proteomic data on this species. Seventeen differentially expressed proteins have been identified which display significantly altered levels between gametic and somatic embryos. These proteins are involved in a variety of cellular processes, most of which had been neither previously associated with embryo development nor identified in the genus Quercus. Some of these proteins are involved in stress and pollen development and others play a role in the metabolism of tannins and phenylpropanoids, which represent two of the major pathways for the synthesis of cork chemical components. Furthermore, the augmented expression levels found for specific proteins are probably related to the homozygous state of a doubled-haploid sample. Proteins involved in synthesis of cork components can be detected at such early stages of development, showing the potential of the method to be useful in searching for biomarkers related to cork quality. PMID:19662628

  11. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion

    PubMed Central

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W.; Kulozik, Andreas E.

    2016-01-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  12. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

    PubMed

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

    2016-09-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  13. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    PubMed

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  14. Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer

    PubMed Central

    2010-01-01

    Background We have recently described the correlation between quantitative measures of HER2 expression or HER2 homodimers by the HERmark assay and objective response (RR), time-to progression (TTP), and overall survival (OS) in an expanded access cohort of trastuzumab-treated HER2-positive patients with metastatic breast cancer (MBC) who were stringently selected by fluorescence in situ hybridization (FISH). Multivariate analyses suggested a continuum of HER2 expression that correlated with outcome following trastuzumab. Here we investigate the relationship between HER2 expression or HER2 homodimers and OS in a clinic-based population of patients with MBC selected primarily by IHC. Methods HERmark, a proximity-based assay designed to detect and quantitate protein expression and dimerization in formalin-fixed paraffin-embedded (FFPE) tissues, was used to measure HER2 expression and HER2 homodimers in FFPE samples from patients with MBC. Assay results were correlated with OS using univariate Kaplan-Meier, hazard function plots, and multivariate Cox regression analyses. Results Initial analyses revealed a parabolic relationship between continuous measures of HER2 expression and risk of death, suggesting that the assumption of linearity for the HER2 expression measurements may be inappropriate in subsequent multivariate analyses. Cox regression analyses using the categorized variable of HER2 expression level demonstrated that higher HER2 levels predicted better survival outcomes following trastuzumab treatment in the high HER2-expressing group. Conclusions These data suggest that the quantitative amount of HER2 expression measured by Hermark may be a new useful marker to identify a more relevant target population for trastuzumab treatment in patients with MBC. PMID:20178580

  15. Differential Classroom Interactions by Ethnicity: A Quantitative Approach

    ERIC Educational Resources Information Center

    Tennant, Geoff

    2004-01-01

    Concerns regarding differentials in classroom experience and academic attainment by ethnicity have been expressed for some time. This study explores, from a quantitative viewpoint based on fieldwork in 10 London schools, one particular aspect of this, namely classroom interactions between teachers and pupils from different ethnic origins. It was…

  16. Quantitative analysis of differential protein expression in cervical carcinoma cells after zeylenone treatment by stable isotope labeling with amino acids in cell culture.

    PubMed

    Zhang, Leilei; Jin, Jing; Zhang, Lijing; Hu, Ruifeng; Gao, Li; Huo, Xiaowei; Liu, Dongyu; Ma, Xiaoling; Wang, Canhong; Han, Jiayuan; Li, Liyong; Sun, Xiaobo; Cao, Li

    2015-08-01

    Cervical carcinoma is a malignant tumor that poses a serious threat to women's health and survival. Approximately 10-25% of cervical cancers are adenocarcinomas (ACs). AC has high rates of recurrence and mortality, while there is no effective treatment for now. Zeylenone (Zey), which is isolated from an ethanol extract of the leaves of Uvaria grandiflora Roxb. of the family Annonaceae, has shown potent inhibitory activity against various tumor cells, including cervical carcinoma cells. To gain insight into the molecular mechanism underlying the effect of Zey on AC, we quantified protein expression changes in AC cells treated with Zey. We used stable isotope labeling with amino acids in cell culture (SILAC) in combination with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and bioinformatics analysis to compare protein expression profiles in HeLa cells before and after Zey treatment. Of 1805 differentially expressed proteins identified, 229 were screened as key protein molecules and classified into nine categories. Profiling of differentially-expressed proteins contributed to our understanding of the molecular mechanism by which Zey induces HeLa cell apoptosis. Using this method, candidate targets can be identified for developing new drugs against cervical carcinoma. PMID:26130516

  17. Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

    PubMed Central

    Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Park, Ji-Sung; Lee, Seung-Chan; Baregundi Subbarao, Raghavendra; Lee, Sung-Lim; Park, Bong-Wook; King, William Allan; Rho, Gyu-Jin

    2015-01-01

    The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs. PMID:25972899

  18. Differential placental gene expression in severe preeclampsia.

    PubMed

    Sitras, V; Paulssen, R H; Grønaas, H; Leirvik, J; Hanssen, T A; Vårtun, A; Acharya, G

    2009-05-01

    We investigated the global placental gene expression profile in severe preeclampsia. Twenty-one women were randomly selected from 50 participants with uncomplicated pregnancies to match 21 patients with severe preeclampsia. A 30K Human Genome Survey Microarray v.2.0 (Applied Biosystems) was used to evaluate the gene expression profile. After RNA isolation, five preeclamptic placentas were excluded due to poor RNA quality. The series composed of 37 hybridizations in a one-channel detection system of chemiluminescence emitted by the microarrays. An empirical Bayes analysis was applied to find differentially expressed genes. In preeclamptic placentas 213 genes were significantly (fold-change>or=2 and pdifferentially expressed genes were associated with Alzheimer disease, angiogenesis, Notch-, TGFbeta- and VEGF-signalling pathways. Sixteen genes best discriminated preeclamptic from normal placentas. Comparison between early- (<34 weeks) and late-onset preeclampsia showed 168 differentially expressed genes with oxidative stress, inflammation, and endothelin signalling pathways mainly involved in early-onset disease. Validation of the microarray results was performed by RT-PCR, quantitative urine hCG measurement and placental histopathologic examination. In summary, placental gene expression is altered in preeclampsia and we provide a comprehensive list of the differentially expressed genes. Placental gene expression is different between early- and late-onset preeclampsia, suggesting differences in pathophysiology. PMID:19249095

  19. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.

    PubMed Central

    Connor, J; Bucana, C; Fidler, I J; Schroit, A J

    1989-01-01

    Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS--e.g., PS exposure in the outer leaflet of the cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells--serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival of pathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7- to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages. PMID:2717615

  20. Beef quality with different intramuscular fat content and proteomic analysis using isobaric tag for relative and absolute quantitation of differentially expressed proteins.

    PubMed

    Mao, Yanwei; Hopkins, David L; Zhang, Yimin; Li, Peng; Zhu, Lixian; Dong, Pengcheng; Liang, Rongrong; Dai, Jin; Wang, Xiaoyun; Luo, Xin

    2016-08-01

    Intramuscular fat (IMF) is an important trait for beef eating quality. The mechanism of how IMF is deposited in beef cattle muscle is not clear at the molecular level. The muscle (M. longissimus lumborum: LL) of a group of Xiangxi yellow×Angus cattle with high fat levels (HF), was compared to the muscle of a low fat group (LF). The meat quality and the expressed protein patterns were compared. It was shown that LL from the HF animals had a greater fat content (P<0.05) and lower moisture content (P<0.05) than LL from LF animals. Forty seven sarcoplasmic proteins were differentially expressed and identified between the two groups. These proteins are involved in 6 molecular functions and 16 biological processes, and affect the Mitogen-activated protein kinases pathway, insulin pathway and c-Jun N-terminal kinases leading to greater IMF deposition. Cattle in the HF group had greater oxidative capacity and lower glycolytic levels suggesting a greater energetic efficiency. PMID:27064846

  1. Differential Gene Expression in Glaucoma

    PubMed Central

    Jakobs, Tatjana C.

    2014-01-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell–matrix interactions and adhesion, the cell cycle, and the endothelin system. PMID:24985133

  2. Differential expression of cysteine desulfurases in soybean

    PubMed Central

    2011-01-01

    Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression. PMID:22099069

  3. Expression quantitative trait loci: present and future

    PubMed Central

    Nica, Alexandra C.; Dermitzakis, Emmanouil T.

    2013-01-01

    The last few years have seen the development of large efforts for the analysis of genome function, especially in the context of genome variation. One of the most prominent directions has been the extensive set of studies on expression quantitative trait loci (eQTLs), namely, the discovery of genetic variants that explain variation in gene expression levels. Such studies have offered promise not just for the characterization of functional sequence variation but also for the understanding of basic processes of gene regulation and interpretation of genome-wide association studies. In this review, we discuss some of the key directions of eQTL research and its implications. PMID:23650636

  4. Identification of quantitative trait loci affecting ectomycorrhizal symbiosis in an interspecific F1 poplar cross and differential expression of genes in ectomycorrhizas of the two parents: Populus deltoides and Populus trichocarpa

    SciTech Connect

    Labbe, Jessy L; Jorge, Veronique; Vion, Patrice; Marcais, Benoit; Bastien, Catherine; Tuskan, Gerald A; Martin, Francis; Le Tacon, F

    2011-01-01

    A Populus deltoides Populus trichocarpa F1 pedigree was analyzed for quantitative trait loci (QTLs) affecting ectomycorrhizal development and for microarray characterization of gene networks involved in this symbiosis. A 300 genotype progeny set was evaluated for its ability to form ectomycorrhiza with the basidiomycete Laccaria bicolor. The percentage of mycorrhizal root tips was determined on the root systems of all 300 progeny and their two parents. QTL analysis identified four significant QTLs, one on the P. deltoides and three on the P. trichocarpa genetic maps. These QTLs were aligned to the P. trichocarpa genome and each contained several megabases and encompass numerous genes. NimbleGen whole-genome microarray, using cDNA from RNA extracts of ectomycorrhizal root tips from the parental genotypes P. trichocarpa and P. deltoides, was used to narrow the candidate gene list. Among the 1,543 differentially expressed genes (p value 0.05; 5.0-fold change in transcript level) having different transcript levels in mycorrhiza of the two parents, 41 transcripts were located in the QTL intervals: 20 in Myc_d1, 14 in Myc_t1, and seven in Myc_t2, while no significant differences among transcripts were found in Myc_t3. Among these 41 transcripts, 25 were overrepresented in P. deltoides relative to P. trichocarpa; 16 were overrepresented in P. trichocarpa. The transcript showing the highest overrepresentation in P. trichocarpa mycorrhiza libraries compared to P. deltoides mycorrhiza codes for an ethylene-sensitive EREBP-4 protein which may repress defense mechanisms in P. trichocarpa while the highest overrepresented transcripts in P. deltoides code for proteins/genes typically associated with pathogen resistance.

  5. Cell cycle imaging with quantitative differential interference contrast microscopy

    NASA Astrophysics Data System (ADS)

    Kostyk, Piotr; Phelan, Shelley; Xu, Min

    2013-02-01

    We report a microscopic approach for determining cell cycle stages by measuring the nuclear optical path length (OPL) with quantitative differential interference contrast (DIC) microscopy. The approach is validated by the excellent agreement between the proportion of proliferating-to-quiescent cancerous breast epithelial cells obtained from DIC microscopy, and that from a standard immunofluorescence assay.

  6. Inferring differentiation pathways from gene expression

    PubMed Central

    Costa, Ivan G.; Roepcke, Stefan; Hafemeister, Christoph; Schliep, Alexander

    2008-01-01

    Motivation: The regulation of proliferation and differentiation of embryonic and adult stem cells into mature cells is central to developmental biology. Gene expression measured in distinguishable developmental stages helps to elucidate underlying molecular processes. In previous work we showed that functional gene modules, which act distinctly in the course of development, can be represented by a mixture of trees. In general, the similarities in the gene expression programs of cell populations reflect the similarities in the differentiation path. Results: We propose a novel model for gene expression profiles and an unsupervised learning method to estimate developmental similarity and infer differentiation pathways. We assess the performance of our model on simulated data and compare it with favorable results to related methods. We also infer differentiation pathways and predict functional modules in gene expression data of lymphoid development. Conclusions: We demonstrate for the first time how, in principal, the incorporation of structural knowledge about the dependence structure helps to reveal differentiation pathways and potentially relevant functional gene modules from microarray datasets. Our method applies in any area of developmental biology where it is possible to obtain cells of distinguishable differentiation stages. Availability: The implementation of our method (GPL license), data and additional results are available at http://algorithmics.molgen.mpg.de/Supplements/InfDif/ Contact: filho@molgen.mpg.de, schliep@molgen.mpg.de Supplementary information: Supplementary data is available at Bioinformatics online. PMID:18586709

  7. Differential Bacterial Gene Expression During Experimental Pneumococcal Endophthalmitis

    PubMed Central

    Thornton, Justin A.; Tullos, Nathan A.; Sanders, Melissa E.; Ridout, Granger; Wang, Yong-Dong; Taylor, Sidney D.; McDaniel, Larry S.; Marquart, Mary E.

    2015-01-01

    Streptococcus pneumoniae (pneumococcus) is a potential cause of bacterial endophthalmitis in humans that can result in ocular morbidity. We sought to identify pneumococcal genes that are differentially expressed during growth in the vitreous humor of the eye in an experimental endophthalmitis model. Microarray analysis was used to identify genes that were differentially expressed when pneumococci replicated in the vitreous of rabbit eyes as compared with bacteria grown in vitro in Todd Hewitt medium. Array results were verified by quantitative real-time PCR analysis of representative genes. Select genes potentially playing a role in virulence during endophthalmitis were deleted and mutants were tested for reduced eye pathogenesis and altered adhesion to host cells. Array analysis identified 134 genes that were differentially expressed during endophthalmitis. 112 genes demonstrated increased expression during growth in the eye whereas 22 were down-regulated. Real-time analysis verified increased expression of neuraminidase A (SP1693), neuraminidase B (SP1687), and serine protease (SP1954), and decreased expression of RlrA (SP0461) and choline transporter (SP1861). Mutation of neuraminidases A and B had no major effect on pathogenesis. Loss of SP1954 led to increased adherence to host cells. S. pneumoniae enhances and represses expression of a variety of genes during endophthalmitis. While some of these genes reflect changes in metabolic requirements, some appear to play a role in immune evasion and pathogenesis in the eye. PMID:25791614

  8. Differential expression of microRNAs in mouse embryonic bladder

    SciTech Connect

    Liu, Benchun; Cunha, Gerald R.; Baskin, Laurence S.

    2009-08-07

    MicroRNAs (miRNAs) are involved in several biological processes including development, differentiation and proliferation. Analysis of miRNA expression patterns in the process of embryogenesis may have substantial value in determining the mechanism of embryonic bladder development as well as for eventual therapeutic intervention. The miRNA expression profiles are distinct among the cellular types and embryonic stages as demonstrated by microarray technology and validated by quantitative real-time RT-PCR approach. Remarkably, the miRNA expression patterns suggested that unique miRNAs from epithelial and submucosal areas are responsible for mesenchymal cellular differentiation, especially regarding bladder smooth muscle cells. Our data show that miRNA expression patterns are unique in particular cell types of mouse bladder at specific developmental stages, reflecting the apparent lineage and differentiation status within the embryonic bladder. The identification of unique miRNAs expression before and after smooth muscle differentiation in site-specific area of the bladder indicates their roles in embryogenesis and may aid in future clinical intervention.

  9. Expression profiles for six zebrafish genes during gonadal sex differentiation

    PubMed Central

    Jørgensen, Anne; Morthorst, Jane E; Andersen, Ole; Rasmussen, Lene J; Bjerregaard, Poul

    2008-01-01

    Background The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. Results In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish. Conclusion In zebrafish, the first significant peak in gene expression during the investigated period (2–40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex

  10. Introducing Knowledge into Differential Expression Analysis

    PubMed Central

    Biecek, Przemysław; Tiuryn, Jerzy; Vingron, Martin

    2010-01-01

    Abstract Gene expression measurements allow determining sets of up- or down-regulated, or unchanged genes in a particular experimental condition. Additional biological knowledge can suggest examples of genes from one of these sets. For instance, known target genes of a transcriptional activator are expected, but are not certain to go down after this activator is knocked out. Available differential expression analysis tools do not take such imprecise examples into account. Here we put forward a novel partially supervised mixture modeling methodology for differential expression analysis. Our approach, guided by imprecise examples, clusters expression data into differentially expressed and unchanged genes. The partially supervised methodology is implemented by two methods: a newly introduced belief-based mixture modeling, and soft-label mixture modeling, a method proved efficient in other applications. We investigate on synthetic data the input example settings favorable for each method. In our tests, both belief-based and soft-label methods prove their advantage over semi-supervised mixture modeling in correcting for erroneous examples. We also compare them to alternative differential expression analysis approaches, showing that incorporation of knowledge yields better performance. We present a broad range of knowledge sources and data to which our partially supervised methodology can be applied. First, we determine targets of Ste12 based on yeast knockout data, guided by a Ste12 DNA-binding experiment. Second, we distinguish miR-1 from miR-124 targets in human by clustering expression data under transfection experiments of both microRNAs, using their computationally predicted targets as examples. Finally, we utilize literature knowledge to improve clustering of time-course expression profiles. PMID:20726790

  11. Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

    PubMed Central

    Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.

    2015-01-01

    Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499

  12. From inverse problems in mathematical physiology to quantitative differential diagnoses.

    PubMed

    Zenker, Sven; Rubin, Jonathan; Clermont, Gilles

    2007-11-01

    The improved capacity to acquire quantitative data in a clinical setting has generally failed to improve outcomes in acutely ill patients, suggesting a need for advances in computer-supported data interpretation and decision making. In particular, the application of mathematical models of experimentally elucidated physiological mechanisms could augment the interpretation of quantitative, patient-specific information and help to better target therapy. Yet, such models are typically complex and nonlinear, a reality that often precludes the identification of unique parameters and states of the model that best represent available data. Hypothesizing that this non-uniqueness can convey useful information, we implemented a simplified simulation of a common differential diagnostic process (hypotension in an acute care setting), using a combination of a mathematical model of the cardiovascular system, a stochastic measurement model, and Bayesian inference techniques to quantify parameter and state uncertainty. The output of this procedure is a probability density function on the space of model parameters and initial conditions for a particular patient, based on prior population information together with patient-specific clinical observations. We show that multimodal posterior probability density functions arise naturally, even when unimodal and uninformative priors are used. The peaks of these densities correspond to clinically relevant differential diagnoses and can, in the simplified simulation setting, be constrained to a single diagnosis by assimilating additional observations from dynamical interventions (e.g., fluid challenge). We conclude that the ill-posedness of the inverse problem in quantitative physiology is not merely a technical obstacle, but rather reflects clinical reality and, when addressed adequately in the solution process, provides a novel link between mathematically described physiological knowledge and the clinical concept of differential diagnoses

  13. Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP).

    PubMed

    Ali, Shahnaz; Ghosh, Kanjaksha; Shetty, Shrimati

    2015-12-01

    Bengal macrothrombocytopenia (BMTCP) is a giant platelet disorder with mild to moderate thrombocytopenia, clinically characterized by mild bleeding symptoms to totally asymptomatic condition. The pathophysiological mechanism of this condition is not fully understood yet. In the present study, 5 subjects (P1-P5) with BMTCP whose platelet counts ranged between 36140X10(9)/l and mean platelet volume (MPV)13.5-16.1fl were analyzed for differential gene expression of platelets by suppressive subtractive hybridization (SSH) technique. Four genes i.e. myotubularin related protein 9 (MTMR9), iron responsive element binding protein 2 (IREB2), alpha tubulin(TUBA) and tyrosine kinase ligand (TKL) were found to be differentially expressed in patient platelets as compared to that of normal healthy controls which was further confirmed by quantitative RT PCR analysis. The study highlights a multi-factorial etiology for BMTCP which is widely prevalent in the northeastern region of the Indian subcontinent. PMID:26460267

  14. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    PubMed Central

    Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

    2015-01-01

    Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

  15. Quantitative imaging of gene expression in Drosophila embryos.

    PubMed

    Surkova, Svetlana; Myasnikova, Ekaterina; Kozlov, Konstantin N; Pisarev, Andrei; Reinitz, John; Samsonova, Maria

    2013-06-01

    Quantitative measurements derived using sophisticated microscopy techniques are essential for understanding the basic principles that control the behavior of biological systems. Here we describe a data pipeline developed to extract quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of image segmentation, background removal, temporal characterization of an embryo, data registration, and data averaging. This pipeline has been successfully applied to obtain quantitative gene expression data at cellular resolution in space and at 6.5-min resolution in time. It has also enabled the construction of a spatiotemporal atlas of segmentation gene expression. We describe the software used to construct a workflow for extracting quantitative data on segmentation gene expression and the BREReA package, which implements the methods for background removal and registration of segmentation gene expression patterns. PMID:23734022

  16. Identification of differentially expressed genes associated with differential body size in mandarin fish (Siniperca chuatsi).

    PubMed

    Tian, Changxu; Li, Ling; Liang, Xu-Fang; He, Shan; Guo, Wenjie; Lv, Liyuan; Wang, Qingchao; Song, Yi

    2016-08-01

    Body size is an obvious and important characteristic of fish. Mandarin fish Siniperca chuatsi (Basilewsky) is one of the most valuable perciform species widely cultured in China. Individual differences in body size are common in mandarin fish and significantly influence the aquaculture production. However, little is currently known about its genetic control. In this study, digital gene expression profiling and transcriptome sequencing were performed in mandarin fish with differential body size at 30 and 180 days post-hatch (dph), respectively. Body weight, total length and body length of fish with big-size were significantly higher than those with small-size at both 30 and 180 dph (P < 0.05). 2171 and 2014 differentially expressed genes were identified between small-size and big-size fish at 30 and 180 dph, respectively. RT quantitative PCR (qPCR) analysis showed that the differential expression of 10 selected genes in mandarin fish that went through the same training procedure. The genes were involved in the growth hormone-insulin-like growth factor axis, cell proliferation and differentiation, appetite control, glucose metabolism, reproduction and sexual size dimorphism pathways. This study will help toward a comprehensive understanding of the complexity of regulation of body size in mandarin fish individuals and provide valuable information for future research. PMID:27393605

  17. Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei

    PubMed Central

    Dejung, Mario; Subota, Ines; Bucerius, Ferdinand; Dindar, Gülcin; Freiwald, Anja; Engstler, Markus; Boshart, Michael; Butter, Falk; Janzen, Christian J.

    2016-01-01

    Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. PMID:26910529

  18. Differentiation among parkinsonisms using quantitative diffusion kurtosis imaging.

    PubMed

    Ito, Kenji; Sasaki, Makoto; Ohtsuka, Chigumi; Yokosawa, Suguru; Harada, Taisuke; Uwano, Ikuko; Yamashita, Fumio; Higuchi, Satomi; Terayama, Yasuo

    2015-03-25

    Differential diagnoses among Parkinson's disease (PD), multiple system atrophy (MSA), and progressive supranuclear palsy syndrome (PSPS) are often difficult. Hence, we investigated whether diffusion kurtosis imaging (DKI) could detect pathological changes that occur in these disorders and be used to differentiate between such patients. Fourteen patients (five with PD, four MSA, and five PSPS) and six healthy controls were examined using a 1.5-T scanner. Mean kurtosis (MK), fractional anisotropy, and mean diffusivity maps were generated, and these values of the midbrain tegmentum (MBT) and pontine crossing tract (PCT), as well as MBT/PCT ratios, were obtained. We found no significant differences in MBT and PCT values on DKI maps among the groups. In contrast, MBT/PCT ratios from MK maps were significantly increased in the MSA group and decreased in the PSPS group compared with the other groups. MBT/PCT ratios from mean diffusivity maps showed a significant increase in the PSPS group. Therefore, quantitative DKI analyses, particularly the MBT/PCT ratio from MK maps, can differentiate patients with parkinsonisms. PMID:25714421

  19. LC–MS Based Detection of Differential Protein Expression

    PubMed Central

    Tuli, Leepika; Ressom, Habtom W.

    2010-01-01

    While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput. For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data. This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed. For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing

  20. Differential temporal expression of matrix metalloproteinases following sciatic nerve crush

    PubMed Central

    Qin, Jing; Zha, Guang-bin; Yu, Jun; Zhang, Hong-hong; Yi, Sheng

    2016-01-01

    We previously performed transcriptome sequencing and found that genes for matrix metalloproteinases (MMPs), such as MMP7 and 12, seem to be highly upregulated following peripheral nerve injury, and may be involved in nerve repair. In the present study, we systematically determined the expression levels of MMPs and their regulators at 1, 4, 7 and 14 days after sciatic nerve crush injury. The number of differentially expressed genes was elevated at 4 and 7 days after injury, but decreased at 14 days after injury. Among the differentially expressed genes, those most up-regulated showed fold changes of more than 214, while those most down-regulated exhibited fold changes of more than 2−10. Gene sequencing showed that, at all time points after injury, a variety of MMP genes in the “Inhibition of MMPs” pathway were up-regulated, and their inhibitor genes were down-regulated. Expression of key up- and down-regulated genes was verified by quantitative real-time polymerase chain reaction analysis and found to be consistent with transcriptome sequencing. These results suggest that MMP-related genes are strongly involved in the process of peripheral nerve regeneration.

  1. Differential isoform expression and selective muscle involvement in muscular dystrophies.

    PubMed

    Huovinen, Sanna; Penttilä, Sini; Somervuo, Panu; Keto, Joni; Auvinen, Petri; Vihola, Anna; Huovinen, Sami; Pelin, Katarina; Raheem, Olayinka; Salenius, Juha; Suominen, Tiina; Hackman, Peter; Udd, Bjarne

    2015-10-01

    Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies. PMID:26269091

  2. MicroRNA expression during demosponge dissociation, reaggregation, and differentiation and a evolutionarily conserved demosponge miRNA expression profile.

    PubMed

    Robinson, Jeffrey M

    2015-11-01

    Demosponges share eight orthologous microRNAs (miRNAs), with none in common with Bilateria. Biological functions of these demosponge miRNAs are unknown. Bilaterian miRNAs are key regulators of cellular processes including cell cycle, differentiation, and metabolism. Resolving if demosponge miRNAs participate in such biological functions will provide clues whether these functions are convergent, evidence on the mode of evolution of cellular developmental processes. Here, a quantitative PCR (qPCR) assay was developed and used to test for differential miRNA expression during dissociation and reaggregation in Spongosorites, compare expression profiles between choanosome and cortex in Spongosorites, and compare undifferentiated gemmules to differentiated juveniles in Ephydatia. During Spongosorites dissociation and reaggregation, miRNA expression showed a global decrease in expression across a range of reaggregating cell densities. miRNA differential response could be related to various general cellular responses, potentially related to nutrient-poor conditions of the minimal artificial seawater media, stress response from tissue dissociation, or loss of cell-cell or cell-matrix contact. In Ephydatia, overall increase in miRNA expression in gemmule-hatched stage 4/5 juveniles relative to gemmules is observed, indicating that increased miRNA expression may be related to increased cellular activity such as migration, cell cycle, and/or differentiation. Observed differential miRNA expression of miRNA during dissociation in Spongosorites (lowered global expression), and during activation, and differentiation of Ephydatia gemmules (increased global expression) could indicate that miRNA expression is associated with cell cycle, differentiation, or metabolism pathways. Interspecies comparison was performed, results indicating that orthologous miRNAs share similar relative expression pattern between the four species tested (Spongosorites, Cinachyrella, Haliclona, and Ephydatia

  3. Quantitative neuroproteomics: classical and novel tools for studying neural differentiation and function.

    PubMed

    Colucci-D'Amato, Luca; Farina, Annarita; Vissers, Johannes P C; Chambery, Angela

    2011-03-01

    Mechanisms underlying neural stem cell proliferation, differentiation and maturation play a critical role in the formation and wiring of neuronal connections. This process involves the activation of multiple serial events, which guide the undifferentiated cells to different lineages via distinctive developmental programs, forming neuronal circuits and thus shaping the adult nervous system. Furthermore, alterations within these strictly regulated pathways can lead to severe neurological and psychiatric diseases. In this framework, the investigation of the high dynamic protein expression changes and other factors affecting protein functions, for example post-translational modifications, the alterations of protein interaction networks, is of pivotal importance for the understanding of the molecular mechanisms responsible for cell differentiation. More recently, proteomic studies in neuroscience ("neuroproteomics") are receiving increased interest for the primary understanding of the regulatory networks underlying neuronal differentiation processes. Besides the classical two-dimensional-based proteomic strategies, the emerging platforms for LC-MS shotgun proteomic analysis hold great promise in unraveling the molecular basis of neural stem cell differentiation. In this review, recent advancements in label-free LC-MS quantitative neuroproteomics are highlighted as a new tool for the study of neural differentiation and functions, in comparison to mass spectrometry-based labeling approaches. The more commonly used protein profiling strategies and model systems for the analysis of neural differentiation are also discussed, along with the challenging proteomic approaches aimed to analyze the nervous system-specific organelles, the neural cells secretome and the specific protein interaction networks. PMID:20352529

  4. Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells

    PubMed Central

    Yang, Jing; Gu, Ping; Menges, Steven

    2012-01-01

    Purpose Differentiation of neural stem/progenitor cells involves changes in the gene expression of these cells. Less clear is the extent to which incremental changes occur and the time course of such changes, particularly in non-rodents. Methods Using porcine genome microarrays, we analyzed changes in the expression of 23,256 genes in porcine neural progenitor cells (pNPCs) subject to two established differentiation protocols. In addition, we performed sequential quantitative assessment of a defined transcription profile consisting of 15 progenitor- and lineage-associated genes following exposure to the same treatment protocols, to examine the temporal dynamics of phenotypic changes following induction of differentiation. Immunocytochemistry was also used to examine the expression of seven of these phenotypically important genes at the protein level. Initial primary isolates were passaged four times in proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) before differentiation was induced. Differentiation was induced by medium without EGF or bFGF and containing either 10 ng/ml ciliary neurotrophic factor or 10% fetal bovine serum (FBS). Cultures were fed every two days and harvested on days 0, 1, 3, and 5 for quantitative real-time PCR. Results The microarray results illustrated and contrasted the global shifts in the porcine transcriptome associated with both treatment conditions. PCR confirmed dramatic upregulation of transcripts for myelin basic protein (up to 88 fold), claudin 11 (up to 32 fold), glial fibrillary acidic protein (GFAP; up to 26 fold), together with notable (>twofold) increases in message for microtubule associated protein 2 (MAP2) and C-X-C chemokine receptor type 4 (CXCR4), Janus kinase 1 (Jak1), signal transducer and activator of transcription 1 (STAT1), and signal transducer and activator of transcription 3 (STAT3). Transcripts for nestin and Krüppel-like factor 4 (KLF4

  5. Differential gene expression in ripening banana fruit.

    PubMed

    Clendennen, S K; May, G D

    1997-10-01

    During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants. PMID:9342866

  6. Towards quantitative atmospheric water vapor profiling with differential absorption lidar.

    PubMed

    Dinovitser, Alex; Gunn, Lachlan J; Abbott, Derek

    2015-08-24

    Differential Absorption Lidar (DIAL) is a powerful laser-based technique for trace gas profiling of the atmosphere. However, this technique is still under active development requiring precise and accurate wavelength stabilization, as well as accurate spectroscopic parameters of the specific resonance line and the effective absorption cross-section of the system. In this paper we describe a novel master laser system that extends our previous work for robust stabilization to virtually any number of multiple side-line laser wavelengths for the future probing to greater altitudes. In this paper, we also highlight the significance of laser spectral purity on DIAL accuracy, and illustrate a simple re-arrangement of a system for measuring effective absorption cross-section. We present a calibration technique where the laser light is guided to an absorption cell with 33 m path length, and a quantitative number density measurement is then used to obtain the effective absorption cross-section. The same absorption cell is then used for on-line laser stabilization, while microwave beat-frequencies are used to stabilize any number of off-line lasers. We present preliminary results using ∼300 nJ, 1 μs pulses at 3 kHz, with the seed laser operating as a nanojoule transmitter at 822.922 nm, and a receiver consisting of a photomultiplier tube (PMT) coupled to a 356 mm mirror. PMID:26368258

  7. Quantitative orientation-independent differential interference contrast (DIC) microscopy

    NASA Astrophysics Data System (ADS)

    Shribak, Michael; LaFountain, James; Biggs, David; Inoué, Shinya

    2007-02-01

    We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly, Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference microscope.

  8. Quantitative EEG patterns of differential in-flight workload

    NASA Technical Reports Server (NTRS)

    Sterman, M. B.; Mann, C. A.; Kaiser, D. A.

    1993-01-01

    Four test pilots were instrumented for in-flight EEG recordings using a custom portable recording system. Each flew six, two minute tracking tasks in the Calspan NT-33 experimental trainer at Edwards AFB. With the canopy blacked out, pilots used a HUD display to chase a simulated aircraft through a random flight course. Three configurations of flight controls altered the flight characteristics to achieve low, moderate, and high workload, as determined by normative Cooper-Harper ratings. The test protocol was administered by a command pilot in the back seat. Corresponding EEG and tracking data were compared off-line. Tracking performance was measured as deviation from the target aircraft and combined with control difficulty to achieve an estimate of 'cognitive workload'. Trended patterns of parietal EEG activity at 8-12 Hz were sorted according to this classification. In all cases, high workload produced a significantly greater suppression of 8-12 Hz activity than low workload. Further, a clear differentiation of EEG trend patterns was obtained in 80 percent of the cases. High workload produced a sustained suppression of 8-12 Hz activity, while moderate workload resulted in an initial suppression followed by a gradual increment. Low workload was associated with a modulated pattern lacking any periods of marked or sustained suppression. These findings suggest that quantitative analysis of appropriate EEG measures may provide an objective and reliable in-flight index of cognitive effort that could facilitate workload assessment.

  9. Building quantitative, three dimensional atlases of gene expression and morphology at cellular resolution

    PubMed Central

    Knowles, David W.; Biggin, Mark D.

    2013-01-01

    Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy based approaches to establish permanent, quantitative datasets—atlases—that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization and quantitative analysis. PMID:24123936

  10. Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

    PubMed Central

    Li, Jianke; Wu, Jing; Begna Rundassa, Desalegn; Song, Feifei; Zheng, Aijuan; Fang, Yu

    2010-01-01

    Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on

  11. Defining Breast Cancer Intrinsic Subtypes by Quantitative Receptor Expression

    PubMed Central

    Cheang, Maggie C.U.; Martin, Miguel; Nielsen, Torsten O.; Prat, Aleix; Voduc, David; Rodriguez-Lescure, Alvaro; Ruiz, Amparo; Chia, Stephen; Shepherd, Lois; Ruiz-Borrego, Manuel; Calvo, Lourdes; Alba, Emilio; Carrasco, Eva; Caballero, Rosalia; Tu, Dongsheng; Pritchard, Kathleen I.; Levine, Mark N.; Bramwell, Vivien H.; Parker, Joel; Bernard, Philip S.; Ellis, Matthew J.; Perou, Charles M.; Di Leo, Angelo

    2015-01-01

    Purpose. To determine intrinsic breast cancer subtypes represented within categories defined by quantitative hormone receptor (HR) and HER2 expression. Methods. We merged 1,557 cases from three randomized phase III trials into a single data set. These breast tumors were centrally reviewed in each trial for quantitative ER, PR, and HER2 expression by immunohistochemistry (IHC) stain and by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), with intrinsic subtyping by research-based PAM50 RT-qPCR assay. Results. Among 283 HER2-negative tumors with <1% HR expression by IHC, 207 (73%) were basal-like; other subtypes, particularly HER2-enriched (48, 17%), were present. Among the 1,298 HER2-negative tumors, borderline HR (1%–9% staining) was uncommon (n = 39), and these tumors were heterogeneous: 17 (44%) luminal A/B, 12 (31%) HER2-enriched, and only 7 (18%) basal-like. Including them in the definition of triple-negative breast cancer significantly diminished enrichment for basal-like cancer (p < .05). Among 106 HER2-positive tumors with <1% HR expression by IHC, the HER2-enriched subtype was the most frequent (87, 82%), whereas among 127 HER2-positive tumors with strong HR (>10%) expression, only 69 (54%) were HER2-enriched and 55 (43%) were luminal (39 luminal B, 16 luminal A). Quantitative HR expression by RT-qPCR gave similar results. Regardless of methodology, basal-like cases seldom expressed ER/ESR1 or PR/PGR and were associated with the lowest expression level of HER2/ERBB2 relative to other subtypes. Conclusion. Significant discordance remains between clinical assay-defined subsets and intrinsic subtype. For identifying basal-like breast cancer, the optimal HR IHC cut point was <1%, matching the American Society of Clinical Oncology and College of American Pathologists guidelines. Tumors with borderline HR staining are molecularly diverse and may require additional assays to clarify underlying biology. PMID:25908555

  12. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  13. Quantitative modeling of the terminal differentiation of B cells and mechanisms of lymphomagenesis

    PubMed Central

    Martínez, María Rodríguez; Corradin, Alberto; Klein, Ulf; Álvarez, Mariano Javier; Toffolo, Gianna M.; di Camillo, Barbara; Califano, Andrea; Stolovitzky, Gustavo A.

    2012-01-01

    Mature B-cell exit from germinal centers is controlled by a transcriptional regulatory module that integrates antigen and T-cell signals and, ultimately, leads to terminal differentiation into memory B cells or plasma cells. Despite a compact structure, the module dynamics are highly complex because of the presence of several feedback loops and self-regulatory interactions, and understanding its dysregulation, frequently associated with lymphomagenesis, requires robust dynamical modeling techniques. We present a quantitative kinetic model of three key gene regulators, BCL6, IRF4, and BLIMP, and use gene expression profile data from mature human B cells to determine appropriate model parameters. The model predicts the existence of two different hysteresis cycles that direct B cells through an irreversible transition toward a differentiated cellular state. By synthetically perturbing the interactions in this network, we can elucidate known mechanisms of lymphomagenesis and suggest candidate tumorigenic alterations, indicating that the model is a valuable quantitative tool to simulate B-cell exit from the germinal center under a variety of physiological and pathological conditions. PMID:22308355

  14. A Hybrid Approach to Protein Differential Expression in Mass Spectrometry-Based Proteomics

    SciTech Connect

    Wang, Xuan; Anderson, Gordon A.; Smith, Richard D.; Dabney, Alan R.

    2012-04-19

    Motivation: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein's associated spectral peaks. However, typical MS-based proteomics data sets have substantial proportions of missing observations, due at least in part to censoring of low intensities. This complicates intensity-based differential expression analysis. Results: We outline a statistical method for protein differential expression, based on a simple Binomial likelihood. By modeling peak intensities as binary, in terms of 'presence/ absence,' we enable the selection of proteins not typically amendable to quantitative analysis; e.g., 'one-state' proteins that are present in one condition but absent in another. In addition, we present an analysis protocol that combines quantitative and presence/ absence analysis of a given data set in a principled way, resulting in a single list of selected proteins with a single associated FDR.

  15. Screening of differentially expressed genes in pathological scar tissues using expression microarray.

    PubMed

    Huang, L P; Mao, Z; Zhang, L; Liu, X X; Huang, C; Jia, Z S

    2015-01-01

    Pathological scar tissues and normal skin tissues were differentiated by screening for differentially expressed genes in pathologic scar tissues via gene expression microarray. The differentially expressed gene data was analyzed by gene ontology and pathway analyses. There were 5001 up- or down-regulated genes in 2-fold differentially expressed genes, 956 up- or down-regulated genes in 5-fold differentially expressed genes, and 114 up- or down-regulated genes in 20-fold differentially expressed genes. Therefore, significant differences were observed in the gene expression in pathological scar tissues and normal foreskin tissues. The development of pathological scar tissues has been correlated to changes in multiple genes and pathways, which are believed to form a dynamic network connection. PMID:26400303

  16. Ectopic expression of interferon regulatory factor-1 potentiates granulocytic differentiation.

    PubMed Central

    Coccia, E M; Stellacci, E; Valtieri, M; Masella, B; Feccia, T; Marziali, G; Hiscott, J; Testa, U; Peschle, C; Battistini, A

    2001-01-01

    Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of differentiation. PMID:11716756

  17. Quantitative analysis of HSV gene expression during lytic infection

    PubMed Central

    Turner, Anne-Marie W.; Arbuckle, Jesse H.; Kristie, Thomas M.

    2014-01-01

    Herpes Simplex Virus (HSV) is a human pathogen that establishes latency and undergoes periodic reactivation, resulting in chronic recurrent lytic infection. HSV lytic infection is characterized by an organized cascade of three gene classes, however successful transcription and expression of the first, the immediate early class, is critical to the overall success of viral infection. This initial event of lytic infection is also highly dependent on host cell factors. This unit uses RNA interference and small molecule inhibitors to examine the role of host and viral proteins in HSV lytic infection. Methods detailing isolation of viral and host RNA and genomic DNA, followed by quantitative real-time PCR, allow characterization of impacts on viral transcription and replication respectively. Western blot can be used to confirm quantitative PCR results. This combination of protocols represents a starting point for researchers interested in virus-host interactions during HSV lytic infection. PMID:25367270

  18. Tri-mean-based statistical differential gene expression detection.

    PubMed

    Ji, Zhaohua; Wu, Chunguo; Wang, Yao; Guan, Renchu; Tu, Huawei; Wu, Xiaozhou; Liang, Yanchun

    2012-01-01

    Based on the assumption that only a subset of disease group has differential gene expression, traditional detection of differentially expressed genes is under the constraint that cancer genes are up- or down-regulated in all disease samples compared with normal samples. However, in 2005, Tomlins assumed and discussed the situation that only a subset of disease samples would be activated, which are often referred to as outliers. PMID:23155761

  19. Differential Object Marking in Spanish: A Quantitative Variationist Study

    ERIC Educational Resources Information Center

    Tippets, Ian Robert

    2010-01-01

    This dissertation addresses the variable nature of the linguistic phenomenon known as Differential Object Marking (DOM) as it is manifested in Spanish. More commonly known in the literature as the personal "a" or the accusative "a", this phenomenon has been attributed primarily to marking animate, predominantly human, direct objects. However, this…

  20. Identification of Differentially Expressed miRNAs in Appendiceal Mucinous Cystadenocarcinoma from Mucinous Cystadenoma

    PubMed Central

    Wu, Richard Licheng; Ali, Shadan; Sarkar, Fazlul H; Beydoun, Rafic

    2016-01-01

    Objective Mucinous cystadenocarcinoma of appendix is a rare entity. Differentiating mucinous cystadenocarcinoma from mucinous cystadenoma is very challenging and depends on establishing the presence of malignant cells in the appendix wall. The invasion may be very difficult to assess in some cases, especially in early stages of the disease, which could have devastating prognostic effects on patients. Therefore, it is necessary to develop an ancillary test that can differentiate the mucinous cystadenocarcinoma from mucinous cystadenoma. So far, there is no report available about the role of differentially expressed miRNAs in the diagnosis of appendiceal mucinous cystadenocarcinoma. Materials and Methods Six confirmed mucinous appendiceal cystadenocarcinoma and twelve mucinous appendiceal cystadenoma cases were selected. The total RNAs were extracted from the formalin-fixed paraffin-embedded specimen of these cases. The comprehensive miRNA microarray expression profiling from pooled aliquots of RNA samples from these two entities were analyzed to detect the differentially expressed miRNAs in mucinous cystadenocarcinoma. The best seven differentially expressed miRNAs were validated in individual cases by quantitative reverse transcriptase PCR (qRT-PCR). Results The microarray miRNA expression profiling analysis revealed 646 miRNAs that were differentially expressed in the mucinous cystadenocarcinoma. Among these differentially expressed miRNAs, the expression of 80 miRNAs showed statistical difference (p<0.01). The quantitative RT-PCR validated that the expression of miR-1, miR-4328 was significantly down regulated in mucinous cystadenocarcinoma compared to the mucinous cystadenoma (p<0.05). On the other hand, the expression of miR-200b, miR-200c, miR-451, miR-223 and miR-21 were significantly upregulated in mucinous cystadenocarcinoma (p<0.05). Conclusion The expression levels of miRNAs tested were significantly altered in the appendiceal mucinous cystadenocarcinoma

  1. Sebocytes differentially express and secrete adipokines.

    PubMed

    Kovács, Dóra; Lovászi, Marianna; Póliska, Szilárd; Oláh, Attila; Bíró, Tamás; Veres, Imre; Zouboulis, Christos C; Ståhle, Mona; Rühl, Ralph; Remenyik, Éva; Törőcsik, Dániel

    2016-03-01

    In addition to producing sebum, sebocytes link lipid metabolism with inflammation at a cellular level and hence, greatly resemble adipocytes. However, so far no analysis was performed to identify and characterize the adipocyte-associated inflammatory proteins, the members of the adipokine family in sebocytes. Therefore, we determined the expression profile of adipokines [adiponectin, interleukin (IL) 6, resistin, leptin, serpin E1, visfatin, apelin, chemerin, retinol-binding protein 4 (RBP4) and monocyte chemoattractant protein 1 (MCP1)] in sebaceous glands of healthy and various disease-affected (acne, rosacea, melanoma and psoriasis) skin samples. Sebaceous glands in all examined samples expressed adiponectin, IL6, resistin, leptin, serpin E1 and visfatin, but not apelin, chemerin, RBP4 and MCP1. Confirming the presence of the detected adipokines in the human SZ95 sebaceous gland cell line we further characterized their expression and secretion patterns under different stimuli mimicking bacterial invasion [by using Toll-like receptor (TLR)2 and 4 activators], or by 13-cis retinoic acid (13CRA; also known as isotretinoin), a key anti-acne agent. With the exception of resistin, the expression of all of the detected adipokines (adiponectin, IL6, leptin, serpin E1 and visfatin) could be further regulated at the level of gene expression, showing a close correlation with the secreted protein levels. Besides providing further evidence on similarities between adipocytes and sebocytes, our results strongly suggest that sebocytes are not simply targets of inflammation but may exhibit initiatory and modulatory roles in the inflammatory processes of the skin through the expression and secretion of adipokines. PMID:26476096

  2. Rapid and robust association mapping of expression quantitative trait loci.

    PubMed

    Lam, Alex C; Schouten, Michael; Aulchenko, Yurii S; Haley, Chris S; de Koning, Dirk-Jan

    2007-01-01

    We applied a simple and efficient two-step method to analyze a family-based association study of gene expression quantitative trait loci (eQTL) in a mixed model framework. This two-step method produces very similar results to the full mixed model method, with our method being significantly faster than the full model. Using the Genetic Analysis Workshop 15 (GAW15) Problem 1 data, we demonstrated the value of data filtering for reducing the number of tests and controlling the number of false positives. Specifically, we showed that removing non-expressed genes by filtering on expression variability effectively reduced the number of tests by nearly 50%. Furthermore, we demonstrated that filtering on genotype counts substantially reduced spurious detection. Finally, we restricted our analysis to the markers and transcripts that were closely located. We found five times more signals in close proximity (cis-) to transcripts than in our genome-wide analysis. Our results suggest that careful pre-filtering and partitioning of data are crucial for controlling false positives and allowing detection of genuine effects in genetic analysis of gene expression. PMID:18466488

  3. Genes encoding FAD-binding proteins in Volvariella volvacea exhibit differential expression in homokaryons and heterokaryons.

    PubMed

    Meng, Li; Yan, Junjie; Xie, Baogui; Li, Yu; Chen, Bingzhi; Liu, Shuyan; Li, Dan; Yang, Zhiyun; Zeng, Xiancheng; Deng, Youjin; Jiang, Yuji

    2013-10-01

    Flavin adenine dinucleotide (FAD)-binding proteins play a vital role in energy transfer and utilization during fungal growth and mycelia aggregation. We sequenced the genome of Volvariella volvacea, an economically important edible fungus, and discovered 41 genes encoding FAD-binding proteins. Gene expression profiles revealed that the expression levels of four distinctly differentially expressed genes in heterokaryotic strain H1521 were higher than in homokaryotic strains PYd15 and PYd21 combined. These observations were validated by quantitative real-time PCR. The results suggest that the differential expression of FAD-binding proteins may be important in revealing the distinction between homokaryons and heterokaryons on the basis of FAD-binding protein functionality. PMID:23570970

  4. Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility.

    PubMed

    Jiang, S; Robertson, T; Mostajeran, M; Robertson, A J; Qiu, X

    2016-06-01

    Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees. PMID:26919127

  5. Gene expression indicates a zone of heterocyst differentiation within the thallus of the cyanolichen Pseudocyphellaria crocata.

    PubMed

    Chua, Jocelyn P S; Wallace, Emma J S; Yardley, Jessica A; Duncan, Elizabeth J; Dearden, Peter K; Summerfield, Tina C

    2012-11-01

    Development of the symbiotic association in the bipartite lichen Pseudocyphellaria crocata was investigated by characterizing two regions of the thallus. Thallus organization was examined using microscopy. A HIP1-based differential display technique was modified for use on Nostoc strains, including lichenized strains. Northern hybridization and quantitative real-time polymerase chain reaction were used to confirm differential display results, and determine expression levels of key cyanobacterial genes. Photosystem II yield across the thallus was measured using pulse-amplitude modulated fluorescence. Microscopy revealed structural differences in the thallus margins compared with the centre and identified putative heterocysts in both regions. Differential display identified altered transcript levels in both Nostoc punctiforme and a lichenized Nostoc strain. Transcript abundance of cox2, atpA, and ribA was increased in the thallus margin compared with the centre. Expression of cox2 is heterocyst specific and expression of other heterocyst-specific genes (hetR and nifK) was elevated in the margin, whereas, expression of psbB and PSII yield were not. Structural organization of the thallus margin differed from the centre. Both regions contained putative heterocysts but gene expression data indicated increased heterocyst differentiation in the margins where photosystem II yield was decreased. This is consistent with a zone of heterocyst differentiation within the thallus margin. PMID:22931432

  6. Transcriptome Analysis of Differentially Expressed Genes Relevant to Variegation in Peach Flowers

    PubMed Central

    Yu, Faxin; Li, Shuxian; Yin, Tongming

    2014-01-01

    Background Variegation in flower color is commonly observed in many plant species and also occurs on ornamental peaches (Prunus persica f. versicolor [Sieb.] Voss). Variegated plants are highly valuable in the floricultural market. To gain a global perspective on genes differentially expressed in variegated peach flowers, we performed large-scale transcriptome sequencing of white and red petals separately collected from a variegated peach tree. Results A total of 1,556,597 high-quality reads were obtained, with an average read length of 445 bp. The ESTs were assembled into 16,530 contigs and 42,050 singletons. The resulting unigenes covered about 60% of total predicted genes in the peach genome. These unigenes were further subjected to functional annotation and biochemical pathway analysis. Digital expression analysis identified a total of 514 genes differentially expressed between red and white flower petals. Since peach flower coloration is determined by the expression and regulation of structural genes relevant to flavonoid biosynthesis, a detailed examination detected four key structural genes, including C4H, CHS, CHI and F3H, expressed at a significantly higher level in red than in white petal. Except for the structural genes, we also detected 11 differentially expressed regulatory genes relating to flavonoid biosynthesis. Using the differentially expressed structural genes as the test objects, we validated the digital expression results by using quantitative real-time PCR, and the differential expression of C4H, CHS and F3H were confirmed. Conclusion In this study, we generated a large EST collection from flower petals of a variegated peach. By digital expression analysis, we identified an informative list of candidate genes associated with variegation in peach flowers, which offered a unique opportunity to uncover the genetic mechanisms underlying flower color variegation. PMID:24603808

  7. Differential expression of myrosinase gene families.

    PubMed Central

    Lenman, M; Falk, A; Rödin, J; Höglund, A S; Ek, B; Rask, L

    1993-01-01

    In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominatly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axiz contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of

  8. Lentivirus Live Cell Array for Quantitative Assessment of Gene and Pathway Activation during Myogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Tian, Jun; Gaile, Daniel P.; Andreadis, Stelios T.

    2015-01-01

    Stem cell differentiation involves multiple cascades of transcriptional regulation that govern the cell fate. To study the real-time dynamics of this complex process, quantitative and high throughput live cell assays are required. Herein, we developed a lentiviral library of promoters and transcription factor binding sites to quantitatively capture the gene expression dynamics over a period of several days during myogenic differentiation of human mesenchymal stem cells (MSCs) harvested from two different anatomic locations, bone marrow and hair follicle. Our results enabled us to monitor the sequential activation of signaling pathways and myogenic gene promoters at various stages of differentiation. In conjunction with chemical inhibitors, the lentiviral array (LVA) results also revealed the relative contribution of key signaling pathways that regulate the myogenic differentiation. Our study demonstrates the potential of LVA to monitor the dynamics of gene and pathway activation during MSC differentiation as well as serve as a platform for discovery of novel molecules, genes and pathways that promote or inhibit complex biological processes. PMID:26505747

  9. Differential global gene expression in red and white skeletal muscle

    NASA Technical Reports Server (NTRS)

    Campbell, W. G.; Gordon, S. E.; Carlson, C. J.; Pattison, J. S.; Hamilton, M. T.; Booth, F. W.

    2001-01-01

    The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

  10. Differentially Expressed Genes and Signature Pathways of Human Prostate Cancer

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Robbins, Charles J.; Sang, Qing-Xiang Amy

    2015-01-01

    Genomic technologies including microarrays and next-generation sequencing have enabled the generation of molecular signatures of prostate cancer. Lists of differentially expressed genes between malignant and non-malignant states are thought to be fertile sources of putative prostate cancer biomarkers. However such lists of differentially expressed genes can be highly variable for multiple reasons. As such, looking at differential expression in the context of gene sets and pathways has been more robust. Using next-generation genome sequencing data from The Cancer Genome Atlas, differential gene expression between age- and stage- matched human prostate tumors and non-malignant samples was assessed and used to craft a pathway signature of prostate cancer. Up- and down-regulated genes were assigned to pathways composed of curated groups of related genes from multiple databases. The significance of these pathways was then evaluated according to the number of differentially expressed genes found in the pathway and their position within the pathway using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. The “transforming growth factor-beta signaling” and “Ran regulation of mitotic spindle formation” pathways were strongly associated with prostate cancer. Several other significant pathways confirm reported findings from microarray data that suggest actin cytoskeleton regulation, cell cycle, mitogen-activated protein kinase signaling, and calcium signaling are also altered in prostate cancer. Thus we have demonstrated feasibility of pathway analysis and identified an underexplored area (Ran) for investigation in prostate cancer pathogenesis. PMID:26683658

  11. Characterization and differentiation of three solid tumors using quantitative ultrasound

    NASA Astrophysics Data System (ADS)

    Oelze, Michael L.; O'Brien, William D.; Zachary, James F.

    2001-05-01

    Three kinds of solid tumors were acquired and scanned in vivo ultrasonically. The first tumor series (fibroadenoma) was acquired from tumors that had spontaneously developed in rats. The second tumor series was acquired by culturing a carcinoma cell line (4T1-MMT) in culture media and injecting the cells into Balb/c mice. The third tumor was acquired by transplanting a soft-tissue sarcoma cell line (EHS) into C57BL mice. The tumors were allowed to grow to 1 cm in size and then scanned ultrasonically. The scatterer properties of average scatterer diameter and acoustic concentration were estimated using a Gaussian form factor from the backscattered ultrasound measured from the tumors. Parametric images of the tumors were constructed utilizing estimated scatterer properties for regions of interest inside the tumors. The parametric images showed distinct differences between the various tumor types. Quantitatively, the tumors could be distinguished through feature analysis plots of average scatterer size versus acoustic concentration. Comparison with photomicrographs of the tumors showed structures similar in size to the ultrasound estimates. [Work supported by NIH Grant F32 CA96419 to MLO and by the University of Illinois Research Board.

  12. Differential Gene Expression in Benznidazole-Resistant Trypanosoma cruzi Parasites

    PubMed Central

    Villarreal, Diana; Nirdé, Philippe; Hide, Mallorie; Barnabé, Christian; Tibayrenc, Michel

    2005-01-01

    We analyzed the differential gene expression among representative Trypanosoma cruzi stocks in relation to benznidazole exposures using a random differentially expressed sequences (RADES) technique. Studies were carried out with drug pressure both at the natural susceptibility level of the wild-type parasite (50% inhibitory concentration for the wild type) and at different resistance levels. The pattern of differential gene expression performed with resistant stocks was compared to the population structure of this parasite, established by random amplified polymorphic DNA analysis and multilocus enzyme electrophoresis. A RADES band polymorphism was observed, and over- or underexpression was linked to the resistance level of the stock. The analysis of RADES bands suggested that different products may be involved in benznidazole resistance mechanisms. No significant association was found between phylogenetic clustering and benznidazole susceptibility. Benznidazole resistance may involve several mechanisms, depending on the level of drug exposure. PMID:15980339

  13. Differential expression of pentraxin 3 in neutrophils.

    PubMed

    Razvina, Olga; Jiang, Shuying; Matsubara, Koichi; Ohashi, Riuko; Hasegawa, Go; Aoyama, Takashi; Daigo, Kenji; Kodama, Tatsuhiko; Hamakubo, Takao; Naito, Makoto

    2015-02-01

    Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense. PMID:25449330

  14. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

    PubMed Central

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-01-01

    AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

  15. Detection of differentially expressed genes and association with clinicopathological features in laryngeal squamous cell carcinoma.

    PubMed

    Ni, Rong Sheng; Shen, Xiaohui; Qian, Xiaoyun; Yu, Chenjie; Wu, Haiyan; Gao, Xia

    2012-12-01

    Head and neck cancer is a significant health problem worldwide. Early detection and prediction of prognosis will improve patient survival and quality of life. The aim of this study was to identify genes differentially expressed between laryngeal cancer and the corresponding normal tissues as potential biomarkers. A total of 36 patients with laryngeal squamous cell carcinoma were recruited. Four of these cases were randomly selected for cDNA microarray analysis of the entire genome. Using semi-quantitative RT-PCR and western blot analysis, the differential expression of genes and their protein products, respectively, between laryngeal cancer tissues and corresponding adjacent normal tissues was verified in the remaining 32 cases. The expression levels of these genes and proteins were investigated for associations with clinicopathological parameters taken from patient data. The cDNA microarray analysis identified 349 differentially expressed genes between tumor and normal tissues, 112 of which were upregulated and 237 were downregulated in tumors. Seven genes and their protein products were then selected for validation using RT-PCR and western blot analysis, respectively. The data demonstrated that the expression of SENP1, CD109, CKS2, LAMA3, ITGAV and ITGB8 was increased, while LAMA2 was downregulated in laryngeal cancer compared with the corresponding normal tissues. Associations between the expression of these genes and clinicopathological data from the patients were also established, including age, tumor classification, stage, differentiation and lymph node metastasis. Our current study provides the first evidence that these seven genes may be differentially expressed in laryngeal squamous cell carcinoma and also associated with clinicopathological data. Future study is required to further confirm whether detection of their expression can be used as biomarkers for prediction of patient survival or potential treatment targets. PMID:23226807

  16. Identification of genes differentially expressed in ectomycorrhizal roots during the Pinus pinaster-Laccaria bicolor interaction.

    PubMed

    Flores-Monterroso, Aranzazu; Canales, Javier; de la Torre, Fernando; Ávila, Concepción; Cánovas, Francisco M

    2013-06-01

    Ectomycorrhizal associations are of major ecological importance in temperate and boreal forests. The development of a functional ectomycorrhiza requires many genetic and biochemical changes. In this study, suppressive subtraction hybridization was used to identify differentially expressed genes in the roots of maritime pine (Pinus pinaster Aiton) inoculated with Laccaria bicolor, a mycorrhizal fungus. A total number of 200 unigenes were identified as being differentially regulated in maritime pine roots during the development of mycorrhiza. These unigenes were classified into 10 categories according to the function of their homologues in the GenBank database. Approximately, 40 % of the differentially expressed transcripts were genes that coded for unknown proteins in the databases or that had no homology to known genes. A group of these differentially expressed genes was selected to validate the results using quantitative real-time PCR. The transcript levels of the representative genes were compared between the non-inoculated and inoculated plants at 1, 5, 15 and 30 days after inoculation. The observed expression patterns indicate (1) changes in the composition of the wall cell, (2) tight regulation of defence genes during the development of mycorrhiza and (3) changes in carbon and nitrogen metabolism. Ammonium excess or deficiency dramatically affected the stability of ectomycorrhiza and altered gene expression in maritime pine roots. PMID:23543110

  17. Changes in the expression profiles of claudins during gonocyte differentiation and in seminomas.

    PubMed

    Manku, G; Hueso, A; Brimo, F; Chan, P; Gonzalez-Peramato, P; Jabado, N; Gayden, T; Bourgey, M; Riazalhosseini, Y; Culty, M

    2016-01-01

    Testicular germ cell tumors (TGCTs) are the most common type of cancer in young men and their incidence has been steadily increasing for the past decades. TGCTs and their precursor carcinoma in situ (CIS) are thought to arise from the deficient differentiation of gonocytes, precursors of spermatogonial stem cells. However, the mechanisms relating failed gonocyte differentiation to CIS formation remain unknown. The goal of this study was to uncover genes regulated during gonocyte development that would show abnormal patterns of expression in testicular tumors, as prospective links between failed gonocyte development and TGCT. To identify common gene and protein signatures between gonocytes and seminomas, we first performed gene expression analyses of transitional rat gonocytes, spermatogonia, human normal testicular, and TGCT specimens. Gene expression arrays, pathway analysis, and quantitative real-time PCR analysis identified cell adhesion molecules as a functional gene category including genes downregulated during gonocyte differentiation and highly expressed in seminomas. In particular, the mRNA and protein expressions of claudins 6 and 7 were found to decrease during gonocyte transition to spermatogonia, and to be abnormally elevated in seminomas. The dynamic changes in these genes suggest that they may play important physiological roles during gonocyte development. Moreover, our findings support the idea that TGCTs arise from a disruption of gonocyte differentiation, and position claudins as interesting genes to further study in relation to testicular cancer. PMID:26588606

  18. Differential and limited expression of mutant alleles in multiple myeloma

    PubMed Central

    Rashid, Naim U.; Sperling, Adam S.; Bolli, Niccolo; Wedge, David C.; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A.; Fulciniti, Mariateresa; Samur, Mehmet K.; Richardson, Paul G.; Magrangeas, Florence; Minvielle, Stephane; Futreal, P. Andrew; Anderson, Kenneth C.; Avet-Loiseau, Herve; Parmigiani, Giovanni

    2014-01-01

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  19. Differential and limited expression of mutant alleles in multiple myeloma.

    PubMed

    Rashid, Naim U; Sperling, Adam S; Bolli, Niccolo; Wedge, David C; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A; Fulciniti, Mariateresa; Samur, Mehmet K; Richardson, Paul G; Magrangeas, Florence; Minvielle, Stephane; Futreal, P Andrew; Anderson, Kenneth C; Avet-Loiseau, Herve; Campbell, Peter J; Parmigiani, Giovanni; Munshi, Nikhil C

    2014-11-13

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  20. Modeling overdispersion heterogeneity in differential expression analysis using mixtures.

    PubMed

    Bonafede, Elisabetta; Picard, Franck; Robin, Stéphane; Viroli, Cinzia

    2016-09-01

    Next-generation sequencing technologies now constitute a method of choice to measure gene expression. Data to analyze are read counts, commonly modeled using negative binomial distributions. A relevant issue associated with this probabilistic framework is the reliable estimation of the overdispersion parameter, reinforced by the limited number of replicates generally observable for each gene. Many strategies have been proposed to estimate this parameter, but when differential analysis is the purpose, they often result in procedures based on plug-in estimates, and we show here that this discrepancy between the estimation framework and the testing framework can lead to uncontrolled type-I errors. Instead, we propose a mixture model that allows each gene to share information with other genes that exhibit similar variability. Three consistent statistical tests are developed for differential expression analysis. We show through a wide simulation study that the proposed method improves the sensitivity of detecting differentially expressed genes with respect to the common procedures, since it reaches the nominal value for the type-I error, while keeping elevate discriminative power between differentially and not differentially expressed genes. The method is finally illustrated on prostate cancer RNA-Seq data. PMID:26683201

  1. Differential microRNA expression in aristolochic acid-induced upper urothelial tract cancers ex vivo.

    PubMed

    Tao, Le; Zeng, Yigang; Wang, Jun; Liu, Zhihong; Shen, Bing; Ge, Jifu; Liu, Yong; Guo, Yifeng; Qiu, Jianxin

    2015-11-01

    Aristolochic acid (AA) is a carcinogenic, mutagenic and nephrotoxic compound commonly isolated from members of the plant family of Aristolochiaceae (such as Aristolochia and Asarum) and used in Chinese herbal medicine. Use of AA and AA‑containing plants causes chronic kidney disease (CKD) and upper urinary tract carcinoma (UUC); however, the underlying mechanism remains to be defined. miRNAs regulate a number of biological processes, including cell proliferation, differentiation and metabolism. This study explored differentially expressed miRNAs between AA‑induced upper urothelial tract cancer (AAN‑UUC) and non‑AAN‑UUC tissues. Patients with AAN‑UUC and non‑AAN‑UUC (n=20/group) were recruited in the present study. Five tissue samples from each group were used for miRNA microarray profiling and the rest of the tissue samples were subjected to reverse transcription-quantitative polymerase chain reaction analysis including seven selected miRNAs for confirmation. A total of 29 miRNAs were differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues (P<0.05). TargenScan and Gene ontology analyses predicted the functions and targeted genes of these differentially expressed miRNAs, i.e. Akt3, FGFR3, PSEN1, VEGFa and AR. Subsequently, expression of the selected differentially expressed miRNAs (Hsa‑miR‑4795‑5p, Hsa‑miR‑488, Hsa‑miR‑4784, Hsa‑miR‑330, Hsa‑miR‑3916, Hsa‑miR‑4274 and Hsa‑miR‑181c) was validated in another set of tissue samples. A total of 29 miRNAs were identified to be differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues and these miRNA target genes in FGFR3 and Akt pathways, which regulate cell growth and tumor progression, respectively. PMID:26397152

  2. Expression of Molecular Differentiation Markers Does Not Correlate with Histological Differentiation Grade in Intrahepatic Cholangiocarcinoma

    PubMed Central

    Demarez, Céline; Hubert, Catherine; Sempoux, Christine; Lemaigre, Frédéric P.

    2016-01-01

    The differentiation status of tumor cells, defined by histomorphological criteria, is a prognostic factor for survival of patients affected with intrahepatic cholangiocarcinoma (ICC). To strengthen the value of morphological differentiation criteria, we wished to correlate histopathological differentiation grade with expression of molecular biliary differentiation markers and of microRNAs previously shown to be dysregulated in ICC. We analysed a series of tumors that were histologically classified as well, moderately or poorly differentiated, and investigated the expression of cytokeratin 7, 19 and 903 (CK7, CK19, CK903), SRY-related HMG box transcription factors 4 and 9 (SOX4, SOX9), osteopontin (OPN), Hepatocyte Nuclear Factor-1 beta (HNF1β), Yes-associated protein (YAP), Epithelial cell adhesion molecule (EPCAM), Mucin 1 (MUC1) and N-cadherin (NCAD) by qRT-PCR and immunostaining, and of miR-31, miR-135b, miR-132, miR-200c, miR-221 and miR-222. Unexpectedly, except for subcellular location of SOX9 and OPN, no correlation was found between the expression levels of these molecular markers and histopathological differentiation grade. Therefore, our data point toward necessary caution when investigating the evolution and prognosis of ICC on the basis of cell differentiation criteria. PMID:27280413

  3. Gene expression variation and expression quantitative trait mapping of human chromosome 21 genes.

    PubMed

    Deutsch, Samuel; Lyle, Robert; Dermitzakis, Emmanouil T; Attar, Homa; Subrahmanyan, Lakshman; Gehrig, Corinne; Parand, Leila; Gagnebin, Maryline; Rougemont, Jacques; Jongeneel, C Victor; Antonarakis, Stylianos E

    2005-12-01

    Inter-individual differences in gene expression are likely to account for an important fraction of phenotypic differences, including susceptibility to common disorders. Recent studies have shown extensive variation in gene expression levels in humans and other organisms, and that a fraction of this variation is under genetic control. We investigated the patterns of gene expression variation in a 25 Mb region of human chromosome 21, which has been associated with many Down syndrome (DS) phenotypes. Taqman real-time PCR was used to measure expression variation of 41 genes in lymphoblastoid cells of 40 unrelated individuals. For 25 genes found to be differentially expressed, additional analysis was performed in 10 CEPH families to determine heritabilities and map loci harboring regulatory variation. Seventy-six percent of the differentially expressed genes had significant heritabilities, and genomewide linkage analysis led to the identification of significant eQTLs for nine genes. Most eQTLs were in trans, with the best result (P=7.46 x 10(-8)) obtained for TMEM1 on chromosome 12q24.33. A cis-eQTL identified for CCT8 was validated by performing an association study in 60 individuals from the HapMap project. SNP rs965951 located within CCT8 was found to be significantly associated with its expression levels (P=2.5 x 10(-5)) confirming cis-regulatory variation. The results of our study provide a representative view of expression variation of chromosome 21 genes, identify loci involved in their regulation and suggest that genes, for which expression differences are significantly larger than 1.5-fold in control samples, are unlikely to be involved in DS-phenotypes present in all affected individuals. PMID:16251198

  4. Differentially-Expressed Pseudogenes in HIV-1 Infection

    PubMed Central

    Gupta, Aditi; Brown, C. Titus; Zheng, Yong-Hui; Adami, Christoph

    2015-01-01

    Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit. PMID:26426037

  5. Differential expression of androgen, estrogen, and progesterone receptors in benign prostatic hyperplasia

    PubMed Central

    Song, Lingmin; Shen, Wenhao; Zhang, Heng; Wang, Qiwu; Wang, Yongquan; Zhou, Zhansong

    2016-01-01

    This study aimed to identify the differential expression levels of androgen receptor (AR), estrogen receptors (ERα, ERβ), and progesterone receptor (PGR) between normal prostate and benign prostatic hyperplasia (BPH). The combination of immunohistochemistry, quantitative real-time reverse transcription polymerase chain reaction, and Western blotting assay was used to identify the distribution and differential expression of these receptors at the immunoactive biomarker, transcriptional, and protein levels between 5 normal human prostate tissues and 40 BPH tissues. The results were then validated in a rat model of BPH induced by testosterone propionate and estradiol benzoate. In both human and rat prostate tissues, AR was localized mainly to epithelial and stromal cell nuclei; ERα was distributed mainly to stromal cells, but not exclusively; ERβ was interspersed in the basal layer of epithelium, but sporadically in epithelial and stromal cells; PGR was expressed abundantly in cytoplasm of epithelial and stromal cells. There were decreased expression of ERα and increased expression of PGR, but no difference in the expression of ERβ in the BPH compared to the normal prostate of both human and rat. Increased expression of AR in the BPH compared to the normal prostate of human was observed, however, the expression of AR in the rat prostate tissue was decreased. This study identified the activation of AR and PGR and repression of ERα in BPH, which indicate a promoting role of AR and PGR and an inhibitory role of ERα in the pathogenesis of BPH.

  6. Survivin expression is associated with lens epithelial cell proliferation and fiber cell differentiation

    PubMed Central

    Mansergh, Fiona C.; Boulton, Michael E.; Gunhaga, Lena

    2012-01-01

    Purpose Survivin (Birc5) is the smallest member of the inhibitor of apoptosis (IAP) protein family, which regulates the cell cycle/apoptosis balance. The purpose of this study was to examine Survivin expression in the embryonic chick lens, in chick lens epithelial cell cultures, and in the postnatal mouse lens. Methods Survivin expression was examined using a combination of quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. To correlate Survivin expression with the timing of proliferation, we determined the profile of cell proliferation in the developing lens using the cell cycle marker proliferating cell nuclear antigen (PCNA) in quantitative western blotting and immunocytochemistry studies. We also examined the expression of PCNA and the extent of denucleation using terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labeling (TUNEL) of lentoids (lens fiber-like cells) during chick lens epithelial cell differentiation in vitro. Results At embryonic day (ED) 4, Survivin immunostaining was present in two pools in lens epithelial cells and fiber cells: cytoplasmic and nuclear. The nuclear staining became more pronounced as the lens epithelial cells differentiated into lens fiber cells. At ED12, Survivin staining was observed in lens fiber cell nuclei containing marginalized chromatin, indicative of early denucleation events. Using western blotting, Survivin expression peaked at ED6, diminishing thereafter. This profile of expression correlated with the events in chick lens epithelial cell cultures: i) increased Survivin expression was associated with an increase in PCNA staining up to day 6 of culture and ii) downregulation of Survivin expression at day 8 of culture was coincident with a dramatic decrease in PCNA staining and an increase in TdT-mediated biotin-dUTP nick-end labeling in lentoids. In early postnatal mouse lenses, Survivin and PCNA were highly expressed and decreased thereafter during

  7. Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

    PubMed

    Li, Chun Ge; Wang, Hui; Chen, Hong Ju; Zhao, Yan; Fu, Pei Sheng; Ji, Xiang Shan

    2014-01-01

    Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHβ and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species. PMID:25199961

  8. A high throughput screening for rarely transcribed differentially expressed genes.

    PubMed Central

    von Stein, O D; Thies, W G; Hofmann, M

    1997-01-01

    A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined. PMID:9185570

  9. Differential Expression of Proteins and mRNAs from Border Cells and Root Tips of Pea.

    PubMed Central

    Brigham, L. A.; Woo, H. H.; Nicoll, S. M.; Hawes, M. C.

    1995-01-01

    Many plants release large numbers of metabolically active root border cells into the rhizosphere. We have proposed that border cells, cells produced by the root cap meristem that separate from the rest of the root upon reaching the periphery of the cap, are a singularly differentiated part of the root system that modulates the environment of the plant root by producing specific substances to be released into the rhizosphere. Proteins synthesized in border cells exhibit profiles that are very distinct from those of the root tip (root cap, root meristem, and adjacent cells). In vivo-labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip preparations. Twenty-five percent of the proteins synthesized by border cells in a 1-h period are rapidly excreted into the incubation medium. Quantitative variation in levels of specific marker proteins, including glutamine synthetase, heat-shock protein 70, and isoflavone reductase, also occurs between border cells and cells in the root tip. mRNA differential-display assays demonstrate that these large qualitative and quantitative differences in protein expression are correlated with similarly distinct patterns of gene expression. These observations are consistent with the hypothesis that a major switch in gene expression accompanies differentiation into root border cells, as expected for cells with specialized functions in plant development. PMID:12228604

  10. Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

    PubMed Central

    Straccia, Marco; Garcia-Diaz Barriga, Gerardo; Sanders, Phil; Bombau, Georgina; Carrere, Jordi; Mairal, Pedro Belio; Vinh, Ngoc-Nga; Yung, Sun; Kelly, Claire M; Svendsen, Clive N; Kemp, Paul J; Arjomand, Jamshid; Schoenfeld, Ryan C; Alberch, Jordi; Allen, Nicholas D; Rosser, Anne E; Canals, Josep M

    2015-01-01

    A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening. PMID:26417608

  11. Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell-derived medium spiny neurons.

    PubMed

    Straccia, Marco; Garcia-Diaz Barriga, Gerardo; Sanders, Phil; Bombau, Georgina; Carrere, Jordi; Mairal, Pedro Belio; Vinh, Ngoc-Nga; Yung, Sun; Kelly, Claire M; Svendsen, Clive N; Kemp, Paul J; Arjomand, Jamshid; Schoenfeld, Ryan C; Alberch, Jordi; Allen, Nicholas D; Rosser, Anne E; Canals, Josep M

    2015-01-01

    A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening. PMID:26417608

  12. Quantitative characterization of x-ray differential interference contrast microscopy using modulation transfer function

    NASA Astrophysics Data System (ADS)

    Nakamura, Takashi; Chang, Chang

    2011-08-01

    Performance of two types of differential interference contrast objectives, i.e., the XOR pattern and the zone-plate doublet, is quantitatively characterized and compared using modulation transfer function. Effects of partial coherence, finite absorption and phase in a complex object, as well as bias retardation are also examined.

  13. Quantitative characterization of x-ray differential interference contrast microscopy using modulation transfer function.

    PubMed

    Nakamura, Takashi; Chang, Chang

    2011-08-01

    Performance of two types of differential interference contrast objectives, i.e., the XOR pattern and the zone-plate doublet, is quantitatively characterized and compared using modulation transfer function. Effects of partial coherence, finite absorption and phase in a complex object, as well as bias retardation are also examined. PMID:21934894

  14. Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation

    PubMed Central

    Panek, Cecilia Analia; Ramos, Maria Victoria; Mejias, Maria Pilar; Abrey-Recalde, Maria Jimena; Fernandez-Brando, Romina Jimena; Gori, Maria Soledad; Salamone, Gabriela Verónica; Palermo, Marina Sandra

    2015-01-01

    Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1. PMID:25502213

  15. WEBSAGE: a web tool for visual analysis of differentially expressed human SAGE tags.

    PubMed

    Pylouster, Jean; Sénamaud-Beaufort, Catherine; Saison-Behmoaras, Tula Ester

    2005-07-01

    The serial analysis of gene expression (SAGE) is a powerful method to compare gene expression of mRNA populations. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project (CGAP) uses SAGE. Over 7 million SAGE tags, from 171 human cell types have been assembled. The growing number of laboratories involved in SAGE research necessitates the use of software that provides statistical analysis of raw data, allowing the rapid visualization and interpretation of results. We have created the first simple tool that performs statistical analysis on SAGE data, identifies the tags differentially expressed and shows the results in a scatter plot. It is freely available and accessible at http://bioserv.rpbs.jussieu.fr/websage/index.php. PMID:15980565

  16. Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2.

    PubMed

    Avogadri, Francesca; Gnjatic, Sacha; Tassello, Jodie; Frosina, Denise; Hanson, Nicole; Laudenbach, Megan; Ritter, Erika; Merghoub, Taha; Busam, Klaus J; Jungbluth, Achim A

    2016-03-01

    Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100. PMID:26894771

  17. Orostachys japonicus Suppresses Osteoclast Differentiation by Inhibiting NFATc1 Expression.

    PubMed

    Shim, Ki-Shuk; Ha, Hyunil; Kim, Taesoo; Lee, Chung-Jo; Ma, Jin Yeul

    2015-01-01

    The herb Orostachys japonicus has been traditionally used to treat chronic diseases, such as hepatitis, hemorrhoids, and cancer, in Asia. In this study, we investigated the effect of Orostachys japonicus water extract (OJWE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone loss. We found that OJWE inhibited RANKL-induced osteoclast differentiation in a dose-dependent manner without affecting bone resorption in bone marrow-derived macrophage cells. Interestingly, OJWE significantly reduced serum levels of C-terminal telopeptide of type 1 collagen and tartrate-resistant acid phosphatase (TRAP) 5b, markers of bone resorption and osteoclast number, respectively, in an animal model of bone loss. Furthermore, OJWE suppressed the RANKL-induced up-regulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression, and activation of the p38 signaling pathway, but prevented the RANKL-mediated down-regulation of interferon regulatory factor-8 (IRF-8), which is known to be an anti-osteoclastogenic factor that represses NFATc1 expression. We also identified gallic acid and quercetin-3-O-β-D-glucoside as the OJWE components that inhibit RANKL-induced osteoclast differentiation. These results suggest that OJWE inhibits osteoclast differentiation by inhibiting RANKL-induced NFATc1 expression, which prevents osteoclast differentiation and bone loss. The present study elucidated a mechanism of action underlying the inhibitory effect of OJWE on osteoclast differentiation. Our findings suggest that O. japonicus has therapeutic potential for use in the treatment of bone diseases. PMID:26205967

  18. Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes.

    PubMed

    Pan, Hongmiao; Xu, Joshua; Kweon, Oh-Gew; Zou, Wen; Feng, Jinhui; He, Gui-Xin; Cerniglia, Carl E; Chen, Huizhong

    2015-05-01

    We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes. PMID:25720844

  19. [Differential gene expression in the jellyfish Aurelia aurita].

    PubMed

    Matveev, I V

    2005-01-01

    The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium. PMID:16706147

  20. Quantitative comparison of the expression of myogenic regulatory factors in flounder ( Paralichthys olivaceus) embryos and adult tissues

    NASA Astrophysics Data System (ADS)

    Zhang, Yuqing; Tan, Xungang; Xu, Peng; Sun, Wei; Xu, Yongli; Zhang, Peijun

    2010-03-01

    MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions of Myf5, yoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage.

  1. Identification of differentially expressed genes in rat aortic allograft vasculopathy.

    PubMed Central

    Chen, J.; Myllärniemi, M.; Akyürek, L. M.; Häyry, P.; Marsden, P. A.; Paul, L. C.

    1996-01-01

    Graft vasculopathy is an important complication of long-surviving organ transplants, but its pathogenesis has remained elusive. We investigated rat aortic transplants with vasculopathy, aortic transplants without vasculopathy, and normal aortas for differentially expressed mRNA transcripts to gain further insight into the molecular mechanisms involved. Aortic transplants were performed in allogeneic or syngeneic recipients followed by removal after 1 or 5 months, RNA isolation, and differential display to identify mRNA transcripts the expression of which was modulated in conjunction with the transplant procedure and the development of vasculopathy. Using 80 random primers, 57 differentially displayed polymerase chain reaction products were identified, 18 of which were found in allografts but not in syngeneic grafts or normal vessels, whereas 15 were expressed in normal vessels and syngeneic grafts but not in allografts. Of the differentially displayed amplicons, 13 were successfully reamplified and used as probes for Northern analysis; differential expression was confirmed in 6 instances. DNA sequence analysis of these PCR products revealed identity with the immunoglobulin J chain in 2 instances, the ferritin heavy chain, a sequence related but not identical with Ras, and an established sequence tag recently isolated from a human fetal heart library; 1 sequence was not related to any known gene. To assess whether differential mRNA expression of the J-chain gene, a gene expressed in cells of B lymphocyte lineage, was associated with infiltration of the graft by B lymphocytes, tissue sections were stained with an antibody against the B cell marker CD45RA. Although the number of CD45RA-positive cells was low, there was a significant increase in the number of CD45RA-positive cells in the adventitia and intima of grafts with vasculopathy. Furthermore, immunostaining with anti-ferritin antiserum confirmed the presence of ferritin-positive cells within the inner layer of

  2. DEEP--a tool for differential expression effector prediction.

    PubMed

    Degenhardt, Jost; Haubrock, Martin; Dönitz, Jürgen; Wingender, Edgar; Crass, Torsten

    2007-07-01

    High-throughput methods for measuring transcript abundance, like SAGE or microarrays, are widely used for determining differences in gene expression between different tissue types, dignities (normal/malignant) or time points. Further analysis of such data frequently aims at the identification of gene interaction networks that form the causal basis for the observed properties of the systems under examination. To this end, it is usually not sufficient to rely on the measured gene expression levels alone; rather, additional biological knowledge has to be taken into account in order to generate useful hypotheses about the molecular mechanism leading to the realization of a certain phenotype. We present a method that combines gene expression data with biological expert knowledge on molecular interaction networks, as described by the TRANSPATH database on signal transduction, to predict additional--and not necessarily differentially expressed--genes or gene products which might participate in processes specific for either of the examined tissues or conditions. In a first step, significance values for over-expression in tissue/condition A or B are assigned to all genes in the expression data set. Genes with a significance value exceeding a certain threshold are used as starting points for the reconstruction of a graph with signaling components as nodes and signaling events as edges. In a subsequent graph traversal process, again starting from the previously identified differentially expressed genes, all encountered nodes 'inherit' all their starting nodes' significance values. In a final step, the graph is visualized, the nodes being colored according to a weighted average of their inherited significance values. Each node's, or sub-network's, predominant color, ranging from green (significant for tissue/condition A) over yellow (not significant for either tissue/condition) to red (significant for tissue/condition B), thus gives an immediate visual clue on which molecules--differentially

  3. Differential Gene Expression and Protein Abundance Evince Ontogenetic Bias toward Castes in a Primitively Eusocial Wasp

    PubMed Central

    Hunt, James H.; Wolschin, Florian; Henshaw, Michael T.; Newman, Thomas C.; Toth, Amy L.; Amdam, Gro V.

    2010-01-01

    Polistes paper wasps are models for understanding conditions that may have characterized the origin of worker and queen castes and, therefore, the origin of paper wasp sociality. Polistes is “primitively eusocial” by virtue of having context-dependent caste determination and no morphological differences between castes. Even so, Polistes colonies have a temporal pattern in which most female larvae reared by the foundress become workers, and most reared by workers become future-reproductive gynes. This pattern is hypothesized to reflect development onto two pathways, which may utilize mechanisms that regulate diapause in other insects. Using expressed sequence tags (ESTs) for Polistes metricus we selected candidate genes differentially expressed in other insects in three categories: 1) diapause vs. non-diapause phenotypes and/or worker vs. queen differentiation, 2) behavioral subcastes of worker honey bees, and 3) no a priori expectation of a role in worker/gyne development. We also used a non-targeted proteomics screen to test for peptide/protein abundance differences that could reflect larval developmental divergence. We found that foundress-reared larvae (putative worker-destined) and worker-reared larvae (putative gyne-destined) differed in quantitative expression of sixteen genes, twelve of which were associated with caste and/or diapause in other insects, and they also differed in abundance of nine peptides/proteins. Some differentially-expressed genes are involved in diapause regulation in other insects, and other differentially-expressed genes and proteins are involved in the insulin signaling pathway, nutrient metabolism, and caste determination in highly social bees. Differential expression of a gene and a peptide encoding hexameric storage proteins is especially noteworthy. Although not conclusive, our results support hypotheses of 1) larval developmental pathway divergence that can lead to caste bias in adults and 2) nutritional differences as the

  4. ATF3 represses PPARγ expression and inhibits adipocyte differentiation

    SciTech Connect

    Jang, Min-Kyung; Jung, Myeong Ho

    2014-11-07

    Highlights: • ATF3 decrease the expression of PPARγ and its target gene in 3T3-L1 adipocytes. • ATF3 represses the promoter activity of PPARγ2 gene. • ATF/CRE (−1537/−1530) is critical for ATF3-mediated downregulation of PPARγ. • ATF3 binds to the promoter region containing the ATF/CRE. • ER stress inhibits adipocyte differentiation through downregulation of PPARγ by ATF3. - Abstract: Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3 in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of −2.6 Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of −2037 bp promoter, whereas it did not affect the activity of −1458 bp promoter, suggesting that ATF3 responsive element is located between the −2037 and −1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5′-TGACGTTT-3′) between −1537 and −1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated

  5. Differential Expression of Motilin Receptor in Various Parts of Gastrointestinal Tract in Dogs

    PubMed Central

    He, Yu; Wang, Hui; Yang, DongYan; Wang, ChengYan; Yang, LanLan; Jin, Chunxiang

    2015-01-01

    Objectives. The presence of motilin receptor in the GI tract of different animal species has been verified. However, the quantitation of motilin receptor expression in different regions of the GI tract remains unclear. The aim of this study was to investigate the expression of motilin receptor in the GI tract and semiquantitatively compare the expression difference in different GI regions in dogs. Methods. Antrum, duodenum, jejunum, ileum, proximal colon, middle colon, and distal colon were obtained from various parts of the GI tract of six sacrificed dogs. The distribution of motilin receptor was determined by immunohistochemistry. The expression levels of motilin receptor mRNA in different regions were measured by RT-PCR. Results. Motilin receptor was expressed throughout the GI tract in dogs. Multiple comparisons of the mean motilin receptor mRNA expression among various regions were significant (P < 0.05). Motilin receptor mRNA was extensively expressed in duodenum, followed by ileum, jejunum, proximal colon, antrum, middle colon, and distal colon. Immunohistochemistry revealed that motilin receptor immunoreactivity was observed only in the enteric nervous system. Conclusion. Motilin receptor is expressed differentially along the GI tract in dogs. The significantly high expression of motilin receptor mRNA is found in the duodenum. PMID:25918525

  6. Screening for Differentially Expressed Proteins Relevant to the Differential Diagnosis of Sarcoidosis and Tuberculosis

    PubMed Central

    Hu, Yang; Wang, Liu-Sheng; Zhou, Ying; Li, Qiu-Hong; Li, Yan; Du, Yu-Kui; He, Xian; Li, Nan; Yin, Zhao-Fang; Wei, Ya-Ru; Weng, Dong; Li, Hui-Ping

    2015-01-01

    Background In this study, we sought to identify differentially expressed proteins in the serum of patients with sarcoidosis or tuberculosis and to evaluate these proteins as markers for the differential diagnosis of sarcoidosis and sputum-negative tuberculosis. Methods Using protein microarrays, we identified 3 proteins exhibiting differential expression between patients with sarcoidosis and tuberculosis. Elevated expression of these proteins was verified using the enzyme-linked immunosorbent assay (ELISA) and was further confirmed by immunohistochemistry. Receiver operating characteristic (ROC) curve, logistic regression analysis, parallel, and serial tests were used to evaluate the diagnostic efficacy of the proteins. Results Intercellular Adhesion Molecule 1(ICAM-1) and leptin were screened for differentially expressed proteins relevant to sarcoidosis and tuberculosis. Using ROC curves, we found that ICAM-1 (cutoff value: 57740 pg/mL) had an area under the curve (AUC), sensitivity, and specificity of 0.718, 62.3%, and 79.5% respectively, while leptin (cutoff value: 1193.186 pg/mL) had an AUC, sensitivity, and specificity of 0.763, 88.3%, and 65.8%, respectively. Logistic regression analysis revealed that the AUC, sensitivity, and specificity of combined leptin and ICAM-1 were 0.787, 89.6%, and 65.8%, respectively, while those of combined leptin, ICAM-1, and body mass index (BMI) were 0.837, 90.9%, and 64.4%, respectively, which had the greatest diagnostic value. Parallel and serial tests indicated that the BMI-leptin parallel with the ICAM-1 serial was the best diagnostic method, achieving a sensitivity and specificity of 86.5% and 73.1%, respectively. Thus, our results identified elevated expression of ICAM-1 and leptin in serum and granulomas of sarcoidosis patients. Conclusions ICAM-1 and leptin were found to be potential markers for the diagnosis of sarcoidosis and differential diagnosis of sarcoidosis and sputum-negative tuberculosis. PMID:26368286

  7. Differentially expressed genes of Chenopodium amaranticolor in response to cymbidium mosaic virus infection.

    PubMed

    Kim, Su Min; Baek, Eseul; Ryu, Ki Hyun; Choi, Sun Hee

    2016-09-01

    Cymbidium mosaic virus (CymMV)-induced expressed sequence tag (EST) clones from Chenopodium amaranticolor were identified. CymMV was mechanically inoculated onto C. amaranticolor, and local lesion symptoms were observed. Inoculated leaves were collected on serial days post inoculation (dpi) to identify activated or suppressed genes. mRNA isolation and suppression subtractive hybridization (SSH) were then performed to identify differentially expressed genes related to the local lesion response. Fifty-three ESTs, including genes related to defense and stress responses (e.g., lipoxygenase, jasmonate-induced protein, and heat shock protein), were generated. In addition, a large proportion of the ESTs were found to be involved in photosynthesis, as determined by their functional categories. Expression levels of several EST genes were observed using quantitative real-time reverse transcription-polymerase chain reaction, and the evaluated genes showed varying levels of expression during the experimental period. In this study, differentially expressed sequences via SSH were identified from CymMV-infected C. amaranticolor, and profiling and annotation were carried out to determine the expression pattern of CymMV and its interaction with C. amaranticolor. PMID:27364083

  8. Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas.

    PubMed

    Manku, Gurpreet; Culty, Martine

    2015-01-01

    Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor (TGCT) formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs. PMID:25677133

  9. Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas

    PubMed Central

    Manku, Gurpreet; Culty, Martine

    2015-01-01

    Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor (TGCT) formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs. PMID:25677133

  10. Differentiation of embryonic stem cells conditionally expressing neurogenin 3.

    PubMed

    Treff, Nathan R; Vincent, Robert K; Budde, Melisa L; Browning, Victoria L; Magliocca, Joseph F; Kapur, Vivek; Odorico, Jon S

    2006-11-01

    Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression. PMID:16809427

  11. Differentially Expressed Genes Associated with Low-Dose Gamma Radiation

    NASA Astrophysics Data System (ADS)

    Hegyesi, Hargita; Sándor, Nikolett; Schilling, Boglárka; Kis, Enikő; Lumniczky, Katalin; Sáfrány, Géza

    We have studied low dose radiation induced gene expression alterations in a primary human fibroblast cell line using Agilent's whole human genome microarray. Cells were irradiated with 60Co γ-rays (0; 0.1; 0.5 Gy) and 2 hours later total cellular RNA was isolated. We observed differential regulation of approximately 300-500 genes represented on the microarray. Of these, 126 were differentially expressed at both doses, among them significant elevation of GDF-15 and KITLG was confirmed by qRT-PCR. Based on the transcriptional studies we selected GDF-15 to assess its role in radiation response, since GDF-15 is one of the p53 gene targets and is believed to participate in mediating p53 activities. First we confirmed gamma-radiation induced dose-dependent changes in GDF-15 expression by qRT-PCR. Next we determined the effect of GDF-15 silencing on radiosensitivity. Four GDF-15 targeting shRNA expressing lentiviral vectors were transfected into immortalized human fibroblast cells. We obtained efficient GDF-15 silencing in one of the four constructs. RNA interference inhibited GDF-15 gene expression and enhanced the radiosensitivity of the cells. Our studies proved that GDF-15 plays an essential role in radiation response and may serve as a promising target in radiation therapy.

  12. Strontium promotes cementoblasts differentiation through inhibiting sclerostin expression in vitro.

    PubMed

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan; Hu, Min

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. PMID:25003114

  13. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro

    PubMed Central

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. PMID:25003114

  14. Reference genes for accessing differential expression among developmental stages and analysis of differential expression of OBP genes in Anastrepha obliqua

    PubMed Central

    Nakamura, Aline Minali; Chahad-Ehlers, Samira; Lima, André Luís A.; Taniguti, Cristiane Hayumi; Sobrinho Jr., Iderval; Torres, Felipe Rafael; de Brito, Reinaldo Alves

    2016-01-01

    The West Indian fruit fly, Anastrepha obliqua, is an important agricultural pest in the New World. The use of pesticide-free methods to control invasive species such as this reinforces the search for genes potentially useful in their genetic control. Therefore, the study of chemosensory proteins involved with a range of responses to the chemical environment will help not only on the understanding of the species biology but may also help the development of environmentally friendly pest control strategies. Here we analyzed the expression patterns of three OBP genes, Obp19d_2, Obp56a and Obp99c, across different phases of A. obliqua development by qPCR. In order to do so, we tested eight and identified three reference genes for data normalization, rpl17, rpl18 and ef1a, which displayed stability for the conditions here tested. All OBPs showed differential expression on adults and some differential expression among adult stages. Obp99c had an almost exclusive expression in males and Obp56a showed high expression in virgin females. Thereby, our results provide relevant data not only for other gene expression studies in this species, as well as for the search of candidate genes that may help in the development of new pest control strategies. PMID:26818909

  15. Differential gene expression in Pycnoporus coccineus during interspecific mycelial interactions with different competitors.

    PubMed

    Arfi, Yonathan; Levasseur, Anthony; Record, Eric

    2013-11-01

    Fungi compete against each other for environmental resources. These interspecific combative interactions encompass a wide range of mechanisms. In this study, we highlight the ability of the white-rot fungus Pycnoporus coccineus to quickly overgrow or replace a wide range of competitor fungi, including the gray-mold fungus Botrytis cinerea and the brown-rot fungus Coniophora puteana. To gain a better understanding of the mechanisms deployed by P. coccineus to compete against other fungi and to assess whether common pathways are used to interact with different competitors, differential gene expression in P. coccineus during cocultivation was assessed by transcriptome sequencing and confirmed by quantitative reverse transcription-PCR analysis of a set of 15 representative genes. Compared with the pure culture, 1,343 transcripts were differentially expressed in the interaction with C. puteana and 4,253 were differentially expressed in the interaction with B. cinerea, but only 197 transcripts were overexpressed in both interactions. Overall, the results suggest that a broad array of functions is necessary for P. coccineus to replace its competitors and that different responses are elicited by the two competitors, although a portion of the mechanism is common to both. However, the functions elicited by the expression of specific transcripts appear to converge toward a limited set of roles, including detoxification of secondary metabolites. PMID:23974131

  16. Biological network module-based model for the analysis of differential expression in shotgun proteomics.

    PubMed

    Xu, Jia; Wang, Lily; Li, Jing

    2014-12-01

    Protein differential expression analysis plays an important role in the understanding of molecular mechanisms as well as the pathogenesis of complex diseases. With the rapid development of mass spectrometry, shotgun proteomics using spectral counts has become a prevailing method for the quantitative analysis of complex protein mixtures. Existing methods in differential proteomics expression typically carry out analysis at the single-protein level. However, it is well-known that proteins interact with each other when they function in biological processes. In this study, focusing on biological network modules, we proposed a negative binomial generalized linear model for differential expression analysis of spectral count data in shotgun proteomics. In order to show the efficacy of the model in protein expression analysis at the level of protein modules, we conducted two simulation studies using synthetic data sets generated from theoretical distribution of count data and a real data set with shuffled counts. Then, we applied our method to a colorectal cancer data set and a nonsmall cell lung cancer data set. When compared with single-protein analysis methods, the results showed that module-based statistical model which takes account of the interactions among proteins led to more effective identification of subtle but coordinated changes at the systems level. PMID:25327611

  17. Differential Gene Expression in Pycnoporus coccineus during Interspecific Mycelial Interactions with Different Competitors

    PubMed Central

    Levasseur, Anthony; Record, Eric

    2013-01-01

    Fungi compete against each other for environmental resources. These interspecific combative interactions encompass a wide range of mechanisms. In this study, we highlight the ability of the white-rot fungus Pycnoporus coccineus to quickly overgrow or replace a wide range of competitor fungi, including the gray-mold fungus Botrytis cinerea and the brown-rot fungus Coniophora puteana. To gain a better understanding of the mechanisms deployed by P. coccineus to compete against other fungi and to assess whether common pathways are used to interact with different competitors, differential gene expression in P. coccineus during cocultivation was assessed by transcriptome sequencing and confirmed by quantitative reverse transcription-PCR analysis of a set of 15 representative genes. Compared with the pure culture, 1,343 transcripts were differentially expressed in the interaction with C. puteana and 4,253 were differentially expressed in the interaction with B. cinerea, but only 197 transcripts were overexpressed in both interactions. Overall, the results suggest that a broad array of functions is necessary for P. coccineus to replace its competitors and that different responses are elicited by the two competitors, although a portion of the mechanism is common to both. However, the functions elicited by the expression of specific transcripts appear to converge toward a limited set of roles, including detoxification of secondary metabolites. PMID:23974131

  18. Differential long non‑coding RNA and mRNA expression in differentiated human glioblastoma stem cells.

    PubMed

    Li, Hao; Li, Haowen; Hao, Yajing; Jiao, Yuming; Li, Zhicen; Yue, Haiyan; Xu, Zhe; Wang, Shuo; Cao, Yong; Zhao, Jizong

    2016-09-01

    Differentiation of glioblastoma stem cells (GSCs) may lead to inhibition of their self‑renewing ability and tumorigenic potential, as well as increasing their sensitivity to treatment. The critical role of long non‑coding RNAs (lncRNAs) in numerous biological processes has been revealed. However, the involvement of lncRNAs in GSC differentiation remains to be elucidated. In the present study, GSCs were isolated from patient samples and differentiation was induced. Using a high‑throughput microarray, the present study identified a profile of 1,545 lncRNAs and 2,729 mRNAs that differed between GSCs and their non‑differentiated counterparts. To ascertain the association between the altered lncRNAs and mRNAs, a co‑expression network was constructed in which 1,087 lncRNAs and 1,928 mRNAs altered upon GSC differentiation formed a total of 19,642 lncRNA‑mRNA pairs. Based on the co‑expression network, the lncRNA functions were additionally predicted by a cis‑ or trans‑ targeting program. Furthermore, three pairs of lncRNAs and their nearby target mRNAs were selected [ENSG00000261924.1‑regulatory associated protein of MTOR complex 1, ENSG00000235427.1‑caveolin 1 and Tax1 binding protein 3 (TAX1BP3)‑purinergic receptor P2X 5 (P2RX5)‑TAX1BP3] and their expression levels were validated by reverse transcription‑quantitative polymerase chain reaction. The altered lncRNAs were also regulated by various pluripotency transcription factors (POU domain, class 3, transcription factor, sex determining region Y‑box 2, spalt‑like transcription factor 2 and oligodendrocyte lineage transcription factor 2). In conclusion, the results of the present study revealed that lncRNAs may function in GSC differentiation by regulating their target mRNAs, and a set of lncRNAs were identified as candidates for further study concerning the future treatment of GSCs. PMID:27432080

  19. [A new micromethod for differential quantitative assay of zeatin and zeatin riboside].

    PubMed

    Blintsov, A N; Gusakovskaia, M A; Ermakov, I P

    2001-01-01

    A new method is proposed for differential quantitative assay of two major endogenous cytokinin forms. It is based on determination of two effective parameters-concentrations of zeatin and zeatin riboside--with the use of appropriate antigens as standards. The method can be used for determining cytokinins in small samples of plant tissues without extract fractionation. This study pioneers in quantitation of changes in the hormonal status of ovules and ovaries of Triticum aestivum L. at early stages of embryogeny. A gradual increase in the content of the active and storage forms of the hormones from the ovary to the ovule was revealed. PMID:11530676

  20. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

    PubMed Central

    Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

    2016-01-01

    Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes. PMID:27383650

  1. Differential effects of detergents on keratinocyte gene expression.

    PubMed

    van Ruissen, F; Le, M; Carroll, J M; van der Valk, P G; Schalkwijk, J

    1998-04-01

    We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on

  2. Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Zhi, J.; Sommerfeldt, D. W.; Rubin, C. T.; Hadjiargyrou, M.

    2001-01-01

    Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

  3. Nonlinear Dependence in the Discovery of Differentially Expressed Genes

    PubMed Central

    Deller, J. R.; Radha, Hayder; McCormick, J. Justin; Wang, Huiyan

    2012-01-01

    Microarray data are used to determine which genes are active in response to a changing cell environment. Genes are “discovered” when they are significantly differentially expressed in the microarray data collected under the differing conditions. In one prevalent approach, all genes are assumed to satisfy a null hypothesis, ℍ0, of no difference in expression. A false discovery (type 1 error) occurs when ℍ0 is incorrectly rejected. The quality of a detection algorithm is assessed by estimating its number of false discoveries, 𝔉. Work involving the second-moment modeling of the z-value histogram (representing gene expression differentials) has shown significantly deleterious effects of intergene expression correlation on the estimate of 𝔉. This paper suggests that nonlinear dependencies could likewise be important. With an applied emphasis, this paper extends the “moment framework” by including third-moment skewness corrections in an estimator of 𝔉. This estimator combines observed correlation (corrected for sampling fluctuations) with the information from easily identifiable null cases. Nonlinear-dependence modeling reduces the estimation error relative to that of linear estimation. Third-moment calculations involve empirical densities of 3 × 3 covariance matrices estimated using very few samples. The principle of entropy maximization is employed to connect estimated moments to 𝔉 inference. Model results are tested with BRCA and HIV data sets and with carefully constructed simulations. PMID:25937940

  4. Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling.

    PubMed

    Palmblad, Magnus; Mills, Davinia J; Bindschedler, Laurence V

    2008-02-01

    We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing. PMID:18189342

  5. Meta-analysis of differentially expressed genes in ankylosing spondylitis.

    PubMed

    Lee, Y H; Song, G G

    2015-01-01

    The purpose of this study was to identify differentially expressed (DE) genes and biological processes associated with changes in gene expression in ankylosing spondylitis (AS). We performed a meta-analysis using the integrative meta-analysis of expression data program on publicly available microarray AS Gene Expression Omnibus (GEO) datasets. We performed Gene Ontology (GO) enrichment analyses and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes. Four GEO datasets, including 31 patients with AS and 39 controls, were available for the meta-analysis. We identified 65 genes across the studies that were consistently DE in patients with AS vs controls (23 upregulated and 42 downregulated). The upregulated gene with the largest effect size (ES; -1.2628, P = 0.020951) was integral membrane protein 2A (ITM2A), which is expressed by CD4+ T cells and plays a role in activation of T cells. The downregulated gene with the largest ES (1.2299, P = 0.040075) was mitochondrial ribosomal protein S11 (MRPS11). The most significant GO enrichment was in the respiratory electron transport chain category (P = 1.67 x 10-9). Therefore, our meta-analysis identified genes that were consistently DE as well as biological pathways associated with gene expression changes in AS. PMID:26125709

  6. Differential co-expression analysis of venous thromboembolism based on gene expression profile data

    PubMed Central

    MING, ZHIBING; DING, WENBIN; YUAN, RUIFAN; JIN, JIE; LI, XIAOQIANG

    2016-01-01

    The aim of the present study was to screen differentially co-expressed genes and the involved transcription factors (TFs) and microRNAs (miRNAs) in venous thromboembolism (VTE). Microarray data of GSE19151 were downloaded from Gene Expression Omnibus, including 70 patients with VTE and 63 healthy controls. Principal component analysis (PCA) was performed using R software. Differential co-expression analysis was performed using R, followed by screening of modules using Cytoscape. Functional annotation was performed using Database for Annotation, Visualization, and Integrated Discovery. Moreover, Fisher test was used to screen key TFs and miRNAs for the modules. PCA revealed the disease and healthy samples could not be distinguished at the gene expression level. A total of 4,796 upregulated differentially co-expressed genes (e.g. zinc finger protein 264, electron-transfer-flavoprotein, beta polypeptide and Janus kinase 2) and 3,629 downregulated differentially co-expressed genes (e.g. adenylate cyclase 7 and single-stranded DNA binding protein 2) were identified, which were further mined to obtain 17 and eight modules separately. Functional annotation revealed that the largest upregulated module was primarily associated with acetylation and the largest downregulated module was mainly involved in mitochondrion. Moreover, 48 TFs and 62 miRNA families were screened for the 17 upregulated modules, such as E2F transcription factor 4, miR-30 and miR-135 regulating the largest module. Conversely, 35 TFs and 18 miRNA families were identified for the 8 downregulated modules, including mitochondrial ribosomal protein S12 and miR-23 regulating the largest module. Differentially co-expressed genes regulated by TFs and miRNAs may jointly contribute to the abnormal acetylation and mitochondrion presentation in the progression of VTE. PMID:27284300

  7. Expression Change of miR-214 and miR-135 during Muscle Differentiation

    PubMed Central

    Honardoost, Maryam; Soleimani, Masoud; Arefian, Ehsan; Sarookhani, Mohammad reza

    2015-01-01

    Objective MicroRNAs (miRNAs) are a class of small non-coding RNAs that play pivotal roles in many biological processes such as regulating skeletal muscle development where alterations in miRNA expression are reported during myogenesis. In this study, we aimed to investigate the impact of predicted miRNAs and their target genes on the myoblast to myocyte differentiation process. Materials and Methods This experimental study was conducted on the C2C12 cell line. Using a bioinformatics approach, miR-214 and miR-135 were selected according to their targets as potential factors in myoblast to myocyte differentiation induced by 3% horse serum. Immunocytochemistry (ICC) was undertaken to confirm the differentiation process and quantitative real-time polymerase chain reaction (PCR) to determine the expression level of miRNAs and their targets. Results During myoblast to myocyte differentiation, miR-214 was significantly down- regulated while miRNA-135, Irs2, Akt2 and Insr were overexpressed during the process. Conclusion miR-214 and miR-135 are potential regulators of myogenesis and are involved in skeletal muscle development through regulating the IRS/PI3K pathway. PMID:26464817

  8. Differential expression of SKALP/Elafin in human epidermal tumors.

    PubMed Central

    Alkemade, H. A.; Molhuizen, H. O.; van Vlijmen-Willems, I. M.; van Haelst, U. J.; Schalkwijk, J.

    1993-01-01

    Recently we described a new epidermal serine proteinase inhibitor, skin-derived antileukoproteinase (SKALP), also known as elafin. SKALP/elafin was found to be absent in normal human epidermis, but can be induced in vitro and in vivo under hyperproliferative conditions. Here we studied the expression of SKALP/elafin in several types of epidermal tumors (basal cell carcinoma, squamous cell carcinoma, Bowen's disease, actinic keratosis, and keratoacanthoma). Using immunohistochemical staining SKALP/elafin appeared to be differentially expressed in these tumors. Functional measurements of anti-proteinase activity, and Western blotting of tumor extracts confirmed our findings at the histological level. In well differentiated squamous cell carcinoma, SKALP/elafin messenger RNA was demonstrated by non-radioactive in situ hybridization. We conclude that SKALP/elafin is a marker for abnormal or disturbed squamous differentiation. A possible role of SKALP/elafin in the control of tumor cell invasion is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8256855

  9. Improved detection of differentially expressed genes in microarray experiments through multiple scanning and image integration

    PubMed Central

    Romualdi, Chiara; Trevisan, Silvia; Celegato, Barbara; Costa, Germano; Lanfranchi, Gerolamo

    2003-01-01

    The variability of results in microarray technology is in part due to the fact that independent scans of a single hybridised microarray give spot images that are not quite the same. To solve this problem and turn it to our advantage, we introduced the approach of multiple scanning and of image integration of microarrays. To this end, we have developed specific software that creates a virtual image that statistically summarises a series of consecutive scans of a microarray. We provide evidence that the use of multiple imaging (i) enhances the detection of differentially expressed genes; (ii) increases the image homogeneity; and (iii) reveals false-positive results such as differentially expressed genes that are detected by a single scan but not confirmed by successive scanning replicates. The increase in the final number of differentially expressed genes detected in a microarray experiment with this approach is remarkable; 50% more for microarrays hybridised with targets labelled by reverse transcriptase, and 200% more for microarrays developed with the tyramide signal amplification (TSA) technique. The results have been confirmed by semi-quantitative RT–PCR tests. PMID:14627839

  10. Identification of microRNAs differentially expressed involved in male flower development.

    PubMed

    Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

    2015-03-01

    Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees. PMID:25576251

  11. Beyond differential expression: the quest for causal mutations and effector molecules

    PubMed Central

    2012-01-01

    High throughput gene expression technologies are a popular choice for researchers seeking molecular or systems-level explanations of biological phenomena. Nevertheless, there has been a groundswell of opinion that these approaches have not lived up to the hype because the interpretation of the data has lagged behind its generation. In our view a major problem has been an over-reliance on isolated lists of differentially expressed (DE) genes which – by simply comparing genes to themselves – have the pitfall of taking molecular information out of context. Numerous scientists have emphasised the need for better context. This can be achieved through holistic measurements of differential connectivity in addition to, or in replacement, of DE. However, many scientists continue to use isolated lists of DE genes as the major source of input data for common readily available analytical tools. Focussing this opinion article on our own research in skeletal muscle, we outline our resolutions to these problems – particularly a universally powerful way of quantifying differential connectivity. With a well designed experiment, it is now possible to use gene expression to identify causal mutations and the other major effector molecules with whom they cooperate, irrespective of whether they themselves are DE. We explain why, for various reasons, no other currently available experimental techniques or quantitative analyses are capable of reaching these conclusions. PMID:22849396

  12. Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells.

    PubMed

    Enomoto, Kei; Watanabe-Susaki, Kanako; Kowno, Megumi; Takada, Hitomi; Intoh, Atsushi; Yamanaka, Yuko; Hirano, Hisashi; Sugino, Hiromu; Asashima, Makoto; Kurisaki, Akira

    2015-01-01

    Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells. PMID:26399336

  13. THY-1 Receptor Expression Differentiates Cardiosphere-Derived Cells with Divergent Cardiogenic Differentiation Potential

    PubMed Central

    Gago-Lopez, Nuria; Awaji, Obinna; Zhang, Yiqiang; Ko, Christopher; Nsair, Ali; Liem, David; Stempien-Otero, April; MacLellan, W. Robb

    2014-01-01

    Summary Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation. PMID:24936447

  14. Differentially expressed genes for aggressive pecking behaviour in laying hens

    PubMed Central

    2009-01-01

    Background Aggressive behaviour is an important aspect in the daily lives of animals living in groups. Aggressive animals have advantages, such as better access to food or territories, and they produce more offspring than low ranking animals. The social hierarchy in chickens is measured using the 'pecking order' concept, which counts the number of aggressive pecks given and received. To date, little is known about the underlying genetics of the 'pecking order'. Results A total of 60 hens from a high feather pecking selection line were divided into three groups: only receivers (R), only peckers (P) and mixed peckers and receivers (P&R). In comparing the R and P groups, we observed that there were 40 differentially expressed genes [false discovery rate (FDR) P < 0.10]. It was not fully clear how the 40 genes regulated aggressive behaviour; however, gene set analysis detected a number of GO identifiers, which were potentially involved in aggressive behavioural processes. These genes code for synaptosomes (GO:0019797), and proteins involved in the regulation of the excitatory postsynaptic membrane potential (GO:0060079), the regulation of the membrane potential (GO:0042391), and glutamate receptor binding (GO:0035254). Conclusion In conclusion, our study provides new insights into which genes are involved in aggressive behaviours in chickens. Pecking and receiving hens exhibited different gene expression profiles in their brains. Following confirmation, the identification of differentially expressed genes may elucidate how the pecking order forms in laying hens at a molecular level. PMID:19925670

  15. Differential Matrix Metalloprotease (MMP) Expression Profiles Found in Aged Gingiva

    PubMed Central

    Kim, Suhee; Ahn, Sun Hee; Lee, Jin-Sil; Song, Ji-Eun; Cho, Sung-Hyun; Jung, Seunggon; Kim, Seon-Kyu; Kim, Seok-Ho; Lee, Kwang-Pyo

    2016-01-01

    The periodontium undergoes age-related cellular and clinical changes, but the involved genes are not yet known. Here, we investigated age-related genetic changes in gingiva at the transcriptomic level. Genes that were differentially expressed between young and old human gingiva were identified by RNA sequencing and verified by real-time PCR. A total of 1939 mRNA transcripts showed significantly differential expression between young and old gingival tissues. Matrix metalloprotease (MMP) regulation was the top pathway involved in gingival aging. MMP3, MMP9, MMP12, and MMP13 were upregulated in old gingival tissues, concomitantly with interleukin-1 beta (IL1B) expression. In vitro experiments using human gingival fibroblasts (hGFs) showed that MMP12 was upregulated in old hGFs compared to young hGFs. Moreover, the MMP3, MMP9 and IL1B levels were more highly stimulated by infection with the oral bacterium, Fusobacterium nucleatum, in old hGFs compared to young hGFs. Collectively, these findings suggest that, in gingiva, the upregulation of MMP12 may be a molecular hallmark of natural aging, while the upregulations of MMP3, MMM9, and IL1B may indicate externally (e.g., infection)-induced aging. These findings contribute to our understanding of the molecular targets involved in gingival aging. PMID:27391467

  16. Changes in differential gene expression during a fatal stroke.

    PubMed

    Stone, Shelley F; Armstrong, Christopher; van Eeden, Pauline E; Arendts, Glenn; Hankey, Graeme J; Brown, Simon G A; Fatovich, Daniel M

    2016-01-01

    We present a young woman (with an identical twin sister) who arrived at the Emergency Department (ED) within 1 hour of her initial stroke symptoms. Previous microarray studies have demonstrated differential expression of multiple genes between stroke patients and healthy controls. However, for many of these studies there is a significant delay between the initial symptoms and collection of blood samples, potentially leaving the important early activators/regulators of the inflammatory response unrecognised. Blood samples were collected from the patient for an analysis of differential gene expression over time during the evolution of a fatal stroke. The time points for blood collection were ED arrival (T0) and 1, 3 and 24 hours post ED arrival (T1, T3 and T24). This was compared to her identical twin and an additional two age and sex-matched healthy controls. When compared to the controls, the patient had 12 mRNA that were significantly upregulated at T0, and no downregulated mRNA (with a cut off fold change value ±1.5). Of the 12 upregulated mRNA at T0, granzyme B demonstrated the most marked upregulation on arrival, with expression steadily declining over time, whereas S100 calcium-binding protein A12 (S100A12) gene expression increased from T0 to T24, remaining >two-fold above that in the healthy controls at T24. Other genes, such as matrix metalloproteinase 9, high mobility group box 2 and interleukin-18 receptor I were not upregulated at T0, but they demonstrated clear upregulation from T1–T3, with gene expression declining by T24. A greater understanding of the underlying immunopathological mechanisms that are involved during the evolution of ischaemic stroke may help to distinguish between patients with stroke and stroke mimics. PMID:27088144

  17. Differentially expressed regulatory genes in honey bee caste development

    NASA Astrophysics Data System (ADS)

    Hepperle, C.; Hartfelder, K.

    2001-03-01

    In the honey bee, an eminently fertile queen with up to 200 ovarioles per ovary monopolizes colony level reproduction. In contrast, worker bees have only few ovarioles and are essentially sterile. This phenotype divergence is a result of caste-specifically modulated juvenile hormone and ecdysteroid titers in larval development. In this study we employed a differential-display reverse transcription (DDRT)-PCR protocol to detect ecdysteroid-regulated gene expression during a critical phase of caste development. We identified a Ftz-F1 homolog and a Cut-like transcript. Ftz-F1 could be a putative element of the metamorphic ecdysone response cascade of bees, whereas Cut-like proteins are described as transcription factors involved in maintaining cellular differentiation states. The downregulation of both factors can be interpreted as steps in the metamorphic degradation of ovarioles in worker-bee ovaries.

  18. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells

    SciTech Connect

    Timper, Katharina; Seboek, Dalma; Eberhardt, Michael; Linscheid, Philippe; Christ-Crain, Mirjam; Keller, Ulrich; Mueller, Beat; Zulewski, Henryk . E-mail: henryk.zulewski@unibas.ch

    2006-03-24

    Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin.

  19. EVI1 and MDS1/EVI1 Expression During Primary Human Hematopoietic Progenitor Cell Differentiation into Various Myeloid Lineages

    PubMed Central

    Steinleitner, Katarina; Rampetsreiter, Paulina; Köffel, Rene; Ramanathan, Gajalakshmi; Mannhalter, Christine; Strobl, Herbert; Wieser, Rotraud

    2012-01-01

    Background and Aim Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in myeloid leukemia. We therefore studied its expression and function in cluster of differentiation 34 positive (CD 34+) primary human hematopoietic progenitor cells. Materials and Methods CD34+ cells were differentiated into various myeloid lineages using appropriate cytokines. EVI1 expression was measured by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) and intranuclear fluorescence activated cell sorting (FACS). Experimental manipulation of EVI1 levels was achieved using retroviral infection. Results EVI1 mRNA and its variant myelodysplastic syndrome 1 (MDS1)/EVI1, which gives rise to a partially antagonistic protein, were detectable in CD34+ cells, but their levels declined rapidly during differentiation into the granulocytic, monocytic, dendritic, erythroid, and megakaryocytic lineages. Similarly, EVI1 protein levels decreased during myeloid differentiation. Attempts to experimentally express EVI1 in CD34+ and U937 cells indicated that ectopic expression of EVI1 may cause growth arrest, apoptosis and/or senescence of human hematopoietic cells. Conclusion EVI1 is expressed in human hematopoietic progenitor cells, but is down-regulated during differentiation. Ectopic expression of EVI1 may activate cellular safeguards against oncogene activation. PMID:23155256

  20. Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation*

    PubMed Central

    Nicholas, Dequina; Tang, Hui; Zhang, Qiongyi; Rudra, Jai; Xu, Feng; Langridge, William; Zhang, Kangling

    2015-01-01

    The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells. PMID:25316709

  1. Quantitative proteomics reveals a role for epigenetic reprogramming during human monocyte differentiation.

    PubMed

    Nicholas, Dequina; Tang, Hui; Zhang, Qiongyi; Rudra, Jai; Xu, Feng; Langridge, William; Zhang, Kangling

    2015-01-01

    The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells. PMID:25316709

  2. Differential expression profiling of serum proteins and metabolites for biomarker discovery

    NASA Astrophysics Data System (ADS)

    Roy, Sushmita Mimi; Anderle, Markus; Lin, Hua; Becker, Christopher H.

    2004-11-01

    A liquid chromatography-mass spectrometry (LC-MS) proteomics and metabolomics platform is presented for quantitative differential expression analysis. Proteome profiles obtained from 1.5 [mu]L of human serum show ~5000 de-isotoped and quantifiable molecular ions. Approximately 1500 metabolites are observed from 100 [mu]L of serum. Quantification is based on reproducible sample preparation and linear signal intensity as a function of concentration. The platform is validated using human serum, but is generally applicable to all biological fluids and tissues. The median coefficient of variation (CV) for ~5000 proteomic and ~1500 metabolomic molecular ions is approximately 25%. For the case of C-reactive protein, results agree with quantification by immunoassay. The independent contributions of two sources of variance, namely sample preparation and LC-MS analysis, are respectively quantified as 20.4 and 15.1% for the proteome, and 19.5 and 13.5% for the metabolome, for median CV values. Furthermore, biological diversity for ~20 healthy individuals is estimated by measuring the variance of ~6500 proteomic and metabolomic molecular ions in sera for each sample; the median CV is 22.3% for the proteome and 16.7% for the metabolome. Finally, quantitative differential expression profiling is applied to a clinical study comparing healthy individuals and rheumatoid arthritis (RA) patients.

  3. Differential Expression of Borrelia burgdorferi Proteins during Growth In Vitro

    PubMed Central

    Ramamoorthy, Ramesh; Philipp, Mario T.

    1998-01-01

    In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165–1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host. PMID:9784512

  4. Differential gene expression analysis of benzo(a)pyrene toxicity in the clam, Ruditapes philippinarum.

    PubMed

    Liu, Tong; Pan, Luqing; Jin, Qian; Cai, Yuefeng

    2015-05-01

    Polycyclic aromatic hydrocarbons (PAHs) are known for their carcinogenic, teratogenic and mutagenic properties. Benzo(a)pyrene (BaP) possesses the greatest carcinogenic potential among the various PAHs. In this study, digital gene expression (DGE) was performed to investigate the digestive gland transcriptome profile of the clam Ruditapes philippinarum exposed to BaP. A total of 10,508,312 and 11,414,297 clean reads were generated respectively, from control and BaP exposure DGE libraries. One hundred and forty-five differentially expressed genes were detected after comparing two libraries with 58 up-regulated and 87 down-regulated genes. GO annotation and KEGG pathway analyses were performed on all genes to understand their biological functions and processes. The results showed that numerous enriched differentially expressed genes are related to growth and development, antioxidant metabolism, apoptosis and detoxification metabolism. Quantitative real-time PCR was performed to verify the expressed genes of DGE. Our results provide evidences that RNA-seq is a powerful tool for toxicology and capable of generating novel and valuable information at the transcriptome level for characterizing deleterious effects caused by BaP. PMID:25686690

  5. Aberrant Expression of Posterior HOX Genes in Well Differentiated Histotypes of Thyroid Cancers

    PubMed Central

    Cantile, Monica; Scognamiglio, Giosuè; La Sala, Lucia; La Mantia, Elvira; Scaramuzza, Veronica; Valentino, Elena; Tatangelo, Fabiana; Losito, Simona; Pezzullo, Luciano; Chiofalo, Maria Grazia; Fulciniti, Franco; Franco, Renato; Botti, Gerardo

    2013-01-01

    Molecular etiology of thyroid cancers has been widely studied, and several molecular alterations have been identified mainly associated with follicular and papillary histotypes. However, the molecular bases of the complex pathogenesis of thyroid carcinomas remain poorly understood. HOX genes regulate normal embryonic development, cell differentiation and other critical processes in eukaryotic cell life. Several studies have shown that HOX genes play a role in neoplastic transformation of several human tissues. In particular, the genes belonging to HOX paralogous group 13 seem to hold a relevant role in both tumor development and progression. We have identified a significant prognostic role of HOX D13 in pancreatic cancer and we have recently showed the strong and progressive over-expression of HOX C13 in melanoma metastases and deregulation of HOX B13 expression in bladder cancers. In this study we have investigated, by immunohistochemisty and quantitative Real Time PCR, the HOX paralogous group 13 genes/proteins expression in thyroid cancer evolution and progression, also evaluating its ability to discriminate between main histotypes. Our results showed an aberrant expression, both at gene and protein level, of all members belonging to paralogous group 13 (HOX A13, HOX B13, HOX C13 and HOX D13) in adenoma, papillary and follicular thyroid cancers samples. The data suggest a potential role of HOX paralogous group 13 genes in pathogenesis and differential diagnosis of thyroid cancers. PMID:24189220

  6. Differentially expressed microRNAs in colorectal cancer metastasis.

    PubMed

    Abba, Mohammed; Benner, Axel; Patil, Nitin; Heil, Oliver; Allgayer, Heike

    2015-12-01

    Tumor metastasis continues to be the most significant contributor to cancer related mortality, and although several studies have examined expression profiles emanating from patients with metastatic disease, very little information is available about signatures that differentiate metastatic lesions from primary tumors and associated normal tissues, largely because such matched tissue sample series are rare. This study was specifically designed to identify the metastasis relevant microRNAs in colorectal cancer and characterize microRNAs that modulate the metastatic phenotype. Here we describe in detail how the data, deposited in the Gene Expression Omnibus (GEO) with the accession number GSE54088, was generated including the basic analysis as contained in the manuscript published in Cancer Research with the PMID 26069251. PMID:26697326

  7. Differentially expressed microRNAs in colorectal cancer metastasis

    PubMed Central

    Abba, Mohammed; Benner, Axel; Patil, Nitin; Heil, Oliver; Allgayer, Heike

    2015-01-01

    Tumor metastasis continues to be the most significant contributor to cancer related mortality, and although several studies have examined expression profiles emanating from patients with metastatic disease, very little information is available about signatures that differentiate metastatic lesions from primary tumors and associated normal tissues, largely because such matched tissue sample series are rare. This study was specifically designed to identify the metastasis relevant microRNAs in colorectal cancer and characterize microRNAs that modulate the metastatic phenotype. Here we describe in detail how the data, deposited in the Gene Expression Omnibus (GEO) with the accession number GSE54088, was generated including the basic analysis as contained in the manuscript published in Cancer Research with the PMID 26069251. PMID:26697326

  8. Utilization of digital differential display to identify differentially expressed genes related to rumen development.

    PubMed

    Kato, Daichi; Suzuki, Yutaka; Haga, Satoshi; So, KyoungHa; Yamauchi, Eri; Nakano, Miwa; Ishizaki, Hiroshi; Choi, Kichoon; Katoh, Kazuo; Roh, Sang-Gun

    2016-04-01

    This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)-based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5-week-old pre-weaning: n = 3; 15-week-old weaned calves: n = 6). Among the 11 genes, only 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), aldo-keto reductase family 1, member C1-like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre- and post-weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both. © 2015 Japanese Society of Animal Science. PMID:26388291

  9. Differential Gene Expression in HIV-Infected Individuals Following ART

    PubMed Central

    Massanella, Marta; Singhania, Akul; Beliakova-Bethell, Nadejda; Pier, Rose; Lada, Steven; White, Cory H.; Pérez-Santiago, Josué; Blanco, Julià; Richman, Douglas D.; Little, Susan J.; Woelk, Christopher H.

    2013-01-01

    Previous studies of the effect of ART on gene expression in HIV-infected individuals have identified small numbers of modulated genes. Since these studies were underpowered or cross-sectional in design, a paired analysis of peripheral blood mononuclear cells (PBMCs), isolated before and after ART, from a robust number of HIV-infected patients (N=32) was performed. Gene expression was assayed by microarray and 4,157 differentially expressed genes (DEGs) were identified following ART using multivariate permutation tests. Pathways and Gene Ontology (GO) terms over-represented for DEGs reflected the transition from a period of active virus replication before ART to one of viral suppression (e.g., repression of JAK-STAT signaling) and possible prolonged drug exposure (e.g. oxidative phosphorylation pathway) following ART. CMYC was the DEG whose product made the greatest number of interactions at the protein level in protein interaction networks (PINs), which has implications for the increased incidence of Hodgkin’s lymphoma (HL) in HIV-infected patients. The differential expression of multiple genes was confirmed by RT-qPCR including well-known drug metabolism genes (e.g., ALOX12 and CYP2S1). Targets not confirmed by RT-qPCR (i.e., GSTM2 and RPL5) were significantly confirmed by droplet digital (ddPCR), which may represent a superior method when confirming DEGs with low fold changes. In conclusion, a paired design revealed that the number of genes modulated following ART was an order of magnitude higher than previously recognized. PMID:23933117

  10. Differential gene expression in HIV-infected individuals following ART.

    PubMed

    Massanella, Marta; Singhania, Akul; Beliakova-Bethell, Nadejda; Pier, Rose; Lada, Steven M; White, Cory H; Pérez-Santiago, Josué; Blanco, Julià; Richman, Douglas D; Little, Susan J; Woelk, Christopher H

    2013-11-01

    Previous studies of the effect of ART on gene expression in HIV-infected individuals have identified small numbers of modulated genes. Since these studies were underpowered or cross-sectional in design, a paired analysis of peripheral blood mononuclear cells (PBMCs), isolated before and after ART, from a robust number of HIV-infected patients (N=32) was performed. Gene expression was assayed by microarray and 4157 differentially expressed genes (DEGs) were identified following ART using multivariate permutation tests. Pathways and gene ontology (GO) terms over-represented for DEGs reflected the transition from a period of active virus replication before ART to one of viral suppression (e.g., repression of JAK-STAT signaling) and possible prolonged drug exposure (e.g., oxidative phosphorylation pathway) following ART. CMYC was the DEG whose product made the greatest number of interactions at the protein level in protein interaction networks (PINs), which has implications for the increased incidence of Hodgkin's lymphoma (HL) in HIV-infected patients. The differential expression of multiple genes was confirmed by RT-qPCR including well-known drug metabolism genes (e.g., ALOX12 and CYP2S1). Targets not confirmed by RT-qPCR (i.e., GSTM2 and RPL5) were significantly confirmed by droplet digital (ddPCR), which may represent a superior method when confirming DEGs with low fold changes. In conclusion, a paired design revealed that the number of genes modulated following ART was an order of magnitude higher than previously recognized. PMID:23933117

  11. Identification of differentially expressed genes between hepatocytes of Landes geese (Anser anser) and Sichuan White geese (Anser cygnoides).

    PubMed

    Pan, Zhixiong; Wang, Jiwen; Han, Chunchun; Zhai, Ningfeng; Lv, Jia; Zhou, Zehui; Tang, Hui; Xiang, Shuxia; Wang, Jing; Li, Liang

    2010-12-01

    In this study, we examined gene expression in order to identify genes that are differentially expressed between the hepatocytes of Sichuan White geese and Landes geese. We hypothesized that such genes may be involved in the different predispositions between these two species to develop hepatic steatosis. RNA was isolated from primary hepatocytes of the two species, and suppression subtractive hybridization was employed to screen for genes that showed differences in mRNA expression. We built and characterized two reciprocal cDNA libraries that were enriched in genes up-regulated in Landes geese or Sichuan White geese. Using dot blot analysis we identified 128 of 600 randomly selected sequences that demonstrated differential expression between the two species. Of these differentially expressed genes, 115 sequences shared high homology with 46 known genes and 13 sequences corresponded to eight novel expressed sequence tags (ESTs). Based on functional clustering, up and down-regulated genes were mostly related to lipid metabolism, nuclear mRNA splicing, enzyme activity and transcription control. The expression of 18 selected clones was further studied by quantitative PCR. The data showed that eight clones similar to the genes ACSL5, CTGF, CIDEA, PPARγ, PCK, GSTS1, RPS4X, and THBS1 had significantly higher expression levels in the hepatocytes of Landes geese. In contrast, seven clones similar to the genes ADH5, YBX1, ASAH1, UCB, AOPVLDL, SCD-1, and ELOVL-6 had significantly higher expression levels in the hepatocytes of Sichuan White geese. PMID:20238173

  12. Identification of differentially expressed genes between developing seeds of different soybean cultivars.

    PubMed

    Lin, Rongshuang; Glazebrook, Jane; Katagiri, Fumiaki; Orf, James H; Gibson, Susan I

    2015-12-01

    Soybean is a major source of protein and oil and a primary feedstock for biodiesel production. Research on soybean seed composition and yield has revealed that protein, oil and yield are controlled quantitatively and quantitative trait loci (QTL) have been identified for each of these traits. However, very limited information is available regarding the genetic mechanisms controlling seed composition and yield. To help address this deficiency, we used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean cultivars, which differ in seed weight, yield, protein content and oil content. A total of 700 probe sets were found to be expressed at significantly different (defined as having an adjusted p-value below or equal to 0.05 and an at least 2-fold difference) levels between the two cultivars at one or more of the three developmental stages and in at least one of the two years assayed. Comparison of data from soybeans collected in two different years revealed that 97 probe sets were expressed at significantly different levels in both years. Functional annotations were assigned to 78% of these 97 probe sets based on the SoyBase Affymetrix™ GeneChip® Soybean Genome Array Annotation. Genes involved in receptor binding/activity and protein binding are overrepresented among the group of 97 probe sets that were differentially expressed in both years assayed. Probe sets involved in growth/development, signal transduction, transcription, defense/stress response and protein and lipid metabolism were also identified among the 97 probe sets and their possible implications in the regulation of agronomic traits are discussed. As the Minsoy and Archer soybean cultivars differ with respect to seed size, yield, protein content and lipid content, some of the differentially expressed probe sets identified in this study may thus play important roles in controlling these traits. Others of these probe

  13. Quantitative Surface-Enhanced Raman for Gene Expression Estimation

    PubMed Central

    Sun, Lan; Irudayaraj, Joseph

    2009-01-01

    We demonstrate for the first time, to our knowledge, a unique gene expression assay by surface-enhanced Raman scattering (SERS) using nonfluorescent Raman labels to quantify gene expression at the resolution of alternative splicing using RNA extracted from cancer cells without any amplification steps. Our approach capitalizes on the inherent plasmon-phonon mode of SERS substrates as a self-referencing standard for the detection and quantification of genetic materials. A strategy integrating S1 nuclease digestion with SERS detection was developed to quantify the expression levels of splice junction Δ(9,10), a segment of the breast cancer susceptibility gene 1 (BRCA1) from MCF-7 and MDA-MB-231 cells. Quantification results were cross-validated using two Raman tags and qualitatively confirmed by RT-PCR. Our methodology based on SERS technology provides reliable gene expression data with high sensitivity, bypassing the intricacies involved in fabricating a consistent SERS substrate. PMID:19486693

  14. Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

    PubMed

    Frara, Nagat; Abdelmagid, Samir M; Sondag, Gregory R; Moussa, Fouad M; Yingling, Vanessa R; Owen, Thomas A; Popoff, Steven N; Barbe, Mary F; Safadi, Fayez F

    2016-01-01

    Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. PMID:25899717

  15. Early Differential Expression of Oncostatin M in Obstructive Nephropathy

    PubMed Central

    Truong, Luan D.; Tawil, Ahmad; Wang, Wansheng; Dawson, Sara; Lan, Hui Y.; Zhang, Ping; Garcia, Gabriela E.; Smith, C. Wayne

    2010-01-01

    Interstitial fibrosis plays a major role in progression of renal diseases. Oncostatin M (OSM) is a cytokine that regulates cell survival, differentiation, and proliferation. Renal tissue from patients with chronic obstructive nephropathy was examined for OSM expression. The elevated levels in diseased human kidneys suggested possible correlation between OSM level and kidney tissue fibrosis. Indeed, unilateral ureteral obstruction (UUO), a model of renal fibrosis, increased OSM and OSM receptor (OSM-R) expression in a time-dependent manner within hours following UUO. In vitro, OSM overexpression in tubular epithelial cells (TECs) resulted in epithelial-myofibroblast transdifferentiation. cDNA microarray technology identified up-regulated expression of immune modulators in obstructed compared with sham-operated kidneys. In vitro, OSM treatment up-regulated CC chemokine ligand CCL7, and CXC chemokine ligand (CXCL)-14 mRNA in kidney fibroblasts. In vivo, treatment of UUO mice with neutralizing anti-OSM antibody decreased renal chemokines expression. In conclusion, OSM is up-regulated in kidney tissue early after urinary obstruction. Therefore, OSM might play an important role in initiation of renal fibrogenesis, possibly by inducing myofibroblast transdifferentiation of TECs as well as leukocyte infiltration. This process may, in turn, contribute in part to progression of obstructive nephropathy and makes OSM a promising therapeutic target in renal fibrosis. PMID:20626292

  16. Differentially Expressed Genes in EEC and LMS Syndromes

    PubMed Central

    Yin, Wei; Song, Yaling; Du, Yangge; Bian, Zhuan

    2015-01-01

    Objectives Ectrodactyly ectodermal dysplasia cleft lip/palate (EEC) syndrome and limb-mammary syndrome (LMS) share a similar phenotype and the same pathogenic gene, which complicates the ability to distinguish between these diagnoses. The current study aims to identify a potential and practical clinical biomarker to distinguish EEC from LMS. Methods Two EEC pedigrees and one LMS pedigree that have been previously reported were reanalyzed. After confirmation of the causative mutations for these new patients, whole-genome expression microarray analysis was performed to assess the molecular genetic changes in these families. Results Five new patients with classic symptoms were reported, and these individuals exhibited the same mutation as their relatives (c.812 G>C; c.611G>A; and c.680G>A). According to the whole genome expression results, the EEC patients exhibited different gene expression characteristics compared with the LMS patients. More than 5,000 genes were differentially expressed (changes >2 or <0.5-fold) among the EEC patients, LMS patients and healthy individuals. The top three altered pathways have been implicated in apoptosis, the hematopoietic cell lineage and the Toll-like receptor signaling pathway. Conclusion Our results provide additional clinical and molecular information regarding EEC and LMS and suggest that peripheral blood cytokines may represent a promising clinical biomarker for the diagnosis of these syndromes. PMID:26075610

  17. Transcription in space--environmental vs. genetic effects on differential immune gene expression.

    PubMed

    Lenz, Tobias L

    2015-09-01

    Understanding how organisms adapt to their local environment is one of the key goals in molecular ecology. Adaptation can be achieved through qualitative changes in the coding sequence and/or quantitative changes in gene expression, where the optimal dosage of a gene's product in a given environment is being selected for. Differences in gene expression among populations inhabiting distinct environments can be suggestive of locally adapted gene regulation and have thus been studied in different species (Whitehead & Crawford ; Hodgins-Davis & Townsend ). However, in contrast to a gene's coding sequence, its expression level at a given point in time may depend on various factors, including the current environment. Although critical for understanding the extent of local adaptation, it is usually difficult to disentangle the heritable differences in gene regulation from environmental effects. In this issue of Molecular Ecology, Stutz et al. () describe an experiment in which they reciprocally transplanted three-spined sticklebacks (Gasterosteus aculeatus) between independent pairs of small and large lakes. Their experimental design allows them to attribute differences in gene expression among sticklebacks either to lake of origin or destination lake. Interestingly, they find that translocated sticklebacks show a pattern of gene expression more similar to individuals from the destination lake than to individuals from the lake of origin, suggesting that expression of the targeted genes is more strongly regulated by environmental effects than by genetics. The environmental effect by itself is not entirely surprising; however, the relative extent of it is. Especially when put in the context of local adaptation and population differentiation, as done here, these findings cast a new light onto the heritability of differential gene expression and specifically its relative importance during population divergence and ultimately ecological speciation. PMID:26374665

  18. Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation

    PubMed Central

    2013-01-01

    Background MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. Results We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. Conclusions Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation

  19. Host differentially expressed genes during association with its defensive endosymbiont.

    PubMed

    Mathew, Meril; Lopanik, Nicole B

    2014-04-01

    Mutualism, a beneficial relationship between two species, often requires intimate interaction between the host and symbiont to establish and maintain the partnership. The colonial marine bryozoan Bugula neritina harbors an as yet uncultured endosymbiont, "Candidatus Endobugula sertula," throughout its life stages. The bacterial symbiont is the putative source of bioactive complex polyketide metabolites, the bryostatins, which chemically defend B. neritina larvae from predation. Despite the presence of "Ca. Endobugula sertula" in all life stages of the host, deterrent bryostatins appear to be concentrated in reproductive portions of the host colony, suggesting an interaction between the two partners to coordinate production and distribution of the metabolites within the colony. In this study, we identified host genes that were differentially expressed in control colonies and in colonies cured of the symbiont. Genes that code for products similar to glycosyl hydrolase family 9 and family 20 proteins, actin, and a Rho-GDP dissociation inhibitor were significantly downregulated (more than twice) in antibiotic-cured non-reproductive zooids compared to control symbiotic ones. Differential expression of these genes leads us to hypothesize that the host B. neritina may regulate the distribution of the symbiont within the colony via mechanisms of biofilm degradation and actin rearrangement, and consequently, influences bryostatin localization to bestow symbiont-associated protection to larvae developing in the reproductive zooids. PMID:24797097

  20. MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells

    PubMed Central

    Ren, Jiaqiang; Jin, Ping; Wang, Ena; Marincola, Francesco M; Stroncek, David F

    2009-01-01

    Background The unique features of human embryonic stem (hES) cells make them the best candidate resource for both cell replacement therapy and development research. However, the molecular mechanisms responsible for the simultaneous maintenance of their self-renewal properties and undifferentiated state remain unclear. Non-coding microRNAs (miRNA) which regulate mRNA cleavage and inhibit encoded protein translation exhibit temporal or tissue-specific expression patterns and they play an important role in development timing. Results In this study, we analyzed miRNA and gene expression profiles among samples from 3 hES cell lines (H9, I6 and BG01v), differentiated embryoid bodies (EB) derived from H9 cells at different time points, and 5 adult cell types including Human Microvascular Endothelial Cells (HMVEC), Human Umbilical Vein Endothelial Cells (HUVEC), Umbilical Artery Smooth Muscle Cells (UASMC), Normal Human Astrocytes (NHA), and Lung Fibroblasts (LFB). This analysis rendered 104 miRNAs and 776 genes differentially expressed among the three cell types. Selected differentially expressed miRNAs and genes were further validated and confirmed by quantitative real-time-PCR (qRT-PCR). Especially, members of the miR-302 cluster on chromosome 4 and miR-520 cluster on chromosome 19 were highly expressed in undifferentiated hES cells. MiRNAs in these two clusters displayed similar expression levels. The members of these two clusters share a consensus 7-mer seed sequence and their targeted genes had overlapping functions. Among the targeted genes, genes with chromatin structure modification function are enriched suggesting a role in the maintenance of chromatin structure. We also found that the expression level of members of the two clusters, miR-520b and miR-302c, were negatively correlated with their targeted genes based on gene expression analysis Conclusion We identified the expression patterns of miRNAs and gene transcripts in the undifferentiation of human embryonic

  1. Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity.

    PubMed

    Pickworth, C L; Loerch, S C; Velleman, S G; Pate, J L; Poole, D H; Fluharty, F L

    2011-02-01

    Genetic regulation of the site of fat deposition is not well defined. The objective of this study was to investigate adipogenic differentiation state-specific gene expression in feedlot cattle (>75% Angus; <25% Simmental parentage) of varying adipose accretion patterns. Four groups of 4 steers were selected via ultrasound for the following adipose tissue characteristics: low subcutaneous-low intramuscular (LSQ-LIM), low subcutaneous-high intramuscular (LSQ-HIM), high subcutaneous-low intramuscular (HSQ-LIM), and high subcutaneous-high intramuscular (HSQ-HIM). Adipose tissue from the subcutaneous (SQ) and intramuscular (IM) depots was collected at slaughter. The relative expression of adipogenic genes was evaluated using quantitative PCR. Data were analyzed using the mixed model of SAS, and gene expression data were analyzed using covariate analysis with ribosomal protein L19 as the covariate. No interactions (P > 0.10) were observed between IM and SQ adipose tissue depots for any of the variables measured. Therefore, only the main effects of high and low accretion within a depot and the effects of depot are reported. Steers with LIM had smaller mean diameter IM adipocytes (P < 0.001) than HIM steers. Steers with HSQ had larger mean diameter SQ adipocytes (P < 0.001) than LSQ. However, there were no differences (P > 0.10) in any of the genes measured due to high or low adipose accretion. Preadipogenic delta-like kinase1 mRNA was greater in the IM than the SQ adipose tissue; conversely, differentiating and adipogenic genes, lipoprotein lipase, PPARγ, fatty acid synthetase, and fatty acid binding protein 4 were greater (P < 0.001) in the SQ than the IM depot. Intramuscular adipocytes were smaller than SQ adipocytes and had greater expression of the preadipogenic gene, indicating that more hyperplasia was occurring. Meanwhile, SQ adipose tissue contained much larger (P < 0.001) adipocytes that had a greater expression (P < 0.001) of differentiating and adipogenic

  2. Quantitative expression of candidate genes affecting eggshell color.

    PubMed

    Zheng, Chuanwei; Li, Zesheng; Yang, Ning; Ning, Zhonghua

    2014-05-01

    There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin-zinc chelate. Protoporphyrin is the main pigment in brown and light-brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate-limiting enzymes for the heme-biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme-responsive gene-1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg-producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high-level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High-level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color. PMID:24612318

  3. Differential expression and interaction of host factors augment HIV-1 gene expression in neonatal mononuclear cells

    SciTech Connect

    Sundaravaradan, Vasudha; Mehta, Roshni; Harris, David T.; Zack, Jerome A.; Ahmad, Nafees

    2010-04-25

    We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

  4. Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells.

    PubMed

    Bua, Gloria; Manaresi, Elisabetta; Bonvicini, Francesca; Gallinella, Giorgio

    2016-01-01

    The pathogenic Parvovirus B19 (B19V) is characterized by a strict adaptation to erythroid progenitor cells (EPCs), a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi) and lower levels at day 18 (+1.5 Log from 2 to 48 hpi). B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18). Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6-15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage. PMID:26845771

  5. Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells

    PubMed Central

    Bua, Gloria; Manaresi, Elisabetta; Bonvicini, Francesca; Gallinella, Giorgio

    2016-01-01

    The pathogenic Parvovirus B19 (B19V) is characterized by a strict adaptation to erythroid progenitor cells (EPCs), a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi) and lower levels at day 18 (+1.5 Log from 2 to 48 hpi). B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18). Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6–15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage. PMID:26845771

  6. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Quinn, Kyle P.; Sridharan, Gautham V.; Hayden, Rebecca S.; Kaplan, David L.; Lee, Kyongbum; Georgakoudi, Irene

    2013-12-01

    The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

  7. Identification of differentially expressed genes in ovaries of chicken attaining sexual maturity at different ages.

    PubMed

    Kang, Li; Zhang, Yujie; Zhang, Ningbo; Zang, Li; Wang, Meng; Cui, Xinxing; Jiang, Yunliang

    2012-03-01

    In poultry as well as in other birds, sexual maturity is one of the important factors influencing female reproduction and egg production. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) differential display approach was used to identify genes related to sexual maturity. Using 54 EcoR I/Mse I selective primer combinations, totally 403 differentially expressed transcript-derived fragments (TDFs) were isolated, 27 of which belong to 25 unigenes. By real-time quantitative PCR (qPCR), the expression pattern of 13 genes was confirmed; among them, four genes including ZNF183 (P < 0.01), KIAA0700, CCT6A, and 23e 15 (P < 0.05) are significantly up-regulated and one gene (Loc418883) is significantly down-regulated (P < 0.01) in sexually mature ovaries compared to immature ones. The mRNA expression dynamics of ZNF183, CCT6A, 23e 15 and Loc418883 were further investigated in ovaries of 70-, 300- and 500-day-old commercial egg-laying hens: the expression level of CCT6A was the highest in 300-day-old hens (P < 0.05), while that of Loc418883 in 500-day-old hens was significantly higher than the other two stages (P < 0.01). The expression levels of ZNF183 and 23e 15 in ovary increase significantly from 70-day-old hens (P < 0.01) and 300-day-old (P < 0.05) to 500-day-old hens, respectively. The consistence of CCT6A expression and egg-laying performance suggests that CCT6A likely plays important role in sexual maturity in hens. PMID:21691707

  8. Differential expression of growth factors at the cellular level in virus-infected brain.

    PubMed

    Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S; Roy, Anirban; Phares, Timothy W; Koprowski, Hilary; Hooper, D Craig

    2003-05-27

    The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

  9. Differential expression of growth factors at the cellular level in virus-infected brain

    PubMed Central

    Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

    2003-01-01

    The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

  10. Identification of differentially expressed subnetworks based on multivariate ANOVA

    PubMed Central

    Hwang, Taeyoung; Park, Taesung

    2009-01-01

    Background Since high-throughput protein-protein interaction (PPI) data has recently become available for humans, there has been a growing interest in combining PPI data with other genome-wide data. In particular, the identification of phenotype-related PPI subnetworks using gene expression data has been of great concern. Successful integration for the identification of significant subnetworks requires the use of a search algorithm with a proper scoring method. Here we propose a multivariate analysis of variance (MANOVA)-based scoring method with a greedy search for identifying differentially expressed PPI subnetworks. Results Given the MANOVA-based scoring method, we performed a greedy search to identify the subnetworks with the maximum scores in the PPI network. Our approach was successfully applied to human microarray datasets. Each identified subnetwork was annotated with the Gene Ontology (GO) term, resulting in the phenotype-related functional pathway or complex. We also compared these results with those of other scoring methods such as t statistic- and mutual information-based scoring methods. The MANOVA-based method produced subnetworks with a larger number of proteins than the other methods. Furthermore, the subnetworks identified by the MANOVA-based method tended to consist of highly correlated proteins. Conclusion This article proposes a MANOVA-based scoring method to combine PPI data with expression data using a greedy search. This method is recommended for the highly sensitive detection of large subnetworks. PMID:19405941

  11. Juvenile hormone and colony conditions differentially influence cytochrome P450 gene expression in the termite Reticulitermes flavipes.

    PubMed

    Zhou, X; Song, C; Grzymala, T L; Oi, F M; Scharf, M E

    2006-12-01

    In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression. PMID:17201768

  12. Computational annotation of genes differentially expressed along olive fruit development

    PubMed Central

    Galla, Giulio; Barcaccia, Gianni; Ramina, Angelo; Collani, Silvio; Alagna, Fiammetta; Baldoni, Luciana; Cultrera, Nicolò GM; Martinelli, Federico; Sebastiani, Luca; Tonutti, Pietro

    2009-01-01

    Background Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software. Results mRNA from fruits of the cv. Leccino sampled at three different stages [i.e., initial fruit set (stage 1), completed pit hardening (stage 2) and veraison (stage 3)] was used for the identification of differentially expressed genes putatively involved in main processes along fruit development. Four subtractive hybridization libraries were constructed: forward and reverse between stage 1 and 2 (libraries A and B), and 2 and 3 (libraries C and D). All sequenced clones (1,132 in total) were analyzed through BlastX against non-redundant NCBI databases and about 60% of them showed similarity to known proteins. A total of 89 out of 642 differentially expressed unique sequences was further investigated by Real-Time PCR, showing a validation of the SSH results as high as 69%. Library-specific cDNA repertories were annotated according to the three main vocabularies of the gene ontology (GO): cellular component, biological process and molecular function. BlastX analysis, GO terms mapping and annotation analysis were performed using the Blast2GO software, a research tool designed with the main purpose of enabling GO based data mining on sequence sets for which no GO annotation is yet available. Bioinformatic analysis pointed out a significantly different distribution of the annotated sequences for each GO category, when comparing the three fruit developmental stages. The olive fruit-specific transcriptome dataset was used to query all

  13. Differential pattern of integrin receptor expression in differentiated and anaplastic thyroid cancer cell lines.

    PubMed

    Hoffmann, S; Maschuw, K; Hassan, I; Reckzeh, B; Wunderlich, A; Lingelbach, S; Zielke, A

    2005-09-01

    Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of

  14. Quantitative PPARγ expression affects the balance between tolerance and immunity.

    PubMed

    Liu, Ya-Hui; Tsai, Yau-Sheng; Lin, Shih-Chieh; Liao, Nan-Shih; Jan, Ming-Shiou; Liang, Chung-Tiang; Hsu, Shih-Wen; Chen, Wen-Chung; Sung, Junne-Ming; Maeda, Nobuyo; Tsai, Pei-Jane

    2016-01-01

    PPARγ modulates energy metabolism and inflammation. However, its specific functions in the balance of immunity in vivo have been explored incompletely. In this study, by the age of 14 mo, Pparg(C/-) mice with PPARγ expression at 25% of the normal level exhibited high autoantibody levels and developed mesangial proliferative glomerulonephritis, which resembled systemic lupus erythematosus (SLE)-like autoimmune disease. These symptoms were preceded by splenomegaly at an early age, which was associated with increases in splenocyte accumulation and B-cell activation but not with relocation of hematopoiesis to the spleen. The mechanism of splenic lymphocyte accumulation involved reduced sphingosine-1-phosphate receptor 1 (S1P1) expression and diminished migration toward S1P in the Pparg(C/-) splenocytes, which impeded lymphocyte egression. Mechanistically, increased Th17 polarization and IL-17 signaling in the Pparg(C/-) CD4(+) T cells contributed to B-cell hyperactivation in the spleen. Finally, the activation of the remaining PPARγ in Pparg(C/-) mice by pioglitazone increased S1P1 levels, reduced the Th17 population in the spleen, and ameliorated splenomegaly. Taken together, our data demonstrated that reduction of Pparg expression in T-helper cells is critical for spontaneous SLE-like autoimmune disease development; we also revealed a novel function of PPARγ in lymphocyte trafficking and cross talk between Th17 and B cells. PMID:27221351

  15. Quantitative PPARγ expression affects the balance between tolerance and immunity

    PubMed Central

    Liu, Ya-Hui; Tsai, Yau-Sheng; Lin, Shih-Chieh; Liao, Nan-Shih; Jan, Ming-Shiou; Liang, Chung-Tiang; Hsu, Shih-Wen; Chen, Wen-Chung; Sung, Junne-Ming; Maeda, Nobuyo; Tsai, Pei-Jane

    2016-01-01

    PPARγ modulates energy metabolism and inflammation. However, its specific functions in the balance of immunity in vivo have been explored incompletely. In this study, by the age of 14 mo, PpargC/− mice with PPARγ expression at 25% of the normal level exhibited high autoantibody levels and developed mesangial proliferative glomerulonephritis, which resembled systemic lupus erythematosus (SLE)-like autoimmune disease. These symptoms were preceded by splenomegaly at an early age, which was associated with increases in splenocyte accumulation and B-cell activation but not with relocation of hematopoiesis to the spleen. The mechanism of splenic lymphocyte accumulation involved reduced sphingosine-1-phosphate receptor 1 (S1P1) expression and diminished migration toward S1P in the PpargC/− splenocytes, which impeded lymphocyte egression. Mechanistically, increased Th17 polarization and IL-17 signaling in the PpargC/− CD4+ T cells contributed to B-cell hyperactivation in the spleen. Finally, the activation of the remaining PPARγ in PpargC/− mice by pioglitazone increased S1P1 levels, reduced the Th17 population in the spleen, and ameliorated splenomegaly. Taken together, our data demonstrated that reduction of Pparg expression in T-helper cells is critical for spontaneous SLE-like autoimmune disease development; we also revealed a novel function of PPARγ in lymphocyte trafficking and cross talk between Th17 and B cells. PMID:27221351

  16. Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

    PubMed Central

    Smalley, Matthew J; Iravani, Marjan; Leao, Maria; Grigoriadis, Anita; Kendrick, Howard; Dexter, Tim; Fenwick, Kerry; Regan, Joseph L; Britt, Kara; McDonald, Sarah; Lord, Christopher J; MacKay, Alan; Ashworth, Alan

    2007-01-01

    Introduction To understand which signalling pathways become deregulated in breast cancer, it is necessary to identify functionally significant gene expression patterns in the stem, progenitor, transit amplifying and differentiated cells of the mammary epithelium. We have previously used the markers 33A10, CD24 and Sca-1 to identify mouse mammary epithelial cell subpopulations. We now investigate the relationship between cells expressing these markers and use gene expression microarray analysis to identify genes differentially expressed in the cell populations. Methods Freshly isolated primary mouse mammary epithelial cells were separated on the basis of staining with the 33A10 antibody and an α-Sca-1 antibody. The populations identified were profiled using gene expression microarray analysis. Gene expression patterns were confirmed on normal mouse and human mammary epithelial subpopulations and were examined in a panel of breast cancer samples and cell lines. Results Analysis of the separated populations demonstrated that Sca-1- 33A10High stained cells were estrogen receptor α (Esr1)- luminal epithelial cells, whereas Sca-1+ 33A10Low/- stained cells were a mix of nonepithelial cells and Esr1+ epithelial cells. Analysis of the gene expression data identified the gene Rgs2 (regulator of G-protein signalling 2) as being highly expressed in the Sca-1- 33A10Low/- population, which included myoepithelial/basal cells. RGS2 has previously been described as a regulator of angiotensin II receptor signalling. Gene expression analysis by quantitative real-time RT-PCR of cells separated on the basis of CD24 and Sca-1 expression confirmed that Rgs2 was more highly expressed in mouse myoepithelial/basal mammary cells than luminal cells. This expression pattern was conserved in normal human breast cells. Functional analysis demonstrated RGS2 to be a modulator of oxytocin receptor signalling. The potential significance of RGS2 expression in breast cancer was demonstrated by semi-quantitative

  17. Differentiating between and Synthesizing Quantitative, Qualitative, and Longitudinal Research on Polarized School Cultures: A Comment on Van Houtte (2006)

    ERIC Educational Resources Information Center

    Abraham, John

    2007-01-01

    Van Houtte provides a valuable large-sample quantitative analysis of differentiation and polarization processes between and within school cultures in Belgium. Such research across 34 secondary schools provides greater confidence that the findings in the ethnographic tradition for the differentiation-polarization theory are not due to…

  18. Race and Older Mothers’ Differentiation: A Sequential Quantitative and Qualitative Analysis

    PubMed Central

    Sechrist, Jori; Suitor, J. Jill; Riffin, Catherine; Taylor-Watson, Kadari; Pillemer, Karl

    2011-01-01

    The goal of this paper is to demonstrate a process by which qualitative and quantitative approaches are combined to reveal patterns in the data that are unlikely to be detected and confirmed by either method alone. Specifically, we take a sequential approach to combining qualitative and quantitative data to explore race differences in how mothers differentiate among their adult children. We began with a standard multivariate analysis examining race differences in mothers’ differentiation among their adult children regarding emotional closeness and confiding. Finding no race differences in this analysis, we conducted an in-depth comparison of the Black and White mothers’ narratives to determine whether there were underlying patterns that we had been unable to detect in our first analysis. Using this method, we found that Black mothers were substantially more likely than White mothers to emphasize interpersonal relationships within the family when describing differences among their children. In our final step, we developed a measure of familism based on the qualitative data and conducted a multivariate analysis to confirm the patterns revealed by the in-depth comparison of the mother’s narratives. We conclude that using such a sequential mixed methods approach to data analysis has the potential to shed new light on complex family relations. PMID:21967639

  19. Population size is weakly related to quantitative genetic variation and trait differentiation in a stream fish.

    PubMed

    Wood, Jacquelyn L A; Tezel, Defne; Joyal, Destin; Fraser, Dylan J

    2015-09-01

    How population size influences quantitative genetic variation and differentiation among natural, fragmented populations remains unresolved. Small, isolated populations might occupy poor quality habitats and lose genetic variation more rapidly due to genetic drift than large populations. Genetic drift might furthermore overcome selection as population size decreases. Collectively, this might result in directional changes in additive genetic variation (VA ) and trait differentiation (QST ) from small to large population size. Alternatively, small populations might exhibit larger variation in VA and QST if habitat fragmentation increases variability in habitat types. We explored these alternatives by investigating VA and QST using nine fragmented populations of brook trout varying 50-fold in census size N (179-8416) and 10-fold in effective number of breeders, Nb (18-135). Across 15 traits, no evidence was found for consistent differences in VA and QST with population size and almost no evidence for increased variability of VA or QST estimates at small population size. This suggests that (i) small populations of some species may retain adaptive potential according to commonly adopted quantitative genetic measures and (ii) populations of varying sizes experience a variety of environmental conditions in nature, however extremely large studies are likely required before any firm conclusions can be made. PMID:26207947

  20. Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology.

    PubMed

    Armenta, Jenny M; Hoeschele, Ina; Lazar, Iulia M

    2009-07-01

    An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated. PMID:19345114

  1. Proteomic analysis of the differentially expressed proteins by airborne nanoparticles.

    PubMed

    Park, Seul Ki; Jeon, Yu Mi; Son, Bu Soon; Youn, Hyung Sun; Lee, Mi Young

    2011-07-01

    Airborne nanoparticles with thermodynamic diameters less than 56 nm (PM(0.056)) were collected using a Moudi cascade impactor, and the differentially expressed proteins upon exposure to the airborne nanoparticles were identified in human bronchial epithelial cells. More than 600 protein spots were detected on the two-dimensional gel, and the identified 13 of these proteins showed notable changes. Nine were up-regulated and four were down-regulated following treatment with the airborne nanoparticles. Notably, malignant transformation-associated multiple forms of keratins, epigenetic regulation-related MBD1-containing chromatin associated factor 2, epithelial malignancy-related vimentin and exocytosis-related annexin A2 were changed upon exposure to airborne nanoparticle PM(0.056). PMID:21491466

  2. Inducible Lentivirus-Mediated Expression of the Oct4 Gene Affects Multilineage Differentiation of Adult Human Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Hao, Qiang; An, Jia-Qiang; Hao, Fei; Yang, Chun; Lu, Tao; Qu, Ting-Yu; Zhao, Li-Ru; Duan, Wei-Ming

    2015-10-01

    The octamer-binding transcription factor 4 (Oct4) gene plays an important role in maintaining the undifferentiated state of embryonic stem cells (ESCs) and reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs). In the present study, we transduced human bone marrow-derived mesenchymal stem cells (hMSCs) using tetracycline-on (Tet-On) lentiviruses carrying human Oct4 to examine the effects of regulated expression of human Oct4 on the proliferation and differentiation of hMSCs. hMSCs were efficiently transduced by Tet-On lentiviruses to express regulated levels of human Oct4 with doxycycline (Dox), as examined by immunofluorescent staining, flow cytometry, and quantitative real time-PCR (qRT-PCR) assays. Ectopic expression of Oct4 in transduced hMSCs increased the ability of colony formation. Continued expression of Oct4 further enhanced adipogenic differentiation of hMSCs, and transient expression of Oct4 sufficiently enhanced osteogenic differentiation of hMSCs. qRT-PCR analysis showed that ectopic expression of Oct4 in transduced hMSCs temporally increased the expression of Sox2 and c-Myc. Interestingly, ectopic expression of Oct4 reduced neuronal differentiation of hMSCs when incubated under neuronal differentiation conditions. Our results suggest that ectopic expression of human Oct4 leads to temporal changes in multilineage differentiation of hMSCs and may inhibit neuronal differentiation of hMSCs. PMID:26230571

  3. Phylogenetic ANOVA: The Expression Variance and Evolution Model for Quantitative Trait Evolution

    PubMed Central

    Nielsen, Rasmus

    2015-01-01

    A number of methods have been developed for modeling the evolution of a quantitative trait on a phylogeny. These methods have received renewed interest in the context of genome-wide studies of gene expression, in which the expression levels of many genes can be modeled as quantitative traits. We here develop a new method for joint analyses of quantitative traits within- and between species, the Expression Variance and Evolution (EVE) model. The model parameterizes the ratio of population to evolutionary expression variance, facilitating a wide variety of analyses, including a test for lineage-specific shifts in expression level, and a phylogenetic ANOVA that can detect genes with increased or decreased ratios of expression divergence to diversity, analogous to the famous Hudson Kreitman Aguadé (HKA) test used to detect selection at the DNA level. We use simulations to explore the properties of these tests under a variety of circumstances and show that the phylogenetic ANOVA is more accurate than the standard ANOVA (no accounting for phylogeny) sometimes used in transcriptomics. We then apply the EVE model to a mammalian phylogeny of 15 species typed for expression levels in liver tissue. We identify genes with high expression divergence between species as candidates for expression level adaptation, and genes with high expression diversity within species as candidates for expression level conservation and/or plasticity. Using the test for lineage-specific expression shifts, we identify several candidate genes for expression level adaptation on the catarrhine and human lineages, including genes putatively related to dietary changes in humans. We compare these results to those reported previously using a model which ignores expression variance within species, uncovering important differences in performance. We demonstrate the necessity for a phylogenetic model in comparative expression studies and show the utility of the EVE model to detect expression divergence

  4. Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

    PubMed Central

    Janot, Mathilde; Audfray, Aymeric; Loriol, Céline; Germot, Agnès; Maftah, Abderrahman; Dupuy, Fabrice

    2009-01-01

    Background Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes. Results Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries), providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation. Conclusion For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis. PMID:19843320

  5. Trace elements as quantitative probes of differentiation processes in planetary interiors

    SciTech Connect

    Drake, M.J.

    1980-02-01

    Abundances of trace elements in extrusive igneous rocks may be used as petrological and geochemical probes of the source regions of the rocks if differentiation processes, partition coefficients, phase equilibria, and initial concentrations in the source region are known. The characteristic trace element signature that each mineral in the source region imparts on the magma forms the conceptual basis for trace element modeling. The task of the trace element geochemist is to solve mathematically the inverse problem. Given trace element abundances in a magma, what is the ode of its source region. The most successful modeling has been performed for small planetary bodies which underwent relatively simple igneous differentiation events. An example is the eucrite parent body, a planet which produced basals at approx. =4.6 Gy. and has been quiescent ever since. This simple differentiation history permits the calculation of its bulk composition (a feldspathic peridotite) and has led to the tentative identification of asteroid 4 Westa as the eucrite parent body. The differentiation of iron meteorite groups in parent body cores is amenable to similar treatment. The 'anomalous' behavior of Cr, suggests that IIIA, B irons and main group pallasites equilibrated with troilite, spinel, ferromagnesian silicates, or some combination thereof. The moon has undergone more complex differentiation, and quantitative geochemical modeling is correspondingly more difficult. Nevertheless, modeling the two-stage evolution of mare basals raises the possibility that the primordial moon did not have chondritic relative abundances of such refractory elements as Ca, Al, U, and the rare-earth elements. The nonchondritic element ratios are characteristic of planetary, not nebular, fractionation processes and are consistent with the derivation of the moon from a precursor planet, possibly the earth.

  6. Differential Expression of miRNA Regulates T Cell Differentiation and Plasticity During Visceral Leishmaniasis Infection.

    PubMed

    Pandey, Rajan Kumar; Sundar, Shyam; Prajapati, Vijay Kumar

    2016-01-01

    Visceral leishmaniasis (VL) is a tropical neglected disease caused by Leishmania donovani, results in significant mortality in the Indian subcontinent. The plasticity of T cell proliferation and differentiation depends on microRNA mediated gene regulation which leads Th1/Th2 or Th17/Treg type of immune response during human VL. This study depicts the identification of target immune signaling molecule and transcription factors, which play a role in T-cell proliferation and differentiation followed by the identification of miRNA controlling their gene expression using three web servers' viz., TargetScan, miRPath and miRDB. This study provides the bioinformatics evidences that seed region present in the miRNAs miR-29-b, miR-29a, have the putative binding site in the 3'-untranslated region (UTR) of TBX21 transcription factor of CD4(+) T helper (Th1), which may suppress the Th1 specific protective immune response. Development of Th2 type specific immune response can be suppressed by binding of miR-135 and miR-126 miRNAs over the 3'-UTR region of GATA-3 transcription factor of Th2 specific CD4(+) T helper cells. MiRNA identified against Th2/Treg immune cells are important and their over expression or administration can be used for developing the Th1/Th17 type of protective immune response during VL infection. This study indicates that miRNAs have the capacity to regulate immune signaling, cytokine production and immune cell migration to control the VL infection in human. This observation warrants further investigation for the development of miRNA based therapy controlling T cell differentiation in human VL. PMID:26941729

  7. Differential Expression of miRNA Regulates T Cell Differentiation and Plasticity During Visceral Leishmaniasis Infection

    PubMed Central

    Pandey, Rajan Kumar; Sundar, Shyam; Prajapati, Vijay Kumar

    2016-01-01

    Visceral leishmaniasis (VL) is a tropical neglected disease caused by Leishmania donovani, results in significant mortality in the Indian subcontinent. The plasticity of T cell proliferation and differentiation depends on microRNA mediated gene regulation which leads Th1/Th2 or Th17/Treg type of immune response during human VL. This study depicts the identification of target immune signaling molecule and transcription factors, which play a role in T-cell proliferation and differentiation followed by the identification of miRNA controlling their gene expression using three web servers’ viz., TargetScan, miRPath and miRDB. This study provides the bioinformatics evidences that seed region present in the miRNAs miR-29-b, miR-29a, have the putative binding site in the 3′-untranslated region (UTR) of TBX21 transcription factor of CD4+ T helper (Th1), which may suppress the Th1 specific protective immune response. Development of Th2 type specific immune response can be suppressed by binding of miR-135 and miR-126 miRNAs over the 3′-UTR region of GATA-3 transcription factor of Th2 specific CD4+ T helper cells. MiRNA identified against Th2/Treg immune cells are important and their over expression or administration can be used for developing the Th1/Th17 type of protective immune response during VL infection. This study indicates that miRNAs have the capacity to regulate immune signaling, cytokine production and immune cell migration to control the VL infection in human. This observation warrants further investigation for the development of miRNA based therapy controlling T cell differentiation in human VL. PMID:26941729

  8. Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus.

    PubMed

    Wang, Dan-Dan; Zhang, Gui-Rong; Wei, Kai-Jian; Ji, Wei; Gardner, Jonathan P A; Yang, Rui-Bin; Chen, Kun-Ci

    2015-12-01

    Channa argus is one of the most commercially important fish species in China. Studies show that males of C. argus grow faster than females at the same age. In order to explore the sex differentiation mechanism of C. argus, we isolated the full length of the sex-related gene Foxl2 cDNA and analysed its expression patterns during gonadal sex differentiation. Alignment of known Foxl2 amino acid sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame, especially the forkhead domain and C-terminal region. Quantitative RT-PCR revealed that Foxl2 is predominantly expressed in brain, pituitary, gill and ovary, with its highest level in ovary but low levels in testis and other tissues, reflecting a potential role for Foxl2 in the brain-pituitary-gonad axis in C. argus. Our ontogenetic stage data showed that C. argus Foxl2 expression was significantly upregulated from 1 to 11 days posthatching (dph) and that the initiation of expression preceded the first anatomical ovarian differentiation (27 dph), suggesting that Foxl2 might play a potential role in early gonadal sex differentiation in C. argus. In addition, the Foxl2 protein was primarily located in granulosa cells surrounding the oocytes of mature C. argus, implying that Foxl2 may have a basic function in granulosa cell differentiation and the maintenance of oocytes. PMID:26159319

  9. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70.

    PubMed

    Lafarge, Sandrine T; Hou, Sen; Pauls, Samantha D; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2015-07-01

    Chronic lymphocytic leukemia is a malignancy driven by abberant B cell signaling and survival. Leukemic B cells accumulate in the peripheral blood and the lymphoid organs where contact with stromal cells and T cells provide critical survival signals. Clinical severity of CLL is associated with several prognostic markers including expression of the kinase ZAP-70. ZAP-70 expression enhances signaling via the B cell antigen receptor and is associated with increased cell adhesion and migration capacity. Here we report that ZAP-70-positive CLL patients display significantly higher expression of the TNF superfamily receptor and memory marker CD27 than do ZAP-70 negative patients. CD27 expression by CLL was acutely elevated upon BCR cross-linking, or upon ectopic expression of ZAP-70. CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma. These results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment. PMID:26002513

  10. Construction and Quantitative Validation of Chicken CXCR4 Expression Reporter.

    PubMed

    Es-Haghi, Masoumeh; Bassami, Mohammadreza; Dehghani, Hesam

    2016-03-01

    Site directional migration is an important biological event and an essential behavior for latent migratory cells. A migratory cell maintains its motility, survival, and proliferation abilities by a network of signaling pathways where CXCR4/SDF signaling route plays crucial role for directed homing of a polarized cell. The chicken embryo due to its specific vasculature modality has been used as a valuable model for organogenesis, migration, cancer, and metastasis. In this research, the regulatory regions of chicken CXCR4 gene have been characterized in a chicken hematopoietic lymphoblast cell line (MSB1). A region extending from -2000 bp upstream of CXCR4 gene to +68 after its transcriptional start site, in addition to two other mutant fragments were constructed and cloned in a promoter-less reporter vector. Promoter activity was analyzed by quantitative real-time RT-PCR and flow cytometry techniques. Our findings show that the full sequence from -2000 to +68 bp of CXCR4 regulatory region is required for maximum promoter functionality, while the mutant CXCR4 promoter fragments show a partial promoter activity. The chicken CXCR4 promoter validated in this study could be used for characterization of directed migratory cells in chicken development and disease models. PMID:26809356

  11. Parameterized algorithms for quantitative differentials in spectrally equivalent medical diagnostic x-ray beams.

    PubMed

    Okunade, Akintunde Akangbe

    2005-06-01

    Qualitative and quantitative equivalence of spectra transmitted by two different elemental filters require a good match in terms of shape and size over the entire energy range of 0-150 keV used in medical diagnostic radiology. However, the photoelectric absorptions and Compton scattering involved in the interaction of x rays with matter at these relatively low photon energies differ in a nonuniform manner with energy and atomic number. By careful choice of thicknesses for filter materials with an atomic number between 12 and 39, when compared with aluminum, it is possible to obtain transmitted beams of the same shape (quality) but not of the same size (quantity). In this paper, calculations have been carried out for the matching of the shapes and sizes of beams transmitted through specified thicknesses of aluminium filter and spectrally equivalent thicknesses of other filter materials (different from aluminium) using FORTRAN source codes traceable to the American Association of Physics in Medicine (AAPM), College Park, MD, USA. Parametrized algorithms for the evaluation of quantitative differentials (deficit or surplus) in radiation output (namely, photon fluence, exposure, kerma, energy imparted, absorbed dose, and effective dose) from these transmitted spectrally equivalent beams were developed. These differentials range between 1%, and 4% at 1 mm Al filtration and between 8%, and 25% for filtration of 6 mm Al for different filter materials in comparison with aluminum. Also developed were models for factors for converting measures of photon fluence, exposure-area product, (EAP), and kerma-area product (KAP) to risk related quantities such as energy imparted, absorbed dose, and effective dose from the spectrally equivalent beams. The thicknesses of other filter materials that are spectrally equivalent to given thicknesses of aluminum filter were characterized using polynomial functions. The fact that the use of equivalent spectra in radiological practice can

  12. Parameterized algorithms for quantitative differentials in spectrally equivalent medical diagnostic x-ray beams

    SciTech Connect

    Okunade, Akintunde Akangbe

    2005-06-15

    Qualitative and quantitative equivalence of spectra transmitted by two different elemental filters require a good match in terms of shape and size over the entire energy range of 0-150 keV used in medical diagnostic radiology. However, the photoelectric absorptions and Compton scattering involved in the interaction of x rays with matter at these relatively low photon energies differ in a nonuniform manner with energy and atomic number. By careful choice of thicknesses for filter materials with an atomic number between 12 and 39, when compared with aluminum, it is possible to obtain transmitted beams of the same shape (quality) but not of the same size (quantity). In this paper, calculations have been carried out for the matching of the shapes and sizes of beams transmitted through specified thicknesses of aluminium filter and spectrally equivalent thicknesses of other filter materials (different from aluminium) using FORTRAN source codes traceable to the American Association of Physics in Medicine (AAPM), College Park, MD, USA. Parametrized algorithms for the evaluation of quantitative differentials (deficit or surplus) in radiation output (namely, photon fluence, exposure, kerma, energy imparted, absorbed dose, and effective dose) from these transmitted spectrally equivalent beams were developed. These differentials range between 1%, and 4% at 1 mm Al filtration and between 8%, and 25% for filtration of 6 mm Al for different filter materials in comparison with aluminum. Also developed were models for factors for converting measures of photon fluence, exposure-area product, (EAP), and kerma-area product (KAP) to risk related quantities such as energy imparted, absorbed dose, and effective dose from the spectrally equivalent beams. The thicknesses of other filter materials that are spectrally equivalent to given thicknesses of aluminum filter were characterized using polynomial functions. The fact that the use of equivalent spectra in radiological practice can

  13. Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling.

    PubMed

    Liu, Ming; Zhu, Jin-Gai; Yu, Zhang-Bin; Song, Gui-Xian; Shen, Ya-Hui; Liu, Yao-Qiu; Zhu, Chun; Qian, Ling-Mei

    2014-06-01

    Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration. PMID:24699800

  14. Differential Expression of Claudin Family Members during Osteoblast and Osteoclast Differentiation: Cldn-1 Is a Novel Positive Regulator of Osteoblastogenesis

    PubMed Central

    Alshbool, Fatima Z.; Mohan, Subburaman

    2014-01-01

    Claudins (Cldns), a family of 27 transmembrane proteins, represent major components of tight junctions. Aside from functioning as tight junctions, Cldns have emerging roles as regulators of cell proliferation and differentiation. While Cldns are known to be expressed and have important functions in various tissues, their expression and function in bone cells is ill-defined. In this study, the expression of Cldns was examined during osteoblast and osteoclast differentiation. The expression of Cldn-1, -7, -11, and -15 was downregulated during early stages of osteoclast differentiation, whereas Cldn-6 was upregulated. Moreover, the expression of several Cldns increased 3–7 fold in fully differentiated osteoclasts. As for osteoblasts, the expression of several Cldns was found to increase more than 10-fold during differentiation, with some peaking at early, and others at late stages. By contrast, only expression of Cldn-12, and -15 decreased during osteoblast differentiation. In subsequent studies, we focused on the role of Cldn-1 in osteoblasts as its expression was increased by more than 10 fold during osteoblast differentiation and was found to be regulated by multiple osteoregulatory agents including IGF-1 and Wnt3a. We evaluated the consequence of lentiviral shRNA-mediated knockdown of Cldn-1 on osteoblast proliferation and differentiation using MC3T3-E1 mouse osteoblasts. Cldn-1 knockdown caused a significant reduction in MC3T3-E1 cell proliferation and ALP activity. Accordingly, expression levels of cyclinD1 and ALP mRNA levels were reduced in Cldn-1 shRNA knockdown cells. We next determined if Cldn-1 regulates the expression of Runx-2 and osterix, master transcription factors of osteoblast differentiation, and found that their levels were reduced significantly as a consequence of Cldn-1 knockdown. Moreover, knocking down Cldn-1 reduced β-catenin level. In conclusion, the expression of Cldn family members during bone cell differentiation is complex and

  15. Gene expression profiling reveals differentially expressed genes in ovarian cancer of the hen: support for oviductal origin?

    PubMed

    Treviño, Lindsey S; Giles, James R; Wang, Wei; Urick, Mary Ellen; Johnson, Patricia Ann

    2010-08-01

    Ovarian cancer has a high mortality rate due, in part, to the lack of early detection and incomplete understanding of the origin of the disease. The hen is the only spontaneous model of ovarian cancer and can therefore aid in the identification and testing of early detection strategies and therapeutics. Our aim was to combine the use of the hen animal model and microarray technology to identify differentially expressed genes in ovarian tissue from normal hens compared with hens with ovarian cancer. We found that the transcripts up-regulated in chicken ovarian tumors were enriched for oviduct-related genes. Quantitative real-time PCR and immunohistochemistry confirmed expression of oviduct-related genes in normal oviduct and in ovaries from hens with early- and late-stage ovarian tumors, but not in normal ovarian surface epithelium. In addition, one of the oviduct-related genes identified in our analysis, paired box 2 has been implicated in human ovarian cancer and may serve as a marker of the disease. Furthermore, estrogen receptor 1 mRNA is over-expressed in early-stage tumors, suggesting that expression of the oviduct-related genes may be regulated by estrogen. We have also identified oviduct-related genes that encode secreted proteins that could represent putative serum biomarkers. The expression of oviduct-related genes in early-stage tumors is similar to what is seen in human ovarian cancer, with tumors resembling normal Müllerian epithelium. These data suggest that chicken ovarian tumors may arise from alternative sites, including the oviduct. PMID:21761365

  16. Quantitative bloodstain analysis: differentiation of contact transfer patterns versus spatter patterns on fabric via microscopic inspection.

    PubMed

    Cho, Yuen; Springer, Faye; Tulleners, Frederic A; Ristenpart, William D

    2015-04-01

    In crime scene reconstruction, it is often necessary to differentiate "contact transfer" and "spatter" bloodstain patterns found on clothing. Current methodologies, however, are qualitative and prone to context bias. In this work, we demonstrate that microscopic inspection of the stain orientations provides a quantitative differentiation of bloodstains resulting from spatter versus contact transfer. Specifically, common knitted fabrics are comprised of parallel rows of left loop legs, in an upward diagonal orientation (/), and right loop legs in a downward diagonal orientation (\\). Our microscopic examination of more than 65,000 individual stained loop legs shows that spatter stains are approximately evenly distributed between left and right loop legs, but contact transfer stains are unevenly distributed: depending on the type of surface contacted, as many as 82% of the stains were preferentially located on the left loop legs. We further show that in these fabrics the left loop legs protrude further out than the right loop legs by approximately 50 μm, indicating that the observation of left loop legs preferentially stained over right loop legs is associated with the topography of the fabric. These findings suggest that microscopic quantification of the relative loop leg stain distributions could provide an objective means of differentiating contact transfer versus spatter patterns in crime scene reconstruction. PMID:25723999

  17. Novel differential transcript expression identified by LongSAGE in the mouse endometrium during the implantation window.

    PubMed

    Ding, Yu-Bin; He, Jun-Lin; Chen, Xue-Mei; Liu, Xue-Qing; Wang, Ying-Xiong

    2013-01-01

    Full development of a receptive uterus is necessary for embryo implantation; however, many genes that are required for the endometrial modifications that occur during this process remain unidentified. To identify novel genes that control endometrial modifications during this period, we investigated the differential gene expression profile in the endometrium of mice on days 2 (D2) (pre-implantation) and 4 (D4) of pregnancy (i.e., the implantation window) using 17-bp long serial analysis of gene expression (LongSAGE). One hundred fifty-six tags were annotated as unique transcripts. Of these, 101 tags were significantly upregulated, and 55 tags were downregulated in the D4 library relative to the D2 library. These differentially expressed genes should therefore be of increased importance in the establishment of uterine receptivity. The differential expressions of certain of the identified genes, namely, Hspa8, Tctp, Sparc, Ifitm1, Ik, serbp1 and Dnmt1, were validated by semi-quantitative RT-PCR and/or immunohistochemistry. Functional grouping analysis classified 86 of the mapped tags into 17 categories, which are closely associated with morphological modifications of the endometrium during pregnancy. Ingenuity pathways analysis revealed that the identified differentially expressed genes fell into six primary networks, which themselves contain numerous factors that are related to key modulators of signaling pathways that are vital for endometrial modifications. These findings will aid in the further understanding of the molecular events that underlie the implantation physiology in mice. PMID:23065227

  18. Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

    PubMed Central

    Jokinen, Riikka; Lahtinen, Taina; Marttinen, Paula; Myöhänen, Maarit; Ruotsalainen, Pilvi; Yeung, Nicolas; Shvetsova, Antonina; Kastaniotis, Alexander J.; Hiltunen, J. Kalervo; Öhman, Tiina; Nyman, Tuula A.; Weiler, Hartmut; Battersby, Brendan J.

    2015-01-01

    Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment. PMID:25808953

  19. PTHrP in differentiating human mesenchymal stem cells: transcript isoform expression, promoter methylation, and protein accumulation.

    PubMed

    Longo, Alessandra; Librizzi, Mariangela; Naselli, Flores; Caradonna, Fabio; Tobiasch, Edda; Luparello, Claudio

    2013-10-01

    Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation. PMID:23810909

  20. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  1. Differential protein expression in the estuarine copepod Eurytemora affinis after diuron and alkylphenol exposures.

    PubMed

    Boulangé-Lecomte, Céline; Rocher, Béatrice; Cailleaud, Kévin; Cosette, Pascal; Legrand, Eléna; Devreker, David; Budzinski, Hélène; Souissi, Sami; Forget-Leray, Joëlle

    2016-07-01

    Proteomics was used in the calanoid copepod Eurytemora affinis for screening of protein expression modifications induced by organic contaminants. The copepods were exposed in a continuous flow-through system for 86 h to environmentally relevant concentrations of contaminants representative of the pollution in the Seine Estuary (Haute-Normandie, France; diuron, 500 ng L(-1) ; alkylphenol mixture, 1000 ng L(-1) ). Proteome analysis of whole-body copepod extracts by 2-dimensional gel electrophoresis revealed that the contaminants induced modifications in protein expression, with the highest quantitative variations occurring after diuron exposure. Specifically, 88 and 41 proteins were differentially expressed after diuron and alkylphenol treatments, respectively. After mass spectrometry analysis, 51 (diuron exposure) and 15 (alkylphenol exposure) proteins were identified. The identified proteins were potentially related to energy metabolism, cell growth, nervous signal conductivity, excitotoxicity, oxidative stress response, and antioxidant defense. The data suggest a massive general disturbance of physiological functions of E. affinis after diuron exposure, whereas alkylphenols induced an alteration of a few targeted physiological functions. The protein expression signatures identified after contaminant exposure deserve further investigation in terms of the development of novel potential biomarkers for water quality assessment. Environ Toxicol Chem 2016;35:1860-1871. © 2015 SETAC. PMID:26677818

  2. Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development.

    PubMed

    Guo, D; Li, H L; Tang, X; Peng, S Q

    2014-01-01

    In plants, homeodomain proteins play a critical role in regulating various aspects of plant growth and development. KNOX proteins are members of the homeodomain protein family. The KNOX transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of cassava (Manihot esculenta Krantz) has allowed a genome-wide search for M. esculenta KNOX (MeKNOX) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. In the present study, we identified 12 MeKNOX genes in the cassava genome and grouped them into two distinct subfamilies based on their domain composition and phylogenetic analysis. Furthermore, semi-quantitative reverse transcription polymerase chain reaction analysis was performed to elucidate the expression profiles of these genes in different tissues and during various stages of root development. The analysis of MeKNOX expression profiles of indicated that 12 MeKNOX genes display differential expressions either in their transcript abundance or expression patterns. PMID:25526192

  3. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) expression is downregulated in poorly differentiated breast invasive ductal carcinoma.

    PubMed

    Cejas, P; García-Cabezas, M A; Casado, E; Belda-Iniesta, C; De Castro, J; Fresno, J A; Sereno, M; Barriuso, J; Espinosa, E; Zamora, P; Feliu, J; Redondo, A; Hardisson, D A; Renart, J; González-Barón, M

    2007-06-01

    Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) is the only known enzyme able to reduce lipid peroxides bound to cell membranes. Moreover it has been involved in apoptosis and can influence intracellular signaling. To investigate the possible relationship between PHGPx and human cancer we have quantified PHGPx expression levels by real-time quantitative PCR and immunohistochemistry in tissue samples of human breast invasive ductal carcinoma from 34 patients compared with their own controls of benign breast tissue. PHGPx expression levels were compared with the clinical and pathological data of these patients. The results showed that PHGPx expression levels are downregulated in poorly differentiated (grade 3) breast invasive ductal carcinoma (P = 0.0043). PHGPx expression levels decreased gradually with tumor grade from grade 1 to grade 3. We also found a downregulation of PHGPx in cases that showed p53 accumulation compared with cases without p53 immunostaining (P = 0.0011). PHGPx was also downregulated in cases without progesterone receptors (PR) immunostaining compared with cases with PR immunostaining (P = 0.0165). Grade 3, p53 immunostaining and absence of PR immunostaining are poor prognostic factors. These results suggest that PHGPx downregulation could be related with a poorer prognosis in breast invasive ductal carcinoma. PMID:17516241

  4. Transcriptome sequencing of purple petal spot region in tree peony reveals differentially expressed anthocyanin structural genes

    PubMed Central

    Zhang, Yanzhao; Cheng, Yanwei; Ya, Huiyuan; Xu, Shuzhen; Han, Jianming

    2015-01-01

    The pigmented cells in defined region of a petal constitute the petal spots. Petal spots attract pollinators and are found in many angiosperm families. Several cultivars of tree peony contain a single red or purple spot at the base of petal that makes the flower more attractive for the ornamental market. So far, the understanding of the molecular mechanism of spot formation is inadequate. In this study, we sequenced the transcriptome of the purple spot and the white non-spot of tree peony flower. We assembled and annotated 67,892 unigenes. Comparative analyses of the two transcriptomes showed 1,573 differentially expressed genes, among which 933 were up-regulated, and 640 were down-regulated in the purple spot. Subsequently, we examined four anthocyanin structural genes, including PsCHS, PsF3′H, PsDFR, and PsANS, which expressed at a significantly higher level in the purple spot than in the white non-spot. We further validated the digital expression data using quantitative real-time PCR. Our result uncovered transcriptome variance between the spot and non-spot of tree peony flower, and revealed that the co-expression of four anthocyanin structural genes was responsible for spot pigment in tree peony. The data will further help to unravel the genetic mechanism of peony flower spot formation. PMID:26583029

  5. Differential response to bacteria, and TOLLIP expression, in the human respiratory tract

    PubMed Central

    Moncayo-Nieto, Olga Lucia; Wilkinson, Thomas S; Brittan, Mairi; McHugh, Brian J; Jones, Richard O; Conway Morris, Andrew; Walker, William S; Davidson, Donald J; Simpson, A John

    2014-01-01

    Objectives The observation that pathogenic bacteria are commonly tolerated in the human nose, yet drive florid inflammation in the lung, is poorly understood, partly due to limited availability of primary human cells from each location. We compared responses to bacterial virulence factors in primary human nasal and alveolar cells, and characterised the distribution of Toll-interacting protein (TOLLIP; an inhibitor of Toll-like receptor (TLR) signalling) in the human respiratory tract. Methods Primary cells were isolated from nasal brushings and lung tissue taken from patients undergoing pulmonary resection. Cells were exposed to lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-C DNA or tumour necrosis factor (TNF). Cytokines were measured in cell supernatants. TOLLIP was characterised using quantitative real-time PCR and immunofluorescence. Results In primary alveolar, but not primary nasal, cells peptidoglycan significantly increased secretion of interleukin (IL)-1β, IL-6, IL-8, IL-10 and TNF. TLR2 expression was significantly higher in alveolar cells and correlated with IL-8 production. TOLLIP expression was significantly greater in nasal cells. Conclusion In conclusion, primary human alveolar epithelial cells are significantly more responsive to peptidoglycan than primary nasal epithelial cells. This may partly be explained by differential TLR2 expression. TOLLIP is expressed widely in the human respiratory tract, and may contribute to the regulation of inflammatory responses. PMID:25478190

  6. Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

    PubMed Central

    Daigo, Yataro; Takayama, Ichiro; Ponder, Bruce AJ; Caldas, Carlos; Ward, Sean M; Sanders, Kenton M; Fujino, Masayuki A

    2003-01-01

    Background The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. Method The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. Results Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. Conclusions These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission. PMID:12795813

  7. Quantitative assessment of gene expression network module-validation methods.

    PubMed

    Li, Bing; Zhang, Yingying; Yu, Yanan; Wang, Pengqian; Wang, Yongcheng; Wang, Zhong; Wang, Yongyan

    2015-01-01

    Validation of pluripotent modules in diverse networks holds enormous potential for systems biology and network pharmacology. An arising challenge is how to assess the accuracy of discovering all potential modules from multi-omic networks and validating their architectural characteristics based on innovative computational methods beyond function enrichment and biological validation. To display the framework progress in this domain, we systematically divided the existing Computational Validation Approaches based on Modular Architecture (CVAMA) into topology-based approaches (TBA) and statistics-based approaches (SBA). We compared the available module validation methods based on 11 gene expression datasets, and partially consistent results in the form of homogeneous models were obtained with each individual approach, whereas discrepant contradictory results were found between TBA and SBA. The TBA of the Zsummary value had a higher Validation Success Ratio (VSR) (51%) and a higher Fluctuation Ratio (FR) (80.92%), whereas the SBA of the approximately unbiased (AU) p-value had a lower VSR (12.3%) and a lower FR (45.84%). The Gray area simulated study revealed a consistent result for these two models and indicated a lower Variation Ratio (VR) (8.10%) of TBA at 6 simulated levels. Despite facing many novel challenges and evidence limitations, CVAMA may offer novel insights into modular networks. PMID:26470848

  8. Differential expression of a retrotransposable element, Rex6, in Colossoma macropomum fish from different Amazonian environments.

    PubMed

    Barbosa, Cassiane Martins; Mareco, Edson Assunção; Silva, Maeli Dal Pai; Martins, Cesar; Alves-Costa, Fernanda Antunes

    2014-01-01

    Transposable elements (TEs) are DNA sequences that have the ability to move and replicate within the genomes. TEs can be classified according to their intermediates of transposition, RNA (retrotransposons) or DNA. In some aquatic organisms, it has been observed that environmental factors such as pH, temperature and pollution may stimulate differential transcription and mobilization of retrotransposons. In light of this information, the present study sought to evaluate the expression of Rex6 TE transcripts in Colossoma macropomum, which is a very commercially exploited fish in Brazil. In order to establish a comparative analysis using real-time PCR, the samples were collected from Amazonian rivers with different physical and chemical characteristics (distinguished by clear water and black water). Quantitative RT-PCR analyses revealed a differential pattern of expression between tissues collected from different types of water (clear and black waters). When it came to the hepatic and muscle tissues sampled, the levels of Rex6 transcripts were significantly different between the two Amazonian water types. These results suggest that environmental conditions operate differently in the regulation of Rex6 transcription in C. macropomum, results which have implications in the reshaping of the genome against environmental variations. PMID:25089227

  9. Differential expression of a retrotransposable element, Rex6, in Colossoma macropomum fish from different Amazonian environments

    PubMed Central

    Barbosa, Cassiane Martins; Mareco, Edson Assunção; Silva, Maeli Dal Pai; Martins, Cesar; Alves-Costa, Fernanda Antunes

    2014-01-01

    Transposable elements (TEs) are DNA sequences that have the ability to move and replicate within the genomes. TEs can be classified according to their intermediates of transposition, RNA (retrotransposons) or DNA. In some aquatic organisms, it has been observed that environmental factors such as pH, temperature and pollution may stimulate differential transcription and mobilization of retrotransposons. In light of this information, the present study sought to evaluate the expression of Rex6 TE transcripts in Colossoma macropomum, which is a very commercially exploited fish in Brazil. In order to establish a comparative analysis using real-time PCR, the samples were collected from Amazonian rivers with different physical and chemical characteristics (distinguished by clear water and black water). Quantitative RT-PCR analyses revealed a differential pattern of expression between tissues collected from different types of water (clear and black waters). When it came to the hepatic and muscle tissues sampled, the levels of Rex6 transcripts were significantly different between the two Amazonian water types. These results suggest that environmental conditions operate differently in the regulation of Rex6 transcription in C. macropomum, results which have implications in the reshaping of the genome against environmental variations. PMID:25089227

  10. The impact of amplification on differential expression analyses by RNA-seq

    PubMed Central

    Parekh, Swati; Ziegenhain, Christoph; Vieth, Beate; Enard, Wolfgang; Hellmann, Ines

    2016-01-01

    Currently, quantitative RNA-seq methods are pushed to work with increasingly small starting amounts of RNA that require amplification. However, it is unclear how much noise or bias amplification introduces and how this affects precision and accuracy of RNA quantification. To assess the effects of amplification, reads that originated from the same RNA molecule (PCR-duplicates) need to be identified. Computationally, read duplicates are defined by their mapping position, which does not distinguish PCR- from natural duplicates and hence it is unclear how to treat duplicated reads. Here, we generate and analyse RNA-seq data sets prepared using three different protocols (Smart-Seq, TruSeq and UMI-seq). We find that a large fraction of computationally identified read duplicates are not PCR duplicates and can be explained by sampling and fragmentation bias. Consequently, the computational removal of duplicates does improve neither accuracy nor precision and can actually worsen the power and the False Discovery Rate (FDR) for differential gene expression. Even when duplicates are experimentally identified by unique molecular identifiers (UMIs), power and FDR are only mildly improved. However, the pooling of samples as made possible by the early barcoding of the UMI-protocol leads to an appreciable increase in the power to detect differentially expressed genes. PMID:27156886

  11. Identification and assessment of differentially expressed genes involved in growth regulation in Apostichopus japonicus.

    PubMed

    Zhu, L; Li, C H; Su, X R; Guo, C Y; Wang, Z; Jin, C H; Li, Y; Li, T W

    2013-01-01

    Rapid and efficient growth is a major consideration and challenge for global mariculture. The differential growth rate of the sea cucumber, Apostichopus japonicus, has significantly hampered the total production of the industry. In the present study, forward and reverse suppression subtractive hybridization libraries were constructed and sequenced from a fast-growth group and a slow-growth group of the sea cucumber. A total of 142 differentially expressed sequence tags (ESTs) with insertions longer than 150 bp were identified and further analyzed. Fifty-seven of these ESTs (approximately 40%) were functionally annotated for cell structure, energy metabolism, immunity response, and growth factor categories. Six candidate genes, arginine kinase, cytochrome c oxidase subunit I, HSP70, β-actin, ferritin, and the ADP-ribosylation factor, were further validated by quantitative PCR. Significant differences were found between the fast- and slow-growth groups (P < 0.05) for the expression levels of arginine kinase, cytochrome c oxidase, HSP70, the ADP-ribosylation factor, and β-actin. However, no significant difference was observed for ferritin. Our results provide promising candidate gene markers for practical size screening, and also further promote marker-assisted selective breeding of this species. PMID:24065658

  12. Differential gene expression in femoral bone from red junglefowl and domestic chicken, differing for bone phenotypic traits

    PubMed Central

    Rubin, Carl-Johan; Lindberg, Johan; Fitzsimmons, Carolyn; Savolainen, Peter; Jensen, Per; Lundeberg, Joakim; Andersson, Leif; Kindmark, Andreas

    2007-01-01

    Background Osteoporosis is frequently observed among aging hens from egg-producing strains (layers) of domestic chicken. White Leghorn (WL) has been intensively selected for egg production and it manifests striking phenotypic differences for a number of traits including several bone phenotypes in comparison with the wild ancestor of chicken, the red junglefowl (RJ). Previously, we have identified four Quantitative Trait Loci (QTL) affecting bone mineral density and bone strength in an intercross between RJ and WL. With the aim of further elucidating the genetic basis of bone traits in chicken, we have now utilized cDNA-microarray technology in order to compare global RNA-expression in femoral bone from adult RJ and WL (five of each sex and population). Results When contrasting microarray data for all WL-individuals to that of all RJ-individuals we observed differential expression (False discovery rate adjusted p-values < 0.015) for 604 microarray probes. In corresponding male and female contrasts, differential expression was observed for 410 and 270 probes, respectively. Altogether, the three contrasts between WL and RJ revealed differential expression of 779 unique transcripts, 57 of which are located to previously identified QTL-regions for bone traits. Some differentially expressed genes have previously been attributed roles in bone metabolism and these were: WNT inhibitory factor 1 (WIF1), WD repeat-containing protein 5 (WDR5) and Syndecan 3 (SDC3). Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all 15 had lower expression in WL. Conclusion We report the identification of 779 differentially expressed transcripts, several residing within QTL-regions for bone traits. Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all had lower expression levels in WL. In addition, transcripts encoding four translation initiation and translation

  13. Population proteomics: quantitative variation within and among populations in cardiac protein expression.

    PubMed

    Rees, Bernard B; Andacht, Tracy; Skripnikova, Elena; Crawford, Douglas L

    2011-03-01

    Population analysis of gene expression is typically achieved by quantifying levels of mRNA; however, gene expression is also a function of protein translation and turnover. Therefore, a complete understanding of population variation in gene expression requires quantitative knowledge of protein expression within and among natural populations. We used two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) to quantitatively compare expression of heart ventricle proteins among 18 individuals in three populations of the teleost fish Fundulus. Among populations, expressions between orthologous proteins and mRNAs were generally positively correlated. Additionally, similar to the pattern of cardiac mRNA expression for the same populations, we found considerable variation in protein expression both within and among populations: Of 408 protein features in 2D gels, 34% are significantly different (P < 0.01) among individuals within a population, 9% differ between populations, and 12% have a pattern of expression that suggests they have evolved by natural selection. Although similar to mRNA expression, the frequency of significant differences among populations is larger for proteins. Similar to mRNA expressions, expressions of most proteins are correlated to the expressions of many other proteins. However, the correlations among proteins are more extensive than the correlation for similar RNAs. These correlations suggest a greater coordinate regulation of protein than mRNA expression. The larger frequency of significant differences among populations and the greater frequency of correlated expression among proteins versus among RNAs suggest that the molecular mechanisms affecting protein expression enhance the differences among populations, and these regulatory steps could be a source of variation for adaptation. PMID:21109588

  14. Multiple breast cancer risk variants are associated with differential transcript isoform expression in tumors

    PubMed Central

    Caswell, Jennifer L.; Camarda, Roman; Zhou, Alicia Y.; Huntsman, Scott; Hu, Donglei; Brenner, Steven E.; Zaitlen, Noah; Goga, Andrei; Ziv, Elad

    2015-01-01

    Genome-wide association studies have identified over 70 single-nucleotide polymorphisms (SNPs) associated with breast cancer. A subset of these SNPs are associated with quantitative expression of nearby genes, but the functional effects of the majority remain unknown. We hypothesized that some risk SNPs may regulate alternative splicing. Using RNA-sequencing data from breast tumors and germline genotypes from The Cancer Genome Atlas, we tested the association between each risk SNP genotype and exon-, exon–exon junction- or transcript-specific expression of nearby genes. Six SNPs were associated with differential transcript expression of seven nearby genes at FDR < 0.05 (BABAM1, DCLRE1B/PHTF1, PEX14, RAD51L1, SRGAP2D and STXBP4). We next developed a Bayesian approach to evaluate, for each SNP, the overlap between the signal of association with breast cancer and the signal of association with alternative splicing. At one locus (SRGAP2D), this method eliminated the possibility that the breast cancer risk and the alternate splicing event were due to the same causal SNP. Lastly, at two loci, we identified the likely causal SNP for the alternative splicing event, and at one, functionally validated the effect of that SNP on alternative splicing using a minigene reporter assay. Our results suggest that the regulation of differential transcript isoform expression is the functional mechanism of some breast cancer risk SNPs and that we can use these associations to identify causal SNPs, target genes and the specific transcripts that may mediate breast cancer risk. PMID:26472073

  15. Expression profiling shows differential molecular pathways and provides potential new diagnostic biomarkers for colorectal serrated adenocarcinoma.

    PubMed

    Conesa-Zamora, Pablo; García-Solano, José; García-García, Francisco; Turpin, María Del Carmen; Trujillo-Santos, Javier; Torres-Moreno, Daniel; Oviedo-Ramírez, Isabel; Carbonell-Muñoz, Rosa; Muñoz-Delgado, Encarnación; Rodriguez-Braun, Edith; Conesa, Ana; Pérez-Guillermo, Miguel

    2013-01-15

    Serrated adenocarcinoma (SAC) is a recently recognized colorectal cancer (CRC) subtype accounting for 7.5 to 8.7% of CRCs. It has been shown that SAC has a poorer prognosis and has different molecular and immunohistochemical features compared with conventional carcinoma (CC) but, to date, only one previous study has analyzed its mRNA expression profile by microarray. Using a different microarray platform, we have studied the molecular signature of 11 SACs and compared it with that of 15 matched CC with the aim of discerning the functions which characterize SAC biology and validating, at the mRNA and protein level, the most differentially expressed genes which were also tested using a validation set of 70 SACs and 70 CCs to assess their diagnostic and prognostic values. Microarray data showed a higher representation of morphogenesis-, hypoxia-, cytoskeleton- and vesicle transport-related functions and also an overexpression of fascin1 (actin-bundling protein associated with invasion) and the antiapoptotic gene hippocalcin in SAC all of which were validated both by quantitative real-time PCR (qPCR) and immunohistochemistry. Fascin1 expression was statistically associated with KRAS mutation with 88.6% sensitivity and 85.7% specificity for SAC diagnosis and the positivity of fascin1 or hippocalcin was highly suggestive of SAC diagnosis (sensitivity = 100%). Evaluation of these markers in CRCs showing histological and molecular characteristics of high-level microsatellite instability (MSI-H) also helped to distinguish SACs from MSI-H CRCs. Molecular profiling demonstrates that SAC shows activation of distinct signaling pathways and that immunohistochemical fascin1 and hippocalcin expression can be reliably used for its differentiation from other CRC subtypes. PMID:22696308

  16. Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

    PubMed Central

    Marraccini, Pierre; Vinecky, Felipe; Alves, Gabriel S.C.; Ramos, Humberto J.O.; Elbelt, Sonia; Vieira, Natalia G.; Carneiro, Fernanda A.; Sujii, Patricia S.; Alekcevetch, Jean C.; Silva, Vânia A.; DaMatta, Fábio M.; Ferrão, Maria A.G.; Leroy, Thierry; Pot, David; Vieira, Luiz G.E.; da Silva, Felipe R.; Andrade, Alan C.

    2012-01-01

    The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora. PMID:22511801

  17. Differential gene expression in response to juvenile hormone analog treatment in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Archotermopsidae).

    PubMed

    Cornette, Richard; Hayashi, Yoshinobu; Koshikawa, Shigeyuki; Miura, Toru

    2013-04-01

    Termite societies are characterized by a highly organized division of labor among conspicuous castes, groups of individuals with various morphological specializations. Termite caste differentiation is under control of juvenile hormone (JH), but the molecular mechanism underlying the response to JH and early events triggering caste differentiation are still poorly understood. In order to profile candidate gene expression during early soldier caste differentiation of the damp-wood termite, Hodotermopsis sjostedti, we treated pseudergates (workers) with a juvenile hormone analog (JHA) to induce soldier caste differentiation. We then used Suppressive Subtractive Hybridization to create two cDNA libraries enriched for transcripts that were either up- or downregulated at 24h after treatment. Finally, we used quantitative PCR to confirm temporal expression patterns. Hexamerins represent a large proportion of the genes upregulated following JHA treatment and have an expression pattern that shows roughly an inverse correlation to intrinsic JH titers. This data is consistent with the role of a JH "sink", which was demonstrated for hexamerins in another termite, Reticulitermes flavipes. A putative nuclear protein was also upregulated a few hours after JHA treatment, which suggests a role in the early response to JH and subsequent regulation of transcriptional events associated with soldier caste differentiation. Some digestive enzymes, such as endogenous beta-endoglucanase and chymotrypsin, as well as a protein associated to digestion were identified among genes downregulated after JHA treatment. This suggests that JH may directly influence the pseudergate-specific digestive system. PMID:23481672

  18. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma.

    PubMed

    Song, Qingfeng; Zhao, Chang; Ou, Shengqiu; Meng, Zhibin; Kang, Ping; Fan, Liwei; Qi, Feng; Ma, Yilong

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  19. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma

    PubMed Central

    SONG, QINGFENG; ZHAO, CHANG; OU, SHENGQIU; MENG, ZHIBIN; KANG, PING; FAN, LIWEI; QI, FENG; MA, YILONG

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  20. Quantitative analysis of lentiviral transgene expression in mice over seven generations.

    PubMed

    Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

    2010-10-01

    Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken

  1. Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas

    PubMed Central

    Lau, Tze Pheng; Roslani, April Camilla; Lian, Lay Hoong; Chai, Hwa Chia; Lee, Ping Chin; Hilmi, Ida; Goh, Khean Lee; Chua, Kek Heng

    2014-01-01

    Objectives To characterise the mRNA expression patterns of early and advanced stage colorectal adenocarcinomas of Malaysian patients. Design Comparative expression analysis. Setting and participants We performed a combination of annealing control primer (ACP)-based PCR and reverse transcription-quantitative real-time PCR for the identification of differentially expressed genes (DEGs) associated with early and advanced stage primary colorectal tumours. We recruited four paired samples from patients with colorectal cancer (CRC) of Dukes’ A and B for the preliminary differential expression study, and a total of 27 paired samples, ranging from CRC stages I to IV, for subsequent confirmatory test. The tumouric samples were obtained from the patients with CRC undergoing curative surgical resection without preoperative chemoradiotherapy. The recruited patients with CRC were newly diagnosed with CRC, and were not associated with any hereditary syndromes, previously diagnosed cancer or positive family history of CRC. The paired non-cancerous tissue specimens were excised from macroscopically normal colonic mucosa distally located from the colorectal tumours. Primary and secondary outcome measures The differential mRNA expression patterns of early and advanced stage colorectal adenocarcinomas compared with macroscopically normal colonic mucosa were characterised by ACP-based PCR and reverse transcription-quantitative real-time PCR. Results The RPL35, RPS23 and TIMP1 genes were found to be overexpressed in both early and advanced stage colorectal adenocarcinomas (p<0.05). However, the ARPC2 gene was significantly underexpressed in early colorectal adenocarcinomas, while the advanced stage primary colorectal tumours exhibited an additional overexpression of the C6orf173 gene (p<0.05). Conclusions We characterised two distinctive gene expression patterns to aid in the stratification of primary colorectal neoplasms among Malaysian patients with CRC. Further work can be done to

  2. Effects of homocysteine on adipocyte differentiation and CD36 gene expression in 3T3-L1 adipocytes.

    PubMed

    Mentese, Ahmet; Alver, Ahmet; Sumer, Aysegul; Demir, Selim

    2016-03-01

    The aim of this study was to investigate the effects of homocysteine (Hcy), a risk factor for cardiovascular diseases, hypertension, stroke and obesity, on expression of CD36 that regulates uptake of oxidized low-density lipoprotein (Ox-LDL) by adipocytes and differentiation of 3T3-L1 cells to adipocytes. Cell viability was determined using MTT assay, and density of triglycerides were measured with Oil Red O staining. The expression levels of CD36 were analyzed using SYBR green assay by quantitative RT-PCR. Our results showed that the addition of Hcy inhibited differentiation of 3T3-L1 preadipocytes in a dose-dependent manner without a significant cell toxicity (p < 0.05). Percentage CD36 gene expression increased in the Hcy treatment groups, but not statistically significantly (p > 0.05) compared to differentiated adipocytes. Hcy reduced adipocyte differentiation, but had no effect on the expression level of CD36 in vitro conditions. The effect of Hcy on uptake and clearance of Ox-LDL by adipose tissue now needs to be investigated in vivo. PMID:26691520

  3. Differential sensing for the regio- and stereoselective identification and quantitation of glycerides

    PubMed Central

    Diehl, Katharine L.; Ivy, Michelle Adams; Rabidoux, Scott; Petry, Stefan Matthias; Müller, Günter; Anslyn, Eric V.

    2015-01-01

    Glycerides are of interest to the areas of food science and medicine because they are the main component of fat. From a chemical sensing perspective, glycerides are challenging analytes because they are structurally similar to one another and lack diversity in terms of functional groups. Furthermore, because animal and plant fat consists of a number of stereo- and regioisomeric acylglycerols, their components remain challenging analytes for chromatographic and mass spectrometric determination, particularly the quantitation of species in mixtures. In this study, we demonstrated the use of an array of cross-reactive serum albumins and fluorescent indicators with chemometric analysis to differentiate a panel of mono-, di-, and triglycerides. Due to the difficulties in identifying the regio- and stereochemistry of the unsaturated glycerides, a sample pretreatment consisting of olefin cross-metathesis with an allyl fluorescein species was used before array analysis. Using this simple assay, we successfully discriminated 20 glycerides via principal component analysis and linear discriminant analysis (PCA and LDA, respectively), including stereo- and regioisomeric pairs. The resulting chemometric patterns were used as a training space for which the structural characteristics of unknown glycerides were identified. In addition, by using our array to perform a standard addition analysis on a mixture of triglycerides and using a method introduced herein, we demonstrated the ability to quantitate glyceride components in a mixture. PMID:26175025

  4. Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes.

    PubMed

    Di Napoli, Claudia; Pope, Iestyn; Masia, Francesco; Watson, Peter; Langbein, Wolfgang; Borri, Paola

    2014-05-01

    In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200-2000/cm) and in the CH stretch region (2600-3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods. PMID:24877002

  5. Use of polymerase chain reaction in the quantitation of mdr-1 gene expression

    SciTech Connect

    Murphy, L.D.; Herzog, C.E.; Rudick, J.B.; Fojo, A.T.; Bates, S.E. )

    1990-11-01

    The ability of the polymerase chain reaction (PCR) to quantitate expression of mRNA was examined in the present study. The model chosen was expression of the multidrug resistance gene mdr-1/Pgp in two colon carcinoma cell lines which express mdr-1/Pgp at levels comparable to those found in many clinical samples. PCR was utilized to evaluate differences in mdr-1/Pgp expression in the two cell lines after modulation by sodium butyrate. Thus, comparisons were made across a range of mdr-1/Pgp expression as well as comparisons of small differences. The PCR was found to be both sensitive and quantitative. Accurate quantitation requires demonstration of an exponential range which varies among samples. The exponential range can be determined by carrying out the PCR for a fixed number of cycles on serial dilutions of the RNA reverse transcription product, or by performing the reaction with a varying number of cycles on a fixed quantity of cDNA. By quantitation of the difference in PCR product derived from a given amount of RNA from the sodium butyrate treated and untreated cells, the difference in mRNA expression between samples can be determined. Normalization of the results can be achieved by independent amplification of a control gene, such as {beta}{sub 2}-microglobulin, when the latter is also evaluated in the exponential range. Simultaneous amplification of the control and target genes results in lower levels of PCR products due to competition, which varies from sample to sample. The PCR is thus a labor-intensive but sensitive method of quantitating gene expression in small samples of RNA.

  6. Preliminary identification of differentially expressed tear proteins in keratoconus

    PubMed Central

    Wasinger, Valerie C.; Pye, David C.; Willcox, Mark D. P.

    2013-01-01

    Purpose To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. Methods Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options. PMID:24194634

  7. Colonies in engineered articular cartilage express superior differentiation.

    PubMed

    Selvaratnam, L; Abd Rahim, S; Kamarul, T; Chan, K Y; Sureshan, S; Penafort, R; Ng, C L L

    2005-07-01

    In view of poor regeneration potential of the articular cartilage, in-vitro engineering of cartilage tissue offers a promising option for progressive joint disease. This study aims to develop a biologically engineered articular cartilage for autologous transplantation. The initial work involved determination of chondrocyte yield and viability, and morphological analysis. Cartilage was harvested from the knee, hip and shoulder joints of adult New Zealand white rabbits and chondrocytes were isolated by enzymatic digestion of the extra-cellular matrix before serial cultivation in DMEM/Ham's F12 media as monolayer cultures. No differences were noted in cell yield. Although chondrocytes viability was optimal (>93%) following harvest from native cartilage, their viability tended to be lowered on passaging. Chondrocytes aggregated in isogenous colonies comprising ovoid cells with intimate intracellular contacts and readily exhibited Safranin-O positive matrix; features typically associated with articular cartilage in-vivo. However, chondrocytes also existed concurrently in scattered bipolar/multipolar forms lacking Safranin-O expression. Therefore, early data demonstrated successful serial culture of adult chondrocytes with differentiated morphology seen in established chondrocyte colonies synthesizing matrix proteoglycans. PMID:16381284

  8. Stereolithographic Bone Scaffold Design Parameters: Osteogenic Differentiation and Signal Expression

    PubMed Central

    Kim, Kyobum; Yeatts, Andrew; Dean, David

    2010-01-01

    Scaffold design parameters including porosity, pore size, interconnectivity, and mechanical properties have a significant influence on osteogenic signal expression and differentiation. This review evaluates the influence of each of these parameters and then discusses the ability of stereolithography (SLA) to be used to tailor scaffold design to optimize these parameters. Scaffold porosity and pore size affect osteogenic cell signaling and ultimately in vivo bone tissue growth. Alternatively, scaffold interconnectivity has a great influence on in vivo bone growth but little work has been done to determine if interconnectivity causes changes in signaling levels. Osteogenic cell signaling could be also influenced by scaffold mechanical properties such as scaffold rigidity and dynamic relationships between the cells and their extracellular matrix. With knowledge of the effects of these parameters on cellular functions, an optimal tissue engineering scaffold can be designed, but a proper technology must exist to produce this design to specification in a repeatable manner. SLA has been shown to be capable of fabricating scaffolds with controlled architecture and micrometer-level resolution. Surgical implantation of these scaffolds is a promising clinical treatment for successful bone regeneration. By applying knowledge of how scaffold parameters influence osteogenic cell signaling to scaffold manufacturing using SLA, tissue engineers may move closer to creating the optimal tissue engineering scaffold. PMID:20504065

  9. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  10. Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

    PubMed Central

    Rice, William L.; Kaplan, David L.; Georgakoudi, Irene

    2010-01-01

    Background The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. Methodology/Principal Findings Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. Conclusions/Significance In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables

  11. Differential expression of the lethal gene Luteus-Pa in cacao of the Parinari series.

    PubMed

    Rehem, B C; Almeida, A-A F; Figueiredo, G S F; Gesteira, A S; Santos, S C; Corrêa, R X; Yamada, M M; Valle, R R

    2016-01-01

    The recessive lethal character Luteus-Pa is found in cacao (Theobroma cacao) genotypes of the Parinari series (Pa) and is characterized by expression of leaf chlorosis and seedling death. Several genotypes of the Pa series are bearers of the gene responsible for the expression of the Luteus-Pa character, which can be used as a tool for determining relationships between genotypes of this group. To evaluate this phenomenon, we analyzed the differential expression of genes between mutant seedlings and wild-type hybrid Pa 30 x 169 seedlings, with the aim of elucidating the possible lethal mechanisms of the homozygous recessive character Luteus-Pa. Plant material was harvested from leaves of wild and mutant seedlings at different periods to construct a subtractive library and perform quantitative analysis using real-time PCR. The 649 sequences obtained from the subtractive library had an average length of 500 bp, forming 409 contigs. The probable proteins encoded were grouped into 10 functional categories. Data from ESTs identified genes associated with Rubisco, peroxidases, and other proteins and enzymes related to carbon assimilation, respiration, and photosystem 2. Mutant seedlings were characterized by synthesizing defective PsbO and PsbA proteins, which were overexpressed from 15 to 20 days after seedling emergence. PMID:26910005

  12. Identification of Differentially Expressed Genes Associated with Apple Fruit Ripening and Softening by Suppression Subtractive Hybridization

    PubMed Central

    Zhang, Zongying; Jiang, Shenghui; Wang, Nan; Li, Min; Ji, Xiaohao; Sun, Shasha; Liu, Jingxuan; Wang, Deyun; Xu, Haifeng; Qi, Sumin; Wu, Shujing; Fei, Zhangjun; Feng, Shouqian; Chen, Xuesen

    2015-01-01

    Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from ‘Taishanzaoxia’ apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in ‘Taishanzaoxia’. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening. PMID:26719904

  13. Diagnostic usefulness of PCR profiling of the differentially expressed marker genes in thyroid papillary carcinomas.

    PubMed

    Hamada, Aiko; Mankovskaya, Svetlana; Saenko, Vladimir; Rogounovitch, Tatiana; Mine, Mariko; Namba, Hiroyuki; Nakashima, Masahiro; Demidchik, Yuri; Demidchik, Eugeny; Yamashita, Shunichi

    2005-06-28

    The study was set out to determine whether characteristic changes in the gene expression profile in papillary thyroid carcinoma (PTC) discovered by microarray assays can be used for conventional molecular diagnosis. Expression levels of five reported to be overexpressed and three underexpressed genes were examined in PTC and normal human tissues by real-time PCR and semi-quantitative duplex PCR. Stepwise logistic regression analysis, duplex PCR data evaluation with recursive partition machine algorithm and hierarchical cluster analysis identified SFTPB (upregulated) and TFF3 (downregulated) gene combination as most favorable for differential molecular diagnosis of PTC. Sensitivity, specificity and accuracy obtained in a series of histologically characterized thyroid tumor and normal tissue samples were 88.9, 96.7 and 94.9%, respectively. Applicability of the method to fine needle aspiration biopsy (FNAB) samples was demonstrated using a collection of needle washouts. In spite individual thyroid tumor and normal tissues as well as FNAB samples displayed a substantial degree of variability in the expression levels of analyzed genes, simultaneous molecular analysis of a panel of optimal markers allows making a high probability predictive estimate and may be considered as an informative method of preoperative PTC diagnosis. PMID:15914279

  14. Identification of Differentially Expressed Genes Associated with Apple Fruit Ripening and Softening by Suppression Subtractive Hybridization.

    PubMed

    Zhang, Zongying; Jiang, Shenghui; Wang, Nan; Li, Min; Ji, Xiaohao; Sun, Shasha; Liu, Jingxuan; Wang, Deyun; Xu, Haifeng; Qi, Sumin; Wu, Shujing; Fei, Zhangjun; Feng, Shouqian; Chen, Xuesen

    2015-01-01

    Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from 'Taishanzaoxia' apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in 'Taishanzaoxia'. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening. PMID:26719904

  15. Differential Granulosa Cell Gene Expression in Young Women with Diminished Ovarian Reserve

    PubMed Central

    Greenseid, Keri; Jindal, Sangita; Hurwitz, Joshua; Santoro, Nanette; Pal, Lubna

    2011-01-01

    Objective: To investigate if a diagnosis of diminished ovarian reserve (DOR) is associated with a differential gene profile of ovarian granulosa cells (GCs) in infertile women undergoing in vitro fertilization (IVF). Design: Prospective Cohort Study. Setting: Academic IVF Program. Patients: Infertile women <38 years were prospectively enrolled into 2 groups: normal ovarian reserve (NOR, follicle-stimulating hormone [FSH] < 10 mIU/mL, n = 4) and DOR (FSH ≥ 10.0 mIU/mL, n = 4). Interventions: Cumulus (C) and mural (M) GCs were isolated at egg retrieval; messenger RNA was extracted and transcribed. Main Outcome Measure(s): Differential gene expression in cerebellar granule cells (CGCs) in the 2 groups was assessed by cDNA microarray. Microarray findings were validated by quantitative real-time polymerase chain reaction (qRTPCR) in CGCs and explored in multinucleated giant cells (MGCs). Results: Of the 1256 differentially regulated genes identified in CGCs of women with DOR, the insulin-like growth factor (IGF) family was a biologically relevant gene family of a priori interest. Downregulation of IGF1 and IGF2 ligands (−3.28- and −2.54–fold, respectively), and their receptors, (−3.53- and −1.32-fold downregulation of IGF1R and IGF2R, respectively) was identified in luteinized CGCs in women with DOR compared to those with NOR. Downregulation of both IGF1 and IGF 2 ligands (−4.35- and 3.89-fold, respectively) was furthermore observed in MGCs in women with DOR compared to those with NOR; no differences in the expression of respective receptors were however observed in MGCs in the 2 groups. Conclusions: Components of the IGF gene family are downregulated in GCs of women with DOR. These findings maybe contributory to the reproductive compromise observed in women with DOR, and merit further exploration. PMID:21846690

  16. Differential Expression of Cell Cycle Regulators During Hyperplastic and Hypertrophic Growth of Broiler Subcutaneous Adipose Tissue.

    PubMed

    Zhang, J; Suh, Y; Choi, Y M; Chen, P R; Davis, M E; Lee, K

    2015-10-01

    Hyperplastic growth and hypertrophic growth within adipose tissue is tightly associated with cell cycle activity. In this study, CCNG2 and CDKN2C were found to be correlated with cell cycle inhibition during fat cell differentiation, whereas CCND3, CCNA1, and ANAPC5 were positively associated with cell cycle activity during fat cell proliferation after selection based on GEO datasets available on the NCBI website. The findings were validated through comparison of expressions of these genes among different tissues/fractions in broiler chickens and time points during primary cell culture using quantitative real-time PCR. Development of broiler subcutaneous adipose tissue was investigated on embryonic days 15 and 17 and on post-hatch days 0, 5, 11, and 33 using H&E staining and PCNA immunostaining with DAPI counter stain. In addition, mRNA expressions of five cell cycle regulators as well as precursor cell and adipocyte markers were measured at those time points. The results suggest that cellular proliferation activity decreased as the fat pad grows, but a population of precursor cells seemed to be maintained until post-hatch day 5 despite increasing differentiation activity. Hypertrophic growth gradually intensified despite a slight cessation on post-hatch day 0 due to increased energy expenditure during hatching and delayed food access. From post-hatch day 5 to day 11, most of the precursor cells may become differentiated. After post-hatch day 11, hyperplastic growth seemed to slow, while hypertrophic growth may become dominant. This study provides further understanding about broiler fat tissue development which is imperative for effective control of fat deposition. PMID:26017028

  17. Genome-wide analysis of differentially expressed genes and splicing isoforms in clear cell renal cell carcinoma.

    PubMed

    Valletti, Alessio; Gigante, Margherita; Palumbo, Orazio; Carella, Massimo; Divella, Chiara; Sbisà, Elisabetta; Tullo, Apollonia; Picardi, Ernesto; D'Erchia, Anna Maria; Battaglia, Michele; Gesualdo, Loreto; Pesole, Graziano; Ranieri, Elena

    2013-01-01

    Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal epithelial tumor and also the most deadly. To identify molecular changes occurring in ccRCC, in the present study we performed a genome wide analysis of its entire complement of mRNAs. Gene and exon-level analyses were carried out by means of the Affymetrix Exon Array platform. To achieve a reliable detection of differentially expressed cassette exons we implemented a novel methodology that considered contiguous combinations of exon triplets and candidate differentially expressed cassette exons were identified when the expression level was significantly different only in the central exon of the triplet. More detailed analyses were performed for selected genes using quantitative RT-PCR and confocal laser scanning microscopy. Our analysis detected over 2,000 differentially expressed genes, and about 250 genes alternatively spliced and showed differential inclusion of specific cassette exons comparing tumor and non-tumoral tissues. We demonstrated the presence in ccRCC of an altered expression of the PTP4A3, LAMA4, KCNJ1 and TCF21 genes (at both transcript and protein level). Furthermore, we confirmed, at the mRNA level, the involvement of CAV2 and SFRP genes that have previously been identified. At exon level, among potential candidates we validated a differentially included cassette exon in DAB2 gene with a significant increase of DAB2 p96 splice variant as compared to the p67 isoform. Based on the results obtained, and their robustness according to both statistical analysis and literature surveys, we believe that a combination of gene/isoform expression signature may remarkably contribute, after suitable validation, to a more effective and reliable definition of molecular biomarkers for ccRCC early diagnosis, prognosis and prediction of therapeutic response. PMID:24194935

  18. Validation of MIMGO: a method to identify differentially expressed GO terms in a microarray dataset

    PubMed Central

    2012-01-01

    Background We previously proposed an algorithm for the identification of GO terms that commonly annotate genes whose expression is upregulated or downregulated in some microarray data compared with in other microarray data. We call these “differentially expressed GO terms” and have named the algorithm “matrix-assisted identification method of differentially expressed GO terms” (MIMGO). MIMGO can also identify microarray data in which genes annotated with a differentially expressed GO term are upregulated or downregulated. However, MIMGO has not yet been validated on a real microarray dataset using all available GO terms. Findings We combined Gene Set Enrichment Analysis (GSEA) with MIMGO to identify differentially expressed GO terms in a yeast cell cycle microarray dataset. GSEA followed by MIMGO (GSEA + MIMGO) correctly identified (p < 0.05) microarray data in which genes annotated to differentially expressed GO terms are upregulated. We found that GSEA + MIMGO was slightly less effective than, or comparable to, GSEA (Pearson), a method that uses Pearson’s correlation as a metric, at detecting true differentially expressed GO terms. However, unlike other methods including GSEA (Pearson), GSEA + MIMGO can comprehensively identify the microarray data in which genes annotated with a differentially expressed GO term are upregulated or downregulated. Conclusions MIMGO is a reliable method to identify differentially expressed GO terms comprehensively. PMID:23232071

  19. Successful personalized chemotherapy for metastatic gastric cancer based on quantitative BRCA1 mRNA expression level: A case report

    PubMed Central

    HUANG, YING; WU, PUYUAN; LIU, BAORUI; DU, JUAN

    2016-01-01

    Personalized chemotherapy is based on the specific genetic profile of individual patients and is replacing the traditional ‘one size fits all’ medicine. Breast cancer 1 (BRCA1) plays a central role in the chemotherapy-induced DNA damage response. It has been repeatedly demonstrated that BRCA1 mRNA levels were negatively associated with cisplatin sensitivity, but positively associated with docetaxel sensitivity in patients with gastric cancer in experimental and clinical studies. This feature leads to customized chemotherapy based on the BRCA1 mRNA expression level and results in a high efficacy of treatment. The present study describes the case of a 77-year-old patient with metastatic gastric cancer who was treated with personalized chemotherapy based on quantitative BRCA1 mRNA expression level. This study and the available literature data suggest that the expression level of BRCA1 mRNA is dynamic to BRCA1-based chemotherapy. More importantly, de novo assessment of BRCA1 status is a preferable option for ciscisplatin- or docetaxel-resistant patients, since the expression levels of BRCA1 mRNA in certain patients may alter significantly following treatment. Therefore, BRCA1 expression should be assessed for predicting differential chemosensitivity and tailoring chemotherapy in gastric cancer. PMID:27313763

  20. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate falsepositive signals and that the length of the amplicon affects the intensity of...

  1. Expressing Certainty in Discussion Sections of Qualitative and Quantitative Research Articles

    ERIC Educational Resources Information Center

    Dobakhti, Leila

    2013-01-01

    This paper investigates how boosters are used by qualitative and quantitative research article writers to express certainty. Boosters are words such as "definitely," "sure," "demonstrate" which signal writers' assurance in what they say. Drawing on a corpus of 200 research articles in Applied Linguistics, this…

  2. Differential gene expression and lipid metabolism in fatty liver induced by acute ethanol treatment in mice

    SciTech Connect

    Yin Huquan; Kim, Mingoo; Kim, Ju-Han; Kong, Gu; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-IL; Lee, Mi-Ock; Lee, Byung-Hoon

    2007-09-15

    Ethanol induces cumulative liver damage including steatosis, steatohepatitis and cirrhosis. The aim of this study is to investigate the global intrahepatic gene expression profile in the mouse liver treated with ethanol. A single oral dose of 0.5 or 5 g/kg ethanol was administered to male ICR mice, and liver samples were obtained after 6, 24 and 72 h. Histopathological evaluation showed typical fatty livers in the high-dose group at 24 h. Microarray analysis identified 28 genes as being ethanol responsive (two-way ANOVA; p < 0.05), after adjustment by the Benjamini-Hochberg multiple testing correction; these genes displayed {>=} 2-fold induction or repression. The expression of genes that are known to be involved in fatty acid synthesis was examined. The transcript for lipogenic transcription factor, sterol regulatory element (SRE)-binding factor 1 (Srebf1), was upregulated by acute ethanol exposure. Of the genes known to contain SRE or SRE-like sequences and to be regulated by SRE-binding protein 1 (SREBP1), those encoding malic enzyme (Mod1), ATP-citrate lyase (Acly), fatty acid synthase (Fasn) and stearyl-CoA desaturase (Scd1) were induced by ethanol. Quantitative real-time PCR confirmed the changes in the expression levels of the selected genes. The change in the Srebf1 mRNA level correlates well with that of the SREBP1 protein expression as well as its binding to the promoters of the target genes. The present study identifies differentially expressed genes that can be applied to the biomarkers for alcohol-binge-induced fatty liver. These results support the hypothesis by which ethanol-induced steatosis in mice is mediated by the fatty acid synthetic pathway regulated by SREBP1.

  3. Differential expression of bitter taste receptors in non-cancerous breast epithelial and breast cancer cells.

    PubMed

    Singh, Nisha; Chakraborty, Raja; Bhullar, Rajinder Pal; Chelikani, Prashen

    2014-04-01

    The human bitter taste receptors (T2Rs) are chemosensory receptors that belong to the G protein-coupled receptor superfamily. T2Rs are present on the surface of oral and many extra-oral cells. In humans 25 T2Rs are present, and these are activated by hundreds of chemical molecules of diverse structure. Previous studies have shown that many bitter compounds including chloroquine, quinidine, bitter melon extract and cucurbitacins B and E inhibit tumor growth and induce apoptosis in cancer cells. However, the existence of T2Rs in cancer cell is not yet elucidated. In this report using quantitative (q)-PCR and flow cytometry, we characterized the expression of T2R1, T2R4, T2R10, T2R38 and T2R49 in the highly metastatic breast cancer cell line MDA-MB-231, poorly metastatic cell line MCF-7, and non-cancerous mammary epithelial cell line MCF-10A. Among the 5 T2Rs analyzed by qPCR and flow cytometry, T2R4 is expressed at 40-70% in mammary epithelial cells in comparison to commonly used breast cancer marker proteins, estrogen receptor and E-cadherin. Interestingly, the expression of T2R4 was downregulated in breast cancer cells. An increase in intracellular calcium mobilization was observed after the application of bitter agonists, quinine, dextromethorphan, and phenylthiocarbamide that are specific for some of the 5 T2Rs. This suggests that the endogenous T2Rs expressed in these cells are functional. Taken together, our novel findings suggest that T2Rs are differentially expressed in mammary epithelial cells, with some T2Rs downregulated in breast cancer cells. PMID:24613843

  4. Differential Expression of Salt Stress-related Genes in Wild Beta vulgaris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Differential display reverse transcription (DDRT) technique was used to detect differentially expressed genes for wild Beta vulgaris in response to salt stress. Two month-old seedlings were treated with 250 mM Na for 1H, 10H and untreated seedlings were used as controls. A group of differentially di...

  5. Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species.

    PubMed

    Wirthgen, Elisa; Höflich, Christine; Spitschak, Marion; Helmer, Carina; Brand, Bodo; Langbein, Jan; Metzger, Friedrich; Hoeflich, Andreas

    2016-02-01

    The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds. PMID:26597140

  6. Quantitative Expression and Immunogenicity of MAGE-3 and -6 in Upper Aerodigestive Tract Cancer

    PubMed Central

    Andrade Filho, Pedro A.; López-Albaitero, Andrés; Xi, Liqiang; Gooding, William; Godfrey, Tony; Ferris, Robert L.

    2009-01-01

    The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aero-digestive tract (UADT) tumor cells and its association with T cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE specific immunotherapy. Using quantitative RT-PCR (QRT-PCR), we evaluated the expression of MAGE-3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA-A*0201+squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using western blot. HLA-A*0201:MAGE-3(271–279) specific cytotoxic T lymphocytes (MAGE-CTL) from SCCHN patients and healthy donors showed that MAGE-3/6 expression was highly associated with CTL recognition in vitro. Based on MAGE-3/6 expression we could identify 31 (47%) of the 65 UADT tumors which appeared to express MAGE-3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE-3 expression was responsible for CTL recognition, two MAGE-3/6 mRNAhigh SCCHN cell lines, PCI-13 and PCI-30, were subjected to MAGE-3/6 specific knockdown. RNAi–transfected cells showed that MAGE expression, and MAGE-CTL recognition, were significantly reduced. Furthermore, treatment of cells expressing low MAGE-3/6 mRNA with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), increased the expression of MAGE-3/6 and CTL recognition. Thus, using QRT-PCR UADT cancers frequently express MAGE-3/6 at levels sufficient for CTL recognition, supporting the use of a QRT-PCR based assay for the selection of candidates likely to respond to MAGE-3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials. PMID:19610063

  7. Quantitative expression and immunogenicity of MAGE-3 and -6 in upper aerodigestive tract cancer.

    PubMed

    Filho, Pedro A Andrade; López-Albaitero, Andrés; Xi, Liqiang; Gooding, William; Godfrey, Tony; Ferris, Robert L

    2009-10-15

    The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T-cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE-specific immunotherapy. Using quantitative RT-PCR (QRT-PCR), we evaluated the expression of MAGE-3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA-A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA-A*0201:MAGE-3- (271-279) specific cytotoxic T lymphocytes (MAGE-CTL) from SCCHN patients and healthy donors showed that MAGE-3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE-3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE-3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE-3 expression was responsible for CTL recognition, 2 MAGE-3/6 mRNA(high) SCCHN cell lines, PCI-13 and PCI-30, were subjected to MAGE-3/6-specific knockdown. RNAi-transfected cells showed that MAGE expression and MAGE-CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE-3/6 mRNA with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), increased the expression of MAGE-3/6 and CTL recognition. Thus, using QRT-PCR UADT cancers frequently express MAGE-3/6 at levels sufficient for CTL recognition, supporting the use of a QRT-PCR-based assay for the selection of candidates likely to respond to MAGE-3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials. PMID:19610063

  8. MiRNA expression profile and miRNA-mRNA integrated analysis (MMIA) during podocyte differentiation.

    PubMed

    Li, Zhigui; Wang, Lifeng; Xu, Jing; Yang, Zhuo

    2015-06-01

    The podocyte is a prominent cell type, which encases the capillaries of glomerulus. Podocyte-selective deletion of Dicer or Drosha was reported to induce proteinuria and glomerulosclerosis, suggesting the essential role of microRNA (miRNA) in podocytes for renal function. However, no comprehensive miRNA expression or miRNA-mRNA integrated analysis (MMIA) can be found during podocyte differentiation. Herein, miRNA and mRNA microarrays are presented, which were carried out in differentiated and undifferentiated mouse podocyte cell lines (MPC5). A total of 50 abnormal miRNAs (26 down-regulated and 24 up-regulated) were identified in differentiated and undifferentiated podocytes. Using MMIA, 80 of the 743 mRNAs (>twofold change) were predicted for potential crosstalk with 30 miRNAs of the 50 abnormal miRNAs. In addition, the gene ontology of mRNAs and the pathway analysis of miRNAs revealed a new potential-regulated network during podocyte differentiation. The expressions of three remarkably changed miRNAs (miR-34c, miR-200a and miR-467e) and four mRNAs (Runx1t1, Atp2a2, Glrp1, and Mmp15), were randomly chosen for further validation by the quantitative real-time polymerase chain reaction, and their expression trends were consistent with the microarray data. Reference searching was also conducted to confirm our data and to find potential new molecules and miRNA-target pairs involved in the podocyte differentiation. The dual luciferase reporter assay for miR-200a/GLRX and let-7b/ARL4D confirmed the prediction of MMIA. The results of this study provide a detailed integration of mRNA and miRNA during podocyte differentiation. The molecular integration mode will open up new perspectives for a better understanding of the mechanism during podocyte differentiation. PMID:25433550

  9. Differentiated Human Midbrain-Derived Neural Progenitor Cells Express Excitatory Strychnine-Sensitive Glycine Receptors Containing α2β Subunits

    PubMed Central

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

    2012-01-01

    Background Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson’s disease. While glutamate and GABAA receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Methodology/Principal Findings Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na+-K+-Cl− co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. Conclusions/Significance These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro. PMID:22606311

  10. Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry

    PubMed Central

    Mazur, Matthew T.; Cardasis, Helene L.; Spellman, Daniel S.; Liaw, Andy; Yates, Nathan A.; Hendrickson, Ronald C.

    2010-01-01

    Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of “unknown” proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL3. The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 → 3)-Gal-β(1 → 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level. PMID:20388904

  11. Effect of castration on carcass quality and differential gene expression of longissimus muscle between steer and bull.

    PubMed

    Zhou, Zheng-Kui; Gao, Xue; Li, Jun-Ya; Chen, Jin-Bao; Xu, Shang-Zhong

    2011-11-01

    The effect of castration on carcass quality was investigated by ten Chinese Simmental calves. Five calves were castrated randomly at 2 months old and the others were retained as normal intact bulls. All animals were slaughtered at 22 months old. The results showed that bulls carcass had higher weight (P < 0.05), dressing percentages and bigger longissimus muscle areas (P < 0.05) than steers. But steer meat had lower shear force values and was fatter (P < 0.05) than bull. Furthermore, in order to discover genes that were involved in determining steer meat quality, we compared related candidate gene expression in longissimus muscle between steer (tester) and bull (driver) using suppressive subtractive hybridization. Ten genes were identified as preferentially expressed in longissimus muscle of steer. The expression of four selected differentially expressed genes was confirmed by quantitative real-time PCR. Overall, a 1.96, 2.41, 2.89, 2.41-fold increase in expression level was observed in steer compared with bull for actin, gamma 2, smooth muscle, tropomyosin-2, insulin like growth factor 1 and hormone-sensitive lipase, respectively. These results implied that these differentially expressed genes could play an important role in the regulation of steer meat quality. PMID:21253852

  12. Differential DNA methylation and expression of inflammatory and zinc transporter genes defines subgroups of osteoarthritic hip patients

    PubMed Central

    Rushton, Michael D; Young, David A; Loughlin, John; Reynard, Louise N

    2015-01-01

    Objectives We have previously shown that the cartilage DNA methylome delineates two clusters of osteoarthritic (OA) hip patients, characterised by differential methylation of inflammatory genes, while others have demonstrated a link between zinc homeostasis and inflammation in OA. We aimed to investigate these effects at the methylation and gene expression level. Methods We used our previously generated methylation data while quantitative PCR was used to measure gene expression using RNA from the hip cartilage of members of both clusters and from control individuals without hip OA. Results One of the OA clusters is characterised by the promoter hypomethylation and increased expression of inflammation-associated genes including IL1A and TNF. Furthermore, we show that the increase in expression of these genes is accompanied by increased expression of several zinc transporter genes. In addition, the zinc responsive transcription factor MTF1 is also upregulated, which is accompanied by an increase in the expression of its targets the metalloproteinases MMP13 and ADAMTS5. Conclusions We have identified a subgroup of OA hip patients that are epigenetically and transcriptiomically characterised by a cartilage inflammatory phenotype with concurrent differential regulation of zinc regulators. The identification of subgroups enhances stratified phenotyping of OA patients and has important implications for future therapeutic applications. PMID:25854584

  13. Expression of nuclear lamin A and muscle-specific proteins in differentiating muscle cells in ovo and in vitro

    PubMed Central

    1989-01-01

    Primary cultures and tissue samples of chicken embryonic muscle were immunologically probed for the expression of muscle-specific proteins, such as myosin heavy chain and the tropomyosins, as well as for the nuclear lamina protein, lamin A. As determined by quantitative immunoblotting, the expression of lamin A and the muscle-specific proteins were at low levels or absent in predifferentiation myoblasts both in vitro and in ovo. During differentiation, an increase of lamin A expression preceded the induction to high levels of expression of muscle-specific proteins. Immunofluorescence staining of chicken embryonic muscle cells in culture also indicates an accumulation of lamin A before the induction of muscle-specific proteins expression. Furthermore, the accumulation of lamin A reached a plateau before the muscle-specific proteins during muscle development. In two dimensional NEPHGE gel analysis of immunoprecipitated lamin A, no detectable change in the ratio of the acidic/basic isoelectric variants of lamin A was observed during myogenesis. A potential role for lamin A in the mechanisms which underlie the differential and coordinate expression of muscle-specific genes is proposed. PMID:2668298

  14. TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differentiating colonocytes and colon cancer.

    PubMed

    Chapman, Christopher G; Mariani, Christopher J; Wu, Feng; Meckel, Katherine; Butun, Fatma; Chuang, Alice; Madzo, Jozef; Bissonette, Marc B; Kwon, John H; Godley, Lucy A

    2015-01-01

    The formation of differentiated cell types from pluripotent progenitors involves epigenetic regulation of gene expression. DNA hydroxymethylation results from the enzymatic oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) 5-mC dioxygenase enzymes. Previous work has mapped changes in 5-mC during differentiation of intestinal stem cells. However, whether or not 5-hmC regulates colonocyte differentiation is unknown. Here we show that 5-hmC regulates gene expression during colonocyte differentiation and controls gene expression in human colon cancers. Genome-wide profiling of 5-hmC during in vitro colonic differentiation demonstrated that 5-hmC is gained at highly expressed and induced genes and is associated with intestinal transcription factor binding sites, including those for HNF4A and CDX2. TET1 induction occurred during differentiation, and TET1 knockdown altered gene expression and inhibited barrier formation of colonocytes. We find that the 5-hmC distribution in primary human colonocytes parallels the distribution found in differentiated cells in vitro, and that gene-specific 5-hmC changes in human colon cancers are directly correlated with changes in gene expression. Our results support a model in which 5-hmC regulates differentiation of adult human intestine and 5-hmC alterations contribute to the disrupted gene expression in colon cancer. PMID:26631571

  15. Differential expression of the regulator of G protein signaling RGS9 protein in nociceptive pathways of different age rats.

    PubMed

    Kim, Ki Jun; Moriyama, Kumi; Han, Kyung Ream; Sharma, Manohar; Han, Xiaokang; Xie, Guo-xi; Palmer, Pamela Pierce

    2005-11-01

    Regulators of G protein signaling (RGS) proteins are GTPase-activating proteins which act as modulators of G-protein-coupled receptors. RGS9 has two alternative splicing variants. RGS9-1 is expressed in the retina. RGS9-2 is expressed in the brain, especially abundant in the striatum. It is believed to be an essential regulatory component of dopamine and opioid signaling. In this study, we compared the expression of RGS9 proteins in the nervous system of different age groups of rats employing immunocytochemistry. In both 3-week- and 1-year-old rats, RGS9 is expressed abundantly in caudate-putamen, nucleus accumbens, and olfactory tubercle. It is also expressed abundantly in the ventral horn of the spinal cord and the dorsal root ganglion (DRG) cells. Quantitative analysis showed that the intensities of RGS9 expression in 1-year-old rats are higher than those in the 3-week-old rats in caudate-putamen, nucleus accumbens, olfactory tubercle, periaqueductal gray, and gray matter of the spinal cord. In contrast, in thalamic nuclei and locus coeruleus, the intensities of RGS9 immunostaining in 3-week-old rats are higher than in 1-year-old rats. In DRG cells, there is no significant difference between the two age groups. These data suggest that RGS9 is differentially expressed with age. Such differential expression may play an important role in neuronal differentiation and development as well as in neuronal function, such as dopamine and opioid signaling. PMID:16153714

  16. Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

    PubMed Central

    Kamata, Masakazu; Liang, Min; Liu, Shirley; Nagaoka, Yoshiko; Chen, Irvin S. Y.

    2010-01-01

    Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells. PMID:20676373

  17. Developmental Progression in the Coral Acropora digitifera Is Controlled by Differential Expression of Distinct Regulatory Gene Networks

    PubMed Central

    Reyes-Bermudez, Alejandro; Villar-Briones, Alejandro; Ramirez-Portilla, Catalina; Hidaka, Michio; Mikheyev, Alexander S.

    2016-01-01

    Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis during Acropora digitifera’s development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression in A. digitifera is regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages. PMID:26941230

  18. Developmental Progression in the Coral Acropora digitifera Is Controlled by Differential Expression of Distinct Regulatory Gene Networks.

    PubMed

    Reyes-Bermudez, Alejandro; Villar-Briones, Alejandro; Ramirez-Portilla, Catalina; Hidaka, Michio; Mikheyev, Alexander S

    2016-03-01

    Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis duringAcropora digitifera's development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression inA. digitiferais regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages. PMID:26941230

  19. Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells.

    PubMed

    Li, X; Huang, K; Chen, F; Li, W; Sun, S; Shi, X-E; Yang, G

    2016-06-01

    Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition. PMID:26781521

  20. Posttranscriptional deregulation of signaling pathways in meningioma subtypes by differential expression of miRNAs

    PubMed Central

    Ludwig, Nicole; Kim, Yoo-Jin; Mueller, Sabine C.; Backes, Christina; Werner, Tamara V.; Galata, Valentina; Sartorius, Elke; Bohle, Rainer M.; Keller, Andreas; Meese, Eckart

    2015-01-01

    Background Micro (mi)RNAs are key regulators of gene expression and offer themselves as biomarkers for cancer development and progression. Meningioma is one of the most frequent primary intracranial tumors. As of yet, there are limited data on the role of miRNAs in meningioma of different histological subtypes and the affected signaling pathways. Methods In this study, we compared expression of 1205 miRNAs in different meningioma grades and histological subtypes using microarrays and independently validated deregulation of selected miRNAs with quantitative real-time PCR. Clinical utility of a subset of miRNAs as biomarkers for World Health Organization (WHO) grade II meningioma based on quantitative real-time data was tested. Potential targets of deregulated miRNAs were discovered with an in silico analysis. Results We identified 13 miRNAs deregulated between different subtypes of benign meningiomas, and 52 miRNAs deregulated in anaplastic meningioma compared with benign meningiomas. Known and putative target genes of deregulated miRNAs include genes involved in epithelial-to-mesenchymal transition for benign meningiomas, and Wnt, transforming growth factor–β, and vascular endothelial growth factor signaling for higher-grade meningiomas. Furthermore, a 4-miRNA signature (miR-222, -34a*, -136, and -497) shows promise as a biomarker differentiating WHO grade II from grade I meningiomas with an area under the curve of 0.75. Conclusions Our data provide novel insights into the contribution of miRNAs to the phenotypic spectrum in benign meningiomas. By deregulating translation of genes belonging to signaling pathways known to be important for meningioma genesis and progression, miRNAs provide a second in line amplification of growth promoting cellular signals. MiRNAs as biomarkers for diagnosis of aggressive meningiomas might prove useful and should be explored further in a prospective manner. PMID:25681310

  1. Quantitative assessment of the differential impacts of arbuscular and ectomycorrhiza on soil carbon cycling.

    PubMed

    Soudzilovskaia, Nadejda A; van der Heijden, Marcel G A; Cornelissen, Johannes H C; Makarov, Mikhail I; Onipchenko, Vladimir G; Maslov, Mikhail N; Akhmetzhanova, Asem A; van Bodegom, Peter M

    2015-10-01

    A significant fraction of carbon stored in the Earth's soil moves through arbuscular mycorrhiza (AM) and ectomycorrhiza (EM). The impacts of AM and EM on the soil carbon budget are poorly understood. We propose a method to quantify the mycorrhizal contribution to carbon cycling, explicitly accounting for the abundance of plant-associated and extraradical mycorrhizal mycelium. We discuss the need to acquire additional data to use our method, and present our new global database holding information on plant species-by-site intensity of root colonization by mycorrhizas. We demonstrate that the degree of mycorrhizal fungal colonization has globally consistent patterns across plant species. This suggests that the level of plant species-specific root colonization can be used as a plant trait. To exemplify our method, we assessed the differential impacts of AM : EM ratio and EM shrub encroachment on carbon stocks in sub-arctic tundra. AM and EM affect tundra carbon stocks at different magnitudes, and via partly distinct dominant pathways: via extraradical mycelium (both EM and AM) and via mycorrhizal impacts on above- and belowground biomass carbon (mostly AM). Our method provides a powerful tool for the quantitative assessment of mycorrhizal impact on local and global carbon cycling processes, paving the way towards an improved understanding of the role of mycorrhizas in the Earth's carbon cycle. PMID:26011828

  2. Detection of Structural and Metabolic Changes in Traumatically Injured Hippocampus by Quantitative Differential Proteomics

    PubMed Central

    Wu, Ping; Zhao, Yingxin; Haidacher, Sigmund J.; Wang, Enyin; Parsley, Margaret O.; Gao, Junling; Sadygov, Rovshan G.; Starkey, Jonathan M.; Luxon, Bruce A.; Spratt, Heidi; DeWitt, Douglas S.; Prough, Donald S.

    2013-01-01

    Abstract Traumatic brain injury (TBI) is a complex and common problem resulting in the loss of cognitive function. In order to build a comprehensive knowledge base of the proteins that underlie these cognitive deficits, we employed unbiased quantitative mass spectrometry, proteomics, and bioinformatics to identify and quantify dysregulated proteins in the CA3 subregion of the hippocampus in the fluid percussion model of TBI in rats. Using stable isotope 18O-water differential labeling and multidimensional tandem liquid chromatography (LC)-MS/MS with high stringency statistical analyses and filtering, we identified and quantified 1002 common proteins, with 124 increased and 76 decreased. The Ingenuity Pathway Analysis (IPA) bioinformatics tool identified that TBI had profound effects on downregulating global energy metabolism, including glycolysis, the Krebs cycle, and oxidative phosphorylation, as well as cellular structure and function. Widespread upregulation of actin-related cytoskeletal dynamics was also found. IPA indicated a common integrative signaling node, calcineurin B1 (CANB1, CaNBα, or PPP3R1), which was downregulated by TBI. Western blotting confirmed that the calcineurin regulatory subunit, CANB1, and its catalytic binding partner PP2BA, were decreased without changes in other calcineurin subunits. CANB1 plays a critical role in downregulated networks of calcium signaling and homeostasis through calmodulin and calmodulin-dependent kinase II to highly interconnected structural networks dominated by tubulins. This large-scale knowledge base lays the foundation for the identification of novel therapeutic targets for cognitive rescue in TBI. PMID:22757692

  3. Highly sensitive quantitative PCR for the detection and differentiation of Pseudogymnoascus destructans and other Pseudogymnoascus species.

    PubMed

    Shuey, Megan M; Drees, Kevin P; Lindner, Daniel L; Keim, Paul; Foster, Jeffrey T

    2014-03-01

    White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi. PMID:24375140

  4. Transcriptome Analysis of Differentially Expressed Genes Provides Insight into Stolon Formation in Tulipa edulis.

    PubMed

    Miao, Yuanyuan; Zhu, Zaibiao; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

    2016-01-01

    Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

  5. Transcriptome Analysis of Differentially Expressed Genes Provides Insight into Stolon Formation in Tulipa edulis

    PubMed Central

    Miao, Yuanyuan; Zhu, Zaibiao; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

    2016-01-01

    Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

  6. Analysis of genes that are differentially expressed during the Sclerotinia sclerotiorum–Phaseolus vulgaris interaction

    PubMed Central

    Oliveira, Marília B.; de Andrade, Rosângela V.; Grossi-de-Sá, Maria F.; Petrofeza, Silvana

    2015-01-01

    The fungus Sclerotinia sclerotiorum (Lib.) de Bary, one of the most important plant pathogens, causes white mold on a wide range of crops. Crop yield can be dramatically decreased due to this disease, depending on the plant cultivar and environmental conditions. In this study, a suppression subtractive hybridization cDNA library approach was used for the identification of pathogen and plant genes that were differentially expressed during infection of the susceptible cultivar BRS Pérola of Phaseolus vulgaris L. A total of 979 unigenes (430 contigs and 549 singletons) were obtained and classified according to their functional categories. The transcriptional profile of 11 fungal genes related to pathogenicity and virulence were evaluated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Additionally, the temporal expression profile obtained by RT-qPCR was evaluated for the following categories of plant defense-related genes: pathogenesis-related genes (PvPR1, PvPR2, and PvPR3), phenylpropanoid pathway genes (PvIsof, PvFPS1, and 4CL), and genes involved in defense and stress-related categories (PvLox, PvHiprp, PvGST, PvPod, and PvDox). Data obtained in this study provide a starting point for achieving a better understanding of the pathosystem S. sclerotiorum–P. vulgaris. PMID:26579080

  7. Differential expression of long non-coding RNAs in hyperoxia-induced bronchopulmonary dysplasia.

    PubMed

    Bao, Tian-Ping; Wu, Rong; Cheng, Huai-Ping; Cui, Xian-Wei; Tian, Zhao-Fang

    2016-07-01

    Bronchopulmonary dysplasia (BPD) is a common complication of premature birth that seriously affects the survival rate and quality of life among preterm neonates. Long non-coding RNAs (lncRNAs) have been implicated in many human diseases. However, the role of lncRNAs in the pathogenesis of BPD remains poorly understood. Here, we exposed neonatal C57BL/6J mice to 95% concentrations of ambient oxygen and established a mouse lung injury model that mimicked human BPD. Next, we compared lncRNA and messenger RNA (mRNA) expression profiles between BPD and normal lung tissues using a high-throughput mouse lncRNA + mRNA array system. Compared with the control group, 882 lncRNAs were upregulated, and 887 lncRNAs were downregulated in BPD lung tissues. We validated some candidate BPD-associated lncRNAs by real-time quantitative reverse-transcription polymerase chain reaction analysis in eight pairs of BPD and normal lung tissues. Gene ontology, pathway and bioinformatics analyses revealed that a downregulated lncRNA, namely AK033210, associated with tenascin C may be involved in the pathogenesis of BPD. To the best of our knowledge, our study is the first to reveal differential lncRNA expression in BPD, which provides a foundation for further understanding of the molecular mechanism of BPD development. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27137150

  8. Differentially Expressed miRNAs in Tumor, Adjacent, and Normal Tissues of Lung Adenocarcinoma

    PubMed Central

    Tian, Fei; Li, Rui; Chen, Zhenzhu; Shen, Yanting; Lu, Jiafeng; Xie, Xueying; Ge, Qinyu

    2016-01-01

    Lung cancer is the leading cause of cancer deaths. Non-small-cell lung cancer (NSCLC) is the major type of lung cancer. The aim of this study was to characterize the expression profiles of miRNAs in adenocarcinoma (AC), one major subtype of NSCLC. In this study, the miRNAs were detected in normal, adjacent, and tumor tissues by next-generation sequencing. Then the expression levels of differential miRNAs were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In the results, 259, 401, and 389 miRNAs were detected in tumor, adjacent, and normal tissues of pooled AC samples, respectively. In addition, for the first time we have found that miR-21-5p and miR-196a-5p were gradually upregulated from normal to adjacent to tumor tissues; miR-218-5p was gradually downregulated with 2-fold or greater change in AC tissues. These 3 miRNAs were validated by qRT-PCR. Lastly, we predicted target genes of these 3 miRNAs and enriched the potential functions and regulatory pathways. The aberrant miR-21-5p, miR-196a-5p, and miR-218-5p may become biomarkers for diagnosis and prognosis of lung adenocarcinoma. This research may be useful for lung adenocarcinoma diagnosis and the study of pathology in lung cancer. PMID:27247934

  9. Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma

    PubMed Central

    Eisenhofer, Graeme; Huynh, Thanh-Truc; Elkahloun, Abdel; Morris, John C.; Bratslavsky, Gennady; Linehan, W. Marston; Zhuang, Zhengping; Balgley, Brian M.; Lee, Cheng S.; Mannelli, Massimo; Lenders, Jacques W. M.; Bornstein, Stefan R.; Pacak, Karel

    2008-01-01

    Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 ± 0.094 vs. 0.018 ± 0.009 day−1), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease. PMID:18854424

  10. Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma.

    PubMed

    Eisenhofer, Graeme; Huynh, Thanh-Truc; Elkahloun, Abdel; Morris, John C; Bratslavsky, Gennady; Linehan, W Marston; Zhuang, Zhengping; Balgley, Brian M; Lee, Cheng S; Mannelli, Massimo; Lenders, Jacques W M; Bornstein, Stefan R; Pacak, Karel

    2008-11-01

    Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 +/- 0.094 vs. 0.018 +/- 0.009 day(-1)), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease. PMID:18854424

  11. Differential expression of anti-angiogenic factors and guidance genes in the developing macula

    PubMed Central

    Kozulin, Peter; Natoli, Riccardo; O’Brien, Keely M. Bumsted; Madigan, Michele C.

    2009-01-01

    Purpose The primate retina contains a specialized, cone-rich macula, which mediates high acuity and color vision. The spatial resolution provided by the neural retina at the macula is optimized by stereotyped retinal blood vessel and ganglion cell axon patterning, which radiate away from the macula and reduce shadowing of macular photoreceptors. However, the genes that mediate these specializations, and the reasons for the vulnerability of the macula to degenerative disease, remain obscure. The aim of this study was to identify novel genes that may influence retinal vascular patterning and definition of the foveal avascular area. Methods We used RNA from human fetal retinas at 19–20 weeks of gestation (WG; n=4) to measure differential gene expression in the macula, a region nasal to disc (nasal) and in the surrounding retina (surround) by hybridization to 12 GeneChip® microarrays (HG-U133 Plus 2.0). The raw data was subjected to quality control assessment and preprocessing, using GC-RMA. We then used ANOVA analysis (Partek® Genomic Suite™ 6.3) and clustering (DAVID website) to identify the most highly represented genes clustered according to “biological process.” The neural retina is fully differentiated at the macula at 19–20 WG, while neuronal progenitor cells are present throughout the rest of the retina. We therefore excluded genes associated with the cell cycle, and markers of differentiated neurons, from further analyses. Significantly regulated genes (p<0.01) were then identified in a second round of clustering according to molecular/reaction (KEGG) pathway. Genes of interest were verified by quantitative PCR (QRT–PCR), and 2 genes were localized by in situ hybridization. Results We generated two lists of differentially regulated genes: “macula versus surround” and “macula versus nasal.” KEGG pathway clustering of the filtered gene lists identified 25 axon guidance-related genes that are differentially regulated in the macula

  12. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    PubMed Central

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  13. Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction.

    PubMed

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin RNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction, and data analysis. PMID:27207283

  14. Analysis of Differentially Expressed Genes Associated with Coronatine-Induced Laticifer Differentiation in the Rubber Tree by Subtractive Hybridization Suppression.

    PubMed

    Zhang, Shi-Xin; Wu, Shao-Hua; Chen, Yue-Yi; Tian, Wei-Min

    2015-01-01

    The secondary laticifer in the secondary phloem is differentiated from the vascular cambia of the rubber tree (Hevea brasiliensis Muell. Arg.). The number of secondary laticifers is closely related to the rubber yield potential of Hevea. Pharmacological data show that jasmonic acid and its precursor linolenic acid are effective in inducing secondary laticifer differentiation in epicormic shoots of the rubber tree. In the present study, an experimental system of coronatine-induced laticifer differentiation was developed to perform SSH identification of genes with differential expression. A total of 528 positive clones were obtained by blue-white screening, of which 248 clones came from the forward SSH library while 280 clones came from the reverse SSH library. Approximately 215 of the 248 clones and 171 of the 280 clones contained cDNA inserts by colony PCR screening. A total of 286 of the 386 ESTs were detected to be differentially expressed by reverse northern blot and sequenced. Approximately 147 unigenes with an average length of 497 bp from the forward and 109 unigenes with an average length of 514 bp from the reverse SSH libraries were assembled and annotated. The unigenes were associated with the stress/defense response, plant hormone signal transduction and structure development. It is suggested that Ca2+ signal transduction and redox seem to be involved in differentiation, while PGA and EIF are associated with the division of cambium initials for COR-induced secondary laticifer differentiation in the rubber tree. PMID:26147807

  15. Expression of HNFs and C/EBP alpha is correlated with immunocytochemical differentiation of cell lines derived from human hepatocellular carcinomas, hepatoblastomas and immortalized hepatocytes.

    PubMed

    Ishiyama, Tadashi; Kano, Junko; Minami, Yuko; Iijima, Tatsuo; Morishita, Yukio; Noguchi, Masayuki

    2003-09-01

    Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver-enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBP alpha using real-time reverse-transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B-17/Icrj-scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF-3beta, HNF-4 alpha, HNF-1 alpha, and C/EBP alpha were specific to HCCs with well-differentiated function and morphology. Furthermore, among these four transcription factors, HNF-4 alpha and HNF-1 alpha expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF-4 alpha and HNF-1 alpha are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBP alpha in HCCs. PMID:12967472

  16. HNF4α Regulates Claudin-7 Protein Expression during Intestinal Epithelial Differentiation

    PubMed Central

    Farkas, Attila E.; Hilgarth, Roland S.; Capaldo, Christopher T.; Gerner-Smidt, Christian; Powell, Doris R.; Vertino, Paula M.; Koval, Michael; Parkos, Charles A.; Nusrat, Asma

    2016-01-01

    The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt–luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation. PMID:26216285

  17. Selecting key genes associated with osteosarcoma based on a differential expression network.

    PubMed

    Wang, Y B; Jia, N; Xu, C M; Zhao, L; Zhao, Y; Wang, X; Jia, T H

    2015-01-01

    Despite recent advances in osteosarcoma diagnosis and therapy, much remains unclear about the molecular mechanisms involved in the disorder, and the discovery of novel drug-targeted genes is essential. We explored the potential molecular mechanisms and target genes involved in the development and progression of osteosarcoma. First, we identified the differentially expressed genes in osteosarcoma patients and matching normal controls. We then constructed a differential expression network based on differential and non-differential interactions. Pathway-enrichment analysis was performed based on the nodes contained in the main differential expression network. Centrality analysis was used to select hub genes that may play vital roles in the progression of human osteosarcoma. Our research revealed a total of 176 differentially expressed genes including 82 upregulated and 94 downregulated genes. A differential expression network was constructed that included 992 gene pairs (1043 nodes). Pathway-enrichment analysis indicated that the nodes in the differential expression network were mainly enriched in several pathways such as those involved in cancer, cell cycle, ubiquitin-mediated proteolysis, DNA replication, ribosomes, T-cell receptor signaling, spliceosomes, neurotrophin signaling, oxidative phosphorylation, and tight junctions. Six hub genes (APP, UBC, CAND1, RPA, YWHAG, and NEDD8) were discovered; of these, two genes (UBC and RPA) were also found to be disease genes. Our study predicted that UBC and RPA had potential as target genes for the diagnosis and treatment of osteosarcoma. PMID:26782416

  18. A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes.

    PubMed

    Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

    2015-01-01

    In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data. PMID:26201006

  19. A comparison of statistical tests for detecting differential expression using Affymetrix oligonucleotide microarrays.

    PubMed

    Vardhanabhuti, Saran; Blakemore, Steven J; Clark, Steven M; Ghosh, Sujoy; Stephens, Richard J; Rajagopalan, Dilip

    2006-01-01

    Signal quantification and detection of differential expression are critical steps in the analysis of Affymetrix microarray data. Many methods have been proposed in the literature for each of these steps. The goal of this paper is to evaluate several signal quantification methods (GCRMA, RSVD, VSN, MAS5, and Resolver) and statistical methods for differential expression (t test, Cyber-T, SAM, LPE, RankProducts, Resolver RatioBuild). Our particular focus is on the ability to detect differential expression via statistical tests. We have used two different datasets for our evaluation. First, we have used the HG-U133 Latin Square spike in dataset developed by Affymetrix. Second, we have used data from an in-house rat liver transcriptomics study following 30 different drug treatments generated using the Affymetrix RAE230A chip. Our overall recommendation based on this study is to use GCRMA for signal quantification. For detection of differential expression, GCRMA coupled with Cyber-T or SAM is the best approach, as measured by area under the receiver operating characteristic (ROC) curve. The integrated pipeline in Resolver RatioBuild combining signal quantification and detection of differential expression is an equally good alternative for detecting differentially expressed genes. For most of the differential expression algorithms we considered, the performance using MAS5 signal quantification was inferior to that of the other methods we evaluated. PMID:17233564

  20. Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage

    PubMed Central

    Pearson, Mark J.; Philp, Ashleigh M.; Heward, James A.; Roux, Benoit T.; Walsh, David A.; Davis, Edward T.; Lindsay, Mark A.

    2016-01-01

    Objective To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. Methods OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non‐OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase–polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. Results RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin‐1β (IL‐1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50‐associated cyclooxygenase 2–extragenic RNA (PACER) and 2 novel chondrocyte inflammation–associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non‐OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL‐1–stimulated secretion of proinflammatory cytokines. Conclusion The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role

  1. Characterization and identification of differentially expressed microRNAs during the process of the peribiliary fibrosis induced by Clonorchis sinensis.

    PubMed

    Yan, Chao; Shen, Li-Ping; Ma, Rui; Li, Bo; Li, Xiang-Yang; Hua, Hui; Zhang, Bo; Yu, Qian; Wang, Yu-Gang; Tang, Ren-Xian; Zheng, Kui-Yang

    2016-09-01

    Clonorchis sinensis (C. sinensis) infection can lead to biliary fibrosis. MicroRNAs (miRNAs) play important roles in regulation of genes expression in the liver diseases. However, the differential expression of miRNAs that probably regulates the portal fibrogenesis caused by C. sinensis has not yet been investigated. Hepatic miRNAs expression profiles from C. sinensis-infected mice at different time-points were analyzed by miRNA microarray and validated by quantitative real-time PCR (qRT-PCR). 349 miRNAs were differentially expressed in the liver of the C. sinensis-infected mice at 2, 8 or 16weeks post infection (p.i.), compared with those at 0week p.i., and there were 143 down-regulated and 206 up-regulated miRNAs among them. These all dysregulated miRNAs were potentially involved in the pathological processes of clonorchiasis by regulation of cancer-related signaling pathway, TGF-β signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, PI3K /AKT signaling pathway, etc. 169 of these dysregulated miRNAs were predicted to be involved in the TGF/Smads signaling pathway which plays an important role in the biliary fibrosis caused by C. sinensis. Additionally, miRNA-32, miRNA-34a, miRNA-125b and miRNA-497 were negatively correlated with Smad7 expression, indicating these miRNAs may specifically down-regulate Smad7 expression and participate in regulation of biliary fibrosis caused by C. sinensis. The results of the present study for the first time demonstrated that miRNAs were differentially expressed in the liver of mice infected by C. sinensis, and these miRNAs may play important roles in regulation of peribiliary fibrosis caused by C. sinensis, which may provide possible therapeutic targets for clonorchiasis. PMID:27267304

  2. Differential gene expression in anatomical compartments of the human eye

    PubMed Central

    Diehn, Jennifer J; Diehn, Maximilian; Marmor, Michael F; Brown, Patrick O

    2005-01-01

    Background The human eye is composed of multiple compartments, diverse in form, function, and embryologic origin, that work in concert to provide us with our sense of sight. We set out to systematically characterize the global gene expression patterns that specify the distinctive characteristics of the various eye compartments. Results We used DNA microarrays representing approximately 30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. The distinctive patterns of expression in each compartment could be interpreted in relation to the physiology and cellular composition of each tissue. Notably, the sets of genes selectively expressed in the retina and in the lens were particularly large and diverse. Genes with roles in immune defense, particularly complement components, were expressed at especially high levels in the anterior segment tissues. We also found consistent differences between the gene expression patterns of the macula and peripheral retina, paralleling the differences in cell layer densities between these regions. Based on the hypothesis that genes responsible for diseases that affect a particular eye compartment are likely to be selectively expressed in that compartment, we compared our gene expression signatures with genetic mapping studies to identify candidate genes for diseases affecting the cornea, lens, and retina. Conclusion Through genome-scale gene expression profiling, we were able to discover distinct gene expression 'signatures' for each eye compartment and identified candidate disease genes that can serve as a reference database for investigating the physiology and pathophysiology of the eye. PMID:16168081

  3. Identification and transcriptional profiling of differentially expressed genes associated with response to UVA radiation in Drosophila melanogaster (Diptera: Drosophilidae).

    PubMed

    Zhou, Li-Jun; Zhu, Zhi-Hui; Liu, Zhen-Xing; Ma, Wei-Hua; Desneux, Nicolas; Lei, Chao-Liang

    2013-10-01

    Ultraviolet A (UVA) radiation, the major component of solar ultraviolet (UV) radiation reaching the earth's surface, leads to negative effects in insects, such as oxidative stress, photoreceptor damage, and cell death. To better understand the molecular mechanisms of insect response to UVA radiation, suppression subtractive hybridization (SSH) and real-time quantitative polymerase chain reaction approaches were combined to reveal differential transcript expression in Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae). In this study, two subtractive cDNA libraries were constructed and sequenced, obtaining 131 high-quality unique expressed sequence tags (ESTs) that were up- or downregulated in D. melanogaster exposed to UVA radiation for 0.5 h. Of the 131 ESTs, 102 unique ESTs were differentially expressed and classified into 10 functional categories. The results showed that UVA radiation induces expression of genes related to stress and defense response and metabolism. Potential transcription factor binding motifs upstream of these genes are associated with multiple signaling pathways that may help the insect cope with the stress of UVA radiation. To our knowledge, this is the first analysis of insect response to UVA radiation at the transcriptional level. Our results reveal that UVA radiation influences the expression profiles of stress-responsive genes and provide further insights into the mechanisms of adaptive response to UVA radiation stress. PMID:24331622

  4. Identification of differentially expressed genes in hepatopancreas of oriental river prawn, Macrobrachium nipponense exposed to environmental hypoxia.

    PubMed

    Sun, Shengming; Xuan, Fujun; Ge, Xianping; Fu, Hongtuo; Zhu, Jian; Zhang, Shiyong

    2013-10-25

    Hypoxia represents a major physiological challenge for prawn culture, and the hepatopancreas plays an important role in these processes. Here, we applied high-throughput sequencing technology to detect the gene expression profile of the hepatopancreas in M. nipponense in response to hypoxia for 3h and hypoxia for 24h. Gene expression profiling identified 1925 genes that were significantly up- or down-regulated by dissolved oxygen availability. Functional categorization of the differentially expressed genes revealed that oxygen transport, electron transport chain, reactive oxygen species generation/scavenging, and immune response were the differentially regulated processes occurring during environmental hypoxia. Finally, quantitative real-time polymerase chain reaction using six genes independently verified the tag-mapped results. Immunohistochemistry analysis revealed, for the first time, hemocyanin protein expression as significant hypoxia-specific signatures in prawns, which opens the way for in depth molecular studies of hypoxia exposure. The analysis of changes in hepatic gene expression in oriental river prawn provides a preliminary basis for a better understanding of the molecular response to hypoxia exposures. PMID:24513331

  5. mef2 activity levels differentially affect gene expression during Drosophila muscle development

    PubMed Central

    Elgar, Stuart J.; Han, Jun; Taylor, Michael V.

    2008-01-01

    Cell differentiation is controlled by key transcription factors, and a major question is how they orchestrate cell-type-specific genetic programs. Muscle differentiation is a well studied paradigm in which the conserved Mef2 transcription factor plays a pivotal role. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. In this study we used a strategy combining genomics and developmental genetics to address this issue in vivo during Drosophila muscle development. We found that groups of mef2-regulated genes respond differently to changes in mef2 activity levels: some require higher levels for their expression than others. Furthermore, this differential requirement correlates with when the gene is first expressed during the muscle differentiation program. Genes that require higher levels are activated later. These results implicate mef2 in the temporal regulation of muscle gene expression, and, consistent with this, we show that changes in mef2 activity levels can alter the start of gene expression in a predictable manner. Together these results indicate that Mef2 is not an all-or-none regulator; rather, its action is more subtle, and levels of its activity are important in the differential expression of muscle genes. This suggests a route by which mef2 can orchestrate the muscle differentiation program and contribute to the stringent regulation of gene expression during myogenesis. PMID:18198273

  6. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

    SciTech Connect

    Tsukasaki, Masayuki; Yamada, Atsushi; Suzuki, Dai; Aizawa, Ryo; Miyazono, Agasa; Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro; Morimura, Naoko; Yamamoto, Matsuo; Kamijo, Ryutaro

    2011-07-15

    Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.

  7. Transcriptome analysis of differentially expressed genes during embryo sac development in apomeiotic non-parthenogenetic interspecific hybrid of Pennisetum glaucum.

    PubMed

    Sahu, Pranav Pankaj; Gupta, Sarika; Malaviya, D R; Roy, Ajoy Kumar; Kaushal, Pankaj; Prasad, Manoj

    2012-07-01

    Apomixis results in the production of genetically uniform progeny, derived from the fertilization independent development (parthenogenesis) of an unreduced egg cell (apomeiosis). To identify genes involved in the apomeiosis, a comparative transcriptome analysis of differentially expressed genes during embryo sac (ES) development in a sexual Pennisetum glaucum (genotype 81A1) and its apomeiotic (aposporic) non-parthenogenetic interspecific hybrid (BC1GO) was investigated. BC1GO exhibited the partitioned apomeiosis component, whereby the second apomixis component viz., parthenogenesis was completely lacking. A total of 96 non-redundant transcripts were recovered using suppression subtractive hybridization and classified into 11 different categories according to their putative functions. Amongst the identified transcripts, many of them belonged to unknown function (40%) followed by those involved in protein metabolism, stress response, pollen/ovule/embryo development, and translation/protein modification process. A data search of transcriptional profiling in other apomictic species revealed that 75% of the differentially expressed transcripts have not been reported in previous studies. By macroarray analysis, we identified differential expression pattern of 96 transcripts, 45 (47%) of which showed ≥2-fold induction in apomeiotic BC1GO. Further, the obtained results were validated by quantitative real-time polymerase chain reaction to have a comparative expression profiling of eight selected up-regulated transcripts (≥2.5-fold) between BC1GO and 81A1 at different phases of ovule development. In silico mapping demonstrated that 13 transcripts were located onto rice chromosome 2, region syntenic with the apospory locus as reported in Brachiaria brizantha and Paspalum notatum. The expression patterns of these transcripts showed a significant difference at differentiating megaspore mother cell and gametogenesis stages thereby suggesting their involvement in floral

  8. [Gene differential expression of liver tissues in crossbred versus purebred chicken and their relationship with heterosis of meat trait].

    PubMed

    Wang, Dong; Zhang, Yuan; Sun, Dong-Xiao; Yu, Ying; Xu, Gui-Yun; Li, Jun-Ying

    2004-03-01

    The concept of heterosis has already been put forward for a century. The hypothesis of Dominance, Superdominace and Epistasis has also been brought forward to explain the phenomenon of heterosis. As we know, there is spatio-temporal speciality about the expression of gene and only expressed genes contribute to the formation of heterosis. So the study on heterosis in expression level becomes more meaningful. A lot of studies on heterosis in this level have been done in plants, but there is no such study carried on animals in this area. In this study, the technique of mRNA Reverse Transcription Differential Display was used to research the heterosis molecular mechanism of animal. In order to expound the molecular genetic mechanism of animals heterosis, the 4 x 4 completely diallele cross experiment of 4 purebreds chicken was conducted among White Polymouth Rock (EE), Chinese Silk Chicken (CC), CAU Brown (DD) and White Leghorn (AA). The chicken of 16 cross combinations were reared to 8 weeks old, then 30 chicken in each combination were selected randomly and slaughtered. The traits of body weight of 8 weeks, wing weight, eviscerated weight, eviscerated weight with giblet, breast muscle yield, leg muscle yield, body length, abdomen fat weight, intramuscular fat width, tibia length were measured, and in which 8 individuals in each combination were selected randomly to collect the liver tissue samples, which were stored in liquid nitrogen or at -80 degrees C to be used for total RNA (TRNA) extracting. After the total RNA (TRNA) was extracted, 16 TRNA pools were formed in the same quantitative according to the concentration of 8 individual TRNA. They were reversely transcribed with three anchor primers H-T11 A, H-T11 G and H-T11 C. Then the reverse transcription PCR for each transcript product was done in two repeats at the same time with the same anchor primers and 8 random primers. The polyacrylamide gel electrophoresis of each PCR product was run in Bio-Rad Power 3

  9. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

    PubMed Central

    Shang, Jin; Fan, Xin; Shangguan, Lei; Liu, Huan; Zhou, Yue

    2015-01-01

    Low back pain (LBP) is a very prevalent disease and degenerative disc diseases (DDDs) usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0) to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale. PMID:26649308

  10. Expression patterns of gonadotropin hormones and their receptors during early sexual differentiation in Nile tilapia Oreochromis niloticus.

    PubMed

    Yan, Hongwei; Ijiri, Shigeho; Wu, Quan; Kobayashi, Tohru; Li, Shuang; Nakaseko, Taro; Adachi, Shinji; Nagahama, Yoshitaka

    2012-11-01

    In Nile tilapia, sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads at 5-6 days after hatching (dah) is critical for differentiation of the gonads into either ovaries or testes. The factors triggering sexually dimorphic expression of these genes are unknown, and whether the gonadotropin hormones are involved in early gonadal sex differentiation of the Nile tilapia has been unclear. In the present study, we determined the precise timing of expression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary and that of their receptors (fshra and lhcgrbb) in the undifferentiated gonad in both XX and XY tilapia fry by quantitative RT-PCR and immunohistochemical analysis. Expression of fshb mRNA and Fsh protein in the pituitary was detected from the first sampling day (3 dah) to 25 dah in both XX and XY tilapia larvae without sexual dimorphism and increased gradually after 25 dah in the pituitary. fshra mRNA was expressed beginning 5 dah and was present at significantly higher levels in XX gonads than in the XY gonads at 6-25 dah. These results indicate that the level of Fsh protein in the pituitary was not critical for differentiation of gonads into ovaries or testes, but the expression level of its receptor, fshra, in undifferentiated gonads appeared to be involved in determining gonadal sexual differentiation. Based on these observations, it is likely that in XX gonads, up-regulation of fshra may be necessary to induce cyp19a1a expression, which stimulates estradiol-17beta (E(2)) production and subsequent ovarian differentiation. On the other hand, lhb mRNA was not detected until 25 dah in the pituitaries of both sexes, and sexual dimorphism in lhcgrbb mRNA levels appeared later (10-25 dah) than that of fshra in the gonads, indicating the limited role of LH and lhcgrbb in gonadal differentiation of the Nile tilapia. PMID:23018182

  11. MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

    PubMed Central

    Guglielmi, L; Cinnella, C; Nardella, M; Maresca, G; Valentini, A; Mercanti, D; Felsani, A; D'Agnano, I

    2014-01-01

    Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma

  12. Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

    PubMed

    Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

    2015-12-17

    Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. PMID

  13. Morphine sulfate concomitantly decreases neuronal differentiation and opioid receptor expression in mouse embryonic stem cells.

    PubMed

    Dholakiya, Sanjay L; Aliberti, Angela; Barile, Frank A

    2016-04-15

    Opioids have been shown to affect prenatal and postnatal neural development in mammals. The present study investigates the impact of morphine sulfate (MS) treatment on neuronal differentiation as well as μ-opioid receptor (MOR) expression in mouse embryonic stem (mES) cells. Stem cells were manipulated in culture to differentiate in 3 sequential stages: Stage 1, cell transformation to embryoid bodies (EB); Stage 2, EB cell differentiation to neural progenitor (NP) cells; and, Stage 3, NP cell differentiation to neurons/astrocytes co-cultured cells. Using RT-PCR and flow cytometry analyses, cell types were confirmed by monitoring expression of Oct4, nestin, microtubule-associated protein 2 (mtap-2), and glial fibrillary acidic protein (GFAP) as cell-specific markers for stem cells, NP cells, neurons, and astrocytes, respectively. Similarly, gene expression for MOR, κ-opioid receptor (KOR), and δ-opioid receptor (DOR) was confirmed in each cell type. In order to investigate the effects of MS on differentiation, cells were treated with MS (1, 10, 100 μM) at either early (Stage 1) or late (Stage 3) stage of cellular differentiation. At Stage 1 exposure, MOR gene expression and neuroectoderm specific marker expression of nestin were down-regulated in both EB and NP cells. In addition, the opioid down-regulated GFAP in differentiated neurons/astrocytes co-cultured cells. Late stage treatment with MS resulted in a down-regulation of mtap-2 and GFAP in differentiated neurons/astrocytes co-cultured cells. Moreover, late stage treatment with MS and naltrexone inhibited the effect of MS on neuronal differentiation, suggesting that MS treatment interferes with differentiation via MOR activation. Together, the results show that MS exposure at early and late stage of cellular differentiation significantly decreases genotype and phenotype in differentiated neuronal cells. The results of this study have implications regarding the potential effect of opiates on fetal brain

  14. Expression of IL-4/IL-13 receptors in differentiating human airway epithelial cells.

    PubMed

    White, Steven R; Martin, Linda D; Stern, Randi; Laxman, Bharathi; Marroquin, Bertha A

    2010-11-01

    IL-4 and IL-13 elicit several important responses in airway epithelium including chemokine secretion and mucous secretion that may contribute to airway inflammation, cell migration, and differentiation. These cytokines have overlapping but not identical effector profiles likely due to shared subunits in their receptor complexes. These receptors are variably described in epithelial cells, and the relative expression, localization, and function of these receptors in differentiated and repairing epithelial cells are not clear. We examined IL-4/IL-13 receptor expression and localization in primary airway epithelial cells collected from normal human lungs and grown under conditions yielding both undifferentiated and differentiated cells inclusive of basal, goblet, and ciliated cell phenotypes. Gene expression of the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2 receptor subunits increased with differentiation, but different patterns of localization and protein abundance were seen for each subunit based on both differentiation and the cell subtypes present. Increased expression of receptor subunits observed in more differentiated cells was associated with more substantial functional responses to IL-4 stimulation including increased eotaxin-3 expression and accelerated migration after injury. We demonstrate substantial differences in IL-4/IL-13 receptor subunit expression and responsiveness to IL-4 based on the extent of airway epithelial cell differentiation and suggest that these differences may have functional consequences in airway inflammation. PMID:20729386

  15. An Exercise to Estimate Differential Gene Expression in Human Cells

    ERIC Educational Resources Information Center

    Chaudhry, M. Ahmad

    2006-01-01

    The expression of genes in cells of various tissue types varies considerably and is correlated with the function of a particular organ. The pattern of gene expression changes in diseased tissues, in response to therapy or infection and exposure to environmental mutagens, chemicals, ultraviolet light, and ionizing radiation. To better understand…

  16. Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis

    PubMed Central

    Chen, Yongxin; Gelfond, Jonathan AL; McManus, Linda M; Shireman, Paula K

    2009-01-01

    Background MicroRNAs (miRNAs) have critical functions in various biological processes. MiRNA profiling is an important tool for the identification of differentially expressed miRNAs in normal cellular and disease processes. A technical challenge remains for high-throughput miRNA expression analysis as the number of miRNAs continues to increase with in silico prediction and experimental verification. Our study critically evaluated the performance of a novel miRNA expression profiling approach, quantitative RT-PCR array (qPCR-array), compared to miRNA detection with oligonucleotide microchip (microarray). Results High reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array. Conclusion Our studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and q

  17. Differential expression of two scribble isoforms during Drosophila embryogenesis.

    PubMed

    Li, M; Marhold, J; Gatos, A; Török, I; Mechler, B M

    2001-10-01

    The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones. PMID:11578873

  18. Identification of Novel Reference Genes Using Multiplatform Expression Data and Their Validation for Quantitative Gene Expression Analysis

    PubMed Central

    Kwon, Mi Jeong; Oh, Ensel; Lee, Seungmook; Roh, Mi Ra; Kim, Si Eun; Lee, Yangsoon; Choi, Yoon-La; In, Yong-Ho; Park, Taesung; Koh, Sang Seok; Shin, Young Kee

    2009-01-01

    Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required. PMID:19584937

  19. Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines.

    PubMed

    Carroll, K; Ray, K; Helm, B; Carey, E

    2001-04-01

    The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion complex between granule and plasma membrane in a positive and negative manner through interaction with effector proteins. The pattern of Rab3 protein expression may, therefore, provide a subtle means of regulating exocytosis. To investigate the relationship between Rab3 expression and secretory activity, we assessed the differential expression of individual Rab3 proteins in high- and low-secreting clones of the rat basophilic (RBL) cell line. mRNAs for Rab3 isoforms (a-d) were analyzed by constructing cDNA libraries of high- and low-secreting RBL clones. The relative abundance of mRNAs for Rab3 isoforms was initially determined from the clonal frequency of corresponding cDNA clones. RT-PCR using isoform-specific primers was successfully applied to the quantitation of Rab3a mRNA. The presence of individual Rab3 proteins was revealed by SDS-PAGE and immunoblotting, and also by in situ immunofluorescence confocal microscopy. We present evidence that Rab3a and Rab3c are expressed at high levels in the low-secreting variant, while Rab3d is predominant in the high secretor. Levels of the Rab3 effector proteins, Rabphilin and Noc2, are similar in both RBL cell lines. Subcellular fractionation of unstimulated high and low secretor RBL clones revealed that in both cell types Rab3a has a cytoplasmic location while Rab3d is present in a membrane/organelle fraction containing secretory vesicles. Differences in the pattern of expression of Rab3 isoforms in the two RBL cell lines and their localisation may influence the secretory potential. Furthermore, the presence of Rab3 and effector proteins indicates that the mechanism for regulated exocytosis in cells of mast cells/basophil lineage appears similar to that in pre

  20. Differential expression pattern of UBX family genes in Caenorhabditis elegans

    SciTech Connect

    Yamauchi, Seiji; Sasagawa, Yohei; Ogura, Teru . E-mail: ogura@gpo.kumamoto-u.ac.jp; Yamanaka, Kunitoshi . E-mail: yamanaka@gpo.kumamoto-u.ac.jp

    2007-06-29

    UBX (ubiquitin regulatory X)-containing proteins belong to an evolutionary conserved protein family and determine the specificity of p97/VCP/Cdc48p function by binding as its adaptors. Caenorhabditis elegans was found to possess six UBX-containing proteins, named UBXN-1 to -6. However, no general or specific function of them has been revealed. During the course of understanding not only their function but also specified function of p97, we investigated spatial and temporal expression patterns of six ubxn genes in this study. Transcript analyses showed that the expression pattern of each ubxn gene was different throughout worm's development and may show potential developmental dynamics in their function, especially ubxn-5 was expressed specifically in the spermatogenic germline, suggesting a crucial role in spermatogenesis. In addition, as ubxn-4 expression was induced by ER stress, it would function as an ERAD factor in C. elegans. In vivo expression analysis by using GFP translational fusion constructs revealed that six ubxn genes show distinct expression patterns. These results altogether demonstrate that the expression of all six ubxn genes of C. elegans is differently regulated.

  1. Differential expression of the Wnt putative receptors Frizzled during mouse somitogenesis.

    PubMed

    Borello, U; Buffa, V; Sonnino, C; Melchionna, R; Vivarelli, E; Cossu, G

    1999-12-01

    The expression of eight murine Frizzled (1,3-9) genes was studied during mouse somitogenesis, in order to correlate the Wnt-dependent activation of myogenesis with the expression of specific Frizzled putative receptors. Frizzled 1, 3, 6, 7, 8, and 9 have specific expression in the forming and differentiating somites. The genes analyzed have a complex and partly overlapping pattern of expression in other regions of the embryo. PMID:10559494

  2. Differentially expressed proteins associated with Fusarium head blight resistance in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contr...

  3. Differentially-expressed glycoproteins in Locusta migratoria hemolymph infected with Metarhizium anisopliae.

    PubMed

    Wang, Chutao; Cao, Yueqing; Wang, Zhongkang; Yin, Youping; Peng, Guoxiong; Li, Zhenlun; Zhao, Hua; Xia, Yuxian

    2007-11-01

    Glycoproteins play important roles in insect physiology. Infection with pathogen always results in the differential expression of some glycoproteins, which may be involved in host-pathogen interactions. In this report, differentially-expressed glycoproteins from the hemolymph of locusts infected with Metarhizium anisopliae were analyzed by two-dimensional electrophoresis (2-DE) and PDQuest software. The results showed that 13 spots were differentially expressed, of which nine spots were upregulated and four were downregulated. Using MS/MS with de novo sequencing and NCBI database searches, three upregulated proteins were identified as locust transferrin, apolipoprotein precursor, and hexameric storage protein 3. These proteins have been reported to be involved in the insect innate immune response to microbial challenge. Due to the limited available genome information and protein sequences of locusts, the possible functions of the other 10 differentially-expressed spots remain unknown. PMID:17658547

  4. Robust Modeling of Differential Gene Expression Data Using Normal/Independent Distributions: A Bayesian Approach

    PubMed Central

    Ganjali, Mojtaba; Baghfalaki, Taban; Berridge, Damon

    2015-01-01

    In this paper, the problem of identifying differentially expressed genes under different conditions using gene expression microarray data, in the presence of outliers, is discussed. For this purpose, the robust modeling of gene expression data using some powerful distributions known as normal/independent distributions is considered. These distributions include the Student’s t and normal distributions which have been used previously, but also include extensions such as the slash, the contaminated normal and the Laplace distributions. The purpose of this paper is to identify differentially expressed genes by considering these distributional assumptions instead of the normal distribution. A Bayesian approach using the Markov Chain Monte Carlo method is adopted for parameter estimation. Two publicly available gene expression data sets are analyzed using the proposed approach. The use of the robust models for detecting differentially expressed genes is investigated. This investigation shows that the choice of model for differentiating gene expression data is very important. This is due to the small number of replicates for each gene and the existence of outlying data. Comparison of the performance of these models is made using different statistical criteria and the ROC curve. The method is illustrated using some simulation studies. We demonstrate the flexibility of these robust models in identifying differentially expressed genes. PMID:25910040

  5. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  6. Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction

    SciTech Connect

    Noonan, K.E.; Beck, C.; Holzmayer, T.A.; Chin, J.E.; Roninson, I.B. ); Wunder, J.S.; Andrulis, I.L. ); Gazdar, A.F. ); Willman, C.L.; Griffith, B. ); Von Hoff, D.D. )

    1990-09-01

    The resistance of tumor cells ot chemotheraprutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in bitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refactory tumors untreated with chemotherapeutic drugs. The authors have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clincal samples, based on the polymerase chain reaction. They have used this assay to measure MDR1 gene expression in MDR cell lines and >300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were ost common among tumor types known to be relatively responsive to chemotherapy.

  7. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

    PubMed

    Xu, R; Janson, C G; Mastakov, M; Lawlor, P; Young, D; Mouravlev, A; Fitzsimons, H; Choi, K L; Ma, H; Dragunow, M; Leone, P; Chen, Q; Dicker, B; During, M J

    2001-09-01

    This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette. PMID:11571569

  8. Differential gene expression between visceral and subcutaneous fat depots.

    PubMed

    Atzmon, G; Yang, X M; Muzumdar, R; Ma, X H; Gabriely, I; Barzilai, N

    2002-01-01

    Abdominal obesity has been linked to the development of insulin resistance and Type 2 diabetes mellitus (DM2). By surgical removal of visceral fat (VF) in a variety of rodent models, we prevented insulin resistance and glucose intolerance, establishing a cause-effect relationship between VF and the metabolic syndrome. To characterize the biological differences between visceral and peripheral fat depots, we obtained perirenal visceral (VF) and subcutaneous (SC) fat from 5 young rats. We extracted mRNA from the fat tissue and performed gene array hybridization using Affymetrix technology with a platform containing 9 000 genes. Out of the 1 660 genes that were expressed in fat tissue, 297 (17.9 %) genes show a two-fold or higher difference in their expression between the two tissues. We present the 20 genes whose expression is higher in VF fat (by 3 - 7 fold) and the 20 genes whose expression is higher in SC fat (by 3 - 150 fold), many of which are predominantly involved in glucose homeostasis, insulin action, and lipid metabolism. We confirmed the findings of gene array expression and quantified the changes in expression in VF of genes involved in insulin resistance (PPARgamma leptin) and its syndrome (angiotensinogen and plasminogen activating inhibitor-1, PAI-1) by real-time PCR (qRT-PCR) technology. Finally, we demonstrated increased expression of resistin in VF by around 12-fold and adiponectin by around 4-fold, peptides that were not part of the gene expression platform. These results indicate that visceral fat and subcutaneous fat are biologically distinct. PMID:12660871

  9. Ehrlichia chaffeensis Transcriptome in Mammalian and Arthropod Hosts Reveals Differential Gene Expression and Post Transcriptional Regulation

    PubMed Central

    Kuriakose, Jeeba A.; Miyashiro, Simone; Luo, Tian; Zhu, Bing; McBride, Jere W.

    2011-01-01

    Background Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. Methodology/Principal Findings The majority (∼80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30–80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. Conclusions/Significance Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes

  10. Differential expression of salivary glycoproteins in aggressive and chronic periodontitis

    PubMed Central

    ROCHA, Daniela de Morais; ZENÓBIO, Elton Gonçalves; VAN DYKE, Thomas; SILVA, Karine Simões; COSTA, Fernando Oliveira; SOARES, Rodrigo Villamarim

    2012-01-01

    Objectives The aim of this study was to compare the pattern of secretion and the expression of mucin glycoprotein-2 (MG2) and lactoferrin in individuals with or without periodontitis. Material and Methods Five individuals with aggressive periodontitis (APG), 5 with generalized chronic periodontitis (CPG) and 5 without periodontitis (CG) were enrolled after informed consent. Non-stimulated and stimulated submandibular and sublingual saliva was collected and samples analyzed by Western blot probed with specific antibodies. Results Stimulated and non-stimulated salivary flow rates did not differ among groups. Western blot analysis revealed that stimulation led to: an increase in MG2 expression in all groups, and to lactoferrin expression in APG and CPG. In non-stimulated saliva, CG exhibited the highest expression of both glycoproteins. In stimulated saliva, CG exhibited the highest expression of MG2, whereas APG the highest of lactoferrin. Conclusions The pattern of secretion of MG2 and lactoferrin in health and disease is complex. Although the present study analyzed samples from a limited number of participants, the reduced expression of MG2 and lactoferrin in APG and CPG under non-stimulated condition, the predominant circumstance of salivary secretion during the day, suggests that these salivary constituents may play a role in the etiopathogenesis of these diseases. PMID:22666834

  11. Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells

    PubMed Central

    Fathi, Ali; Hatami, Maryam; Hajihosseini, Vahid; Fattahi, Faranak; Kiani, Sahar; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2011-01-01

    Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation. PMID:21829537

  12. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  13. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells

    PubMed Central

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  14. CG Methylation Covaries with Differential Gene Expression between Leaf and Floral Bud Tissues of Brachypodium distachyon

    PubMed Central

    Roessler, Kyria; Takuno, Shohei; Gaut, Brandon S.

    2016-01-01

    DNA methylation has the potential to influence plant growth and development through its influence on gene expression. To date, however, the evidence from plant systems is mixed as to whether patterns of DNA methylation vary significantly among tissues and, if so, whether these differences affect tissue-specific gene expression. To address these questions, we analyzed both bisulfite sequence (BSseq) and transcriptomic sequence data from three biological replicates of two tissues (leaf and floral bud) from the model grass species Brachypodium distachyon. Our first goal was to determine whether tissues were more differentiated in DNA methylation than explained by variation among biological replicates. Tissues were more differentiated than biological replicates, but the analysis of replicated data revealed high (>50%) false positive rates for the inference of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). Comparing methylation to gene expression, we found that differential CG methylation consistently covaried negatively with gene expression, regardless as to whether methylation was within genes, within their promoters or even within their closest transposable element. The relationship between gene expression and either CHG or CHH methylation was less consistent. In total, CG methylation in promoters explained 9% of the variation in tissue-specific expression across genes, suggesting that CG methylation is a minor but appreciable factor in tissue differentiation. PMID:26950546

  15. Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

    NASA Technical Reports Server (NTRS)

    Shalhoub, V.; Conlon, D.; Tassinari, M.; Quinn, C.; Partridge, N.; Stein, G. S.; Lian, J. B.

    1992-01-01

    To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the

  16. Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology

    PubMed Central

    Lensen, Joost F. M.; van der Vlag, Johan; Versteeg, Elly M. M.; Wetzels, Jack F. M.; van den Heuvel, Lambert P. W. J.; Berden, Jo H. M.; van Kuppevelt, Toin H.; Rops, Angelique L. W. M. M.

    2015-01-01

    Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis. PMID:26322947

  17. Differential Protein Expression in Congenital and Acquired Cholesteatomas

    PubMed Central

    Kim, Sung Huhn; Choi, Jae Young

    2015-01-01

    Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ

  18. Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

    PubMed

    Lewis, April M; Rice, Kelly C

    2016-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression. PMID:25646613

  19. Two methods for the quantitative analysis of surface antigen expression in acute myeloid leukemia (AML).

    PubMed

    Woźniak, Jolanta

    2004-01-01

    The expression of lineage molecules (CD13 and CD33), c-Kit receptor (CD117), CD34, HLA-DR and adhesion molecule CD49d was assessed in acute myeloid leukemia (AML) blast cells from 32 cases, using direct and indirect quantitative cytometric analysis. High correlation (r=0.8) was found between antigen expression intensity values calculated by direct analysis method (ABC) and by indirect analysis method (RFI). Moreover, the differences in expression intensity of CD13, CD117 and CD34 antigens were found between leukemic and normal myeloblasts. This may be helpful in identification of leukemic cells in the diagnostics of minimal residual disease after treatment in AML patients. PMID:15493582

  20. MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

    PubMed

    Garzon, R; Pichiorri, F; Palumbo, T; Visentini, M; Aqeilan, R; Cimmino, A; Wang, H; Sun, H; Volinia, S; Alder, H; Calin, G A; Liu, C-G; Andreeff, M; Croce, C M

    2007-06-14

    MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively. PMID:17260024

  1. Identification of differentially expressed transcripts from leaves of the boron tolerant plant Gypsophila perfoliata L.

    PubMed

    Unver, Turgay; Bozkurt, Osman; Akkaya, Mahinur S

    2008-08-01

    Very recently some of the species of Gypsophila genus collected from the boron rich soils in Turkey were shown to be remarkably tolerant to high levels of boron. A limited amount of boron is necessary for the normal development of plants; however, a high level of boron in soil is generally toxic. Nevertheless, the adaptability of plant species allows them to withstand the presence of extreme amounts of metal ion by various strategies. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were transferred into plant nutritional medium in the presence high; approximately 500 (35 mg/kg), 1,000, and 30 microM (considered normal) boron concentrations. We compared the transcriptome of the plant sample treated with the excess levels of boron to that of the samples grown under normal concentration using differential display PCR (DDRT-PCR) method. Thirty bands showing differential expression levels (presence or absence of bands or varying intensities) in either of approximately 500 or 30 microM B concentrations at varying time points were excised, cloned, and sequenced. Among which, 18 of them were confirmed via quantitative reverse transcription real time PCR (qRT-PCR). We are reporting the first preliminary molecular level study of boron tolerance on this organism by attempting to identify putative genes related in the tolerance mechanism. The gene fragments are consistent with the literature data obtained from a proteomics study and a metabolomics study performed in barley under varying boron concentrations. PMID:18504585

  2. A reduction in Npas4 expression results in delayed neural differentiation of mouse embryonic stem cells

    PubMed Central

    2014-01-01

    Introduction Npas4 is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. It is known that in the adult brain Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory, yet very little is known about the role of Npas4 during neurodevelopment. The aim of this study was to examine the expression and function of Npas4 during nervous system development by using a combination of in vivo experiments in the developing mouse embryo and neural differentiation of embryonic stem cells (ESCs) as an in vitro model of the early stages of embryogenesis. Methods Two different neural differentiation paradigms were used to investigate Npas4 expression during neurodevelopment in vitro; adherent monolayer differentiation of mouse ESCs in N2B27 medium and Noggin-induced differentiation of human ESCs. This work was complemented by direct analysis of Npas4 expression in the mouse embryo. The function of Npas4 in the context of neurodevelopment was investigated using loss-of-function experiments in vitro. We created several mouse ESC lines in which Npas4 expression was reduced during neural differentiation through RNA interference and we then analyzed the ability of these Npas4 knockdown mouse ESCs lines to undergo neural differentiation. Results We found that while Npas4 is not expressed in undifferentiated ESCs, it becomes transiently up-regulated during neural differentiation of both mouse and human ESCs at a stage of differentiation that is characterized by proliferation of neural progenitor cells. This was corroborated by analysis of Npas4 expression in the mouse embryo where the Npas4 transcript was detected specifically in the developing forebrain beginning at embryonic day 9.5. Finally, knockdown of Npas4 expression in mouse ESCs undergoing neural differentiation

  3. The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

    PubMed

    Sheffield, Jeanne; Taylor, Nigel; Fauquet, Claude; Chen, Sixue

    2006-03-01

    Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots. PMID:16421938

  4. Differential expression of microRNAs in 2-cell and 4-cell mouse embryos.

    PubMed

    Wang, Pei; Cui, Ji; Zhao, Chun; Zhou, Lin; Guo, Xirong; Shen, Rong; Zhang, Junqiang; Ling, Xiufeng

    2014-11-01

    In vitro fertilized (IVF) human embryos have a high incidence of developmental arrest before the blastocyst stage, therefore characterization of the molecular mechanisms that regulate embryo development is urgently required. Post-transcriptional control by microRNAs (miRNAs) is one of the most investigated RNA control mechanisms, and is hypothesized to be involved actively in developmental arrest in preimplantation embryos. In this study, we extracted total RNA from mouse 2-cell and 4-cell embryos. Using a miRNA microarray, 192 miRNAs were found to be differentially expressed in 4-cell embryos and 2-cell embryos; 122 miRNAs were upregulated and 70 were downregulated in 4-cell embryos. The microarray results were confirmed by real-time quantitative RT-PCR for six miRNAs (mmu-miR-467h, mmu-miR-466d-3p, mmu-miR-292-5p, mmu-miR-154, mmu-miR-2145, and mmu-miR-706). Cdca4 and Tcf12 were identified as miR-154 target genes by target prediction analysis. This study provides a developmental map for a large number of miRNAs in 2-cell and 4-cell embryos. The function of these miRNAs and the mechanisms by which they modulate embryonic developmental arrest require further study. The results of this study have potential applications in the field of reproductive medicine. PMID:23731853

  5. Differential Expression of Long Noncoding RNAs between Sperm Samples from Diabetic and Non-Diabetic Mice

    PubMed Central

    An, Tian; Zhao, Dan-Dan; Yang, Xiu-Yan; Zhang, Dong-Wei; Zhang, Yi; Mu, Qian-Qian; Yu, Na; Ma, Xue-Shan; Gao, Si-Hua

    2016-01-01

    To investigate the potential core reproduction-related genes associated with the development of diabetes, the expression profiles of long noncoding RNA (lncRNA) and messenger RNA (mRNA) in the sperm of diabetic mice were studied. We used microarray analysis to detect the expression of lncRNAs and coding transcripts in six diabetic and six normal sperm samples, and differentially expressed lncRNAs and mRNAs were identified through Volcano Plot filtering. The function of differentially expressed mRNA was determined by pathway and gene ontology (GO) analysis, and the function of lncRNAs was studied by subgroup analysis and their physical or functional relationships with corresponding mRNAs. A total of 7721 lncRNAs and 6097 mRNAs were found to be differentially expressed between the diabetic and normal sperm groups. The diabetic sperm exhibited aberrant expression profiles for lncRNAs and mRNAs, and GO and pathway analyses showed that the functions of differentially expressed mRNAs were closely related with many processes involved in the development of diabetes. Furthermore, potential core genes that might play important roles in the pathogenesis of diabetes-related low fertility were revealed by lncRNA- and mRNA-interaction studies, as well as coding-noncoding gene co-expression analysis based on the microarray expression profiles. PMID:27119337

  6. Differential Expression of Long Noncoding RNAs between Sperm Samples from Diabetic and Non-Diabetic Mice.

    PubMed

    Jiang, Guang-Jian; Zhang, Teng; An, Tian; Zhao, Dan-Dan; Yang, Xiu-Yan; Zhang, Dong-Wei; Zhang, Yi; Mu, Qian-Qian; Yu, Na; Ma, Xue-Shan; Gao, Si-Hua

    2016-01-01

    To investigate the potential core reproduction-related genes associated with the development of diabetes, the expression profiles of long noncoding RNA (lncRNA) and messenger RNA (mRNA) in the sperm of diabetic mice were studied. We used microarray analysis to detect the expression of lncRNAs and coding transcripts in six diabetic and six normal sperm samples, and differentially expressed lncRNAs and mRNAs were identified through Volcano Plot filtering. The function of differentially expressed mRNA was determined by pathway and gene ontology (GO) analysis, and the function of lncRNAs was studied by subgroup analysis and their physical or functional relationships with corresponding mRNAs. A total of 7721 lncRNAs and 6097 mRNAs were found to be differentially expressed between the diabetic and normal sperm groups. The diabetic sperm exhibited aberrant expression profiles for lncRNAs and mRNAs, and GO and pathway analyses showed that the functions of differentially expressed mRNAs were closely related with many processes involved in the development of diabetes. Furthermore, potential core genes that might play important roles in the pathogenesis of diabetes-related low fertility were revealed by lncRNA- and mRNA-interaction studies, as well as coding-noncoding gene co-expression analysis based on the microarray expression profiles. PMID:27119337

  7. Decreased Expression of Met During Differentiation in Rat Lung

    PubMed Central

    Kato, T.; Oka, K.; Nakamura, T.; Ito, A.

    2016-01-01

    Organ-specific stem cells play key roles in maintaining the epithelial cell layers of lung. Bronchioalveolar stem cells (BASCs) are distal lung epithelial stem cells of adult mice. Alveolar type 2 (AT2) cells have important functions and serve as progenitor cells of alveolar type 1 (AT1) cells to repair the epithelium when they are injured. Hepatocyte growth factor (HGF) elicits mitogenic, morphogenic, and anti-apoptotic effects on lung epithelial cells through tyrosine phosphorylation of Met receptor, and thus is recognized as a pulmotrophic factor. To understand which cells HGF targets in lung, we identified the cells expressing Met by immunofluorescence assay. Met was strongly expressed in BASCs, which expressed an AT2 cell marker, pro-SP-C, and a club cell marker, CCSP. In alveoli, we found higher expression of Met in primary AT2 than in AT1 cells, which was confirmed using primary AT2 cells. We further examined the mitogenic activity of HGF in AT2-cell-derived alveolar-like cysts (ALCs) in 3D culture. Multicellular ALCs expressed Met, and HGF enhanced the ALC production. Taking these findings together, BASCs could also be an important target for HGF, and HGF-Met signaling could function more potent on cells that have greater multipotency in adult lung. PMID:26972715

  8. Diagnosis of Prostate Cancer Using Differentially Expressed Genes in Stroma

    PubMed Central

    Jia, Zhenyu; Wang, Yipeng; Sawyers, Anne; Yao, Huazhen; Rahmatpanah, Farahnaz; Xia, Xiao-Qin; Xu, Qiang; Pio, Rebecca; Turan, Tolga; Koziol, James A.; Goodison, Steve; Carpenter, Philip; Wang-Rodriquez, Jessica; Simoneau, Anne; Meyskens, Frank; Sutton, Manuel; Lernhardt, Waldemar; Beach, Thomas; Monforte, Joseph; McClelland, Michael; Mercola, Dan

    2011-01-01

    Over one million prostate biopsies are performed in the U.S. every year. A failure to find cancer is not definitive in a significant percentage of patients due to the presence of equivocal structures or continuing clinical suspicion. We have identified gene expression changes in stroma that can detect tumor nearby. We compared gene expression profiles of 13 biopsies containing stroma near tumor and 15 biopsies from volunteers without prostate cancer. About 3800 significant expression changes were found and thereafter filtered using independent expression profiles to eliminate possible age-related genes and genes expressed at detectable levels in tumor cells. A stroma-specific classifier for nearby tumor was constructed based on 114 candidate genes and tested on 364 independent samples, including 243 tumor-bearing samples and 121 non-tumor samples (normal biopsies, normal autopsies, remote stroma, as well as stroma within a few millimeters of tumor). The classifier predicted the tumor status of patients using tumor-free samples with an average accuracy of 97% (sensitivity = 98% and specificity = 88%) whereas classifiers trained with sets of 100 randomly generated genes had no diagnostic value. These results indicate that the prostate cancer microenvironment exhibits reproducible changes useful for categorizing the presence of tumor in patients when a prostate sample is derived from near the tumor but does not contain any recognizable tumor. PMID:21459804

  9. Role of ATG10 expression quantitative trait loci in non-small cell lung cancer survival.

    PubMed

    Xie, Kaipeng; Liang, Cheng; Li, Qin; Yan, Caiwang; Wang, Cheng; Gu, Yayun; Zhu, Meng; Du, Fangzhi; Wang, Hui; Dai, Juncheng; Liu, Xiao'an; Jin, Guangfu; Shen, Hongbing; Ma, Hongxia; Hu, Zhibin

    2016-10-01

    The aim of this article was to evaluate whether genetic variants in autophagy-related genes affect the overall survival (OS) of non-small cell lung cancer (NSCLC) patients. We analyzed 14 single nucleotide polymorphisms (SNPs) in core autophagy-related genes for OS in 1,001 NSCLC patients. Three promising SNPs in ATG10 were subsequently annotated by the expression quantitative trait loci (eQTL) and methylation quantitative trait loci (meQTL) analyses based on Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. We observed that the variants of rs10514231, rs1864182 and rs1864183 were associated with poor lung cancer survival (HR = 1.33, 95% CI = 1.07-1.65; HR = 1.43, 95% CI = 1.13-1.81; HR = 1.38, 95% CI = 1.14-1.68, respectively) and positively correlated with ATG10 expression (all p < 0.05) from GTEx and TCGA datasets. The elevated expression of ATG10 may predict shorter survival time in lung cancer patients in TCGA dataset (HR = 2.10, 95% CI = 1.33-3.29). Moreover, the variants of rs10514231 and rs1864182 were associated with the increased methylation levels of cg17942617 (meQTL), which in turn contributed to the elevated ATG10 expression and decreased survival time. Further functional assays revealed that ATG10 facilitated lung cancer cell proliferation and migration. Our findings suggest that eQTL/meQTL variations of ATG10 could influence lung cancer survival through regulating ATG10 expression. PMID:27225307

  10. Differential expression of three labial genes during earthworm head regeneration.

    PubMed

    Cho, Sung-Jin; Koh, Ki Seok; Lee, Eun; Park, Soon Cheol

    2009-12-01

    The earthworm provides an excellent model for investigating regeneration. Here we report the full-length cloning of three labial genes (Pex-lab01, Pex-lab02, and Pex-lab03) in the earthworm Perionyx excavatus. To analyze their expression pattern during head and tail regeneration, we used the reverse transcription-polymerase chain reaction. Our results indicate that the three labial genes were expressed only in the head-regenerating tissues. Also, we found that the expression of Pex-lab01 and Pex-lab02 is up-regulated, and this indicates their involvement in wound healing and the blastema formation processes during early head regeneration. PMID:19966495

  11. A High-Throughput Data Mining of Single Nucleotide Polymorphisms in Coffea Species Expressed Sequence Tags Suggests Differential Homeologous Gene Expression in the Allotetraploid Coffea arabica1[W

    PubMed Central

    Vidal, Ramon Oliveira; Mondego, Jorge Maurício Costa; Pot, David; Ambrósio, Alinne Batista; Andrade, Alan Carvalho; Pereira, Luiz Filipe Protasio; Colombo, Carlos Augusto; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

    2010-01-01

    Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed. PMID:20864545

  12. A high-throughput data mining of single nucleotide polymorphisms in Coffea species expressed sequence tags suggests differential homeologous gene expression in the allotetraploid Coffea arabica.

    PubMed

    Vidal, Ramon Oliveira; Mondego, Jorge Maurício Costa; Pot, David; Ambrósio, Alinne Batista; Andrade, Alan Carvalho; Pereira, Luiz Filipe Protasio; Colombo, Carlos Augusto; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

    2010-11-01

    Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed. PMID:20864545

  13. Differential Allelic Expression in the Human Genome: A Robust Approach To Identify Genetic and Epigenetic Cis-Acting Mechanisms Regulating Gene Expression

    PubMed Central

    Serre, David; Gurd, Scott; Ge, Bing; Sladek, Robert; Sinnett, Donna; Harmsen, Eef; Bibikova, Marina; Chudin, Eugene; Barker, David L.; Dickinson, Todd; Fan, Jian-Bing; Hudson, Thomas J.

    2008-01-01

    The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60∶40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms. PMID:18454203

  14. Association of tissue lineage and gene expression: conservatively and differentially expressed genes define common and special functions of tissues

    PubMed Central

    2010-01-01

    Background Embryogenesis is the process by which the embryo is formed, develops, and establishes developmental hierarchies of tissues. The recent advance in microarray technology made it possible to investigate the tissue specific patterns of gene expression and their relationship with tissue lineages. This study is focused on how tissue specific functions, tissue lineage, and cell differentiation are correlated, which is essential to understand embryonic development and organism complexity. Results We performed individual gene and gene set based analysis on multiple tissue expression data, in association with the classic topology of mammalian fate maps of embryogenesis. For each sub-group of tissues on the fate map, conservatively, differentially and correlatively expressed genes or gene sets were identified. Tissue distance was found to correlate with gene expression divergence. Tissues of the ectoderm or mesoderm origins from the same segments on the fate map shared more similar expression pattern than those from different origins. Conservatively expressed genes or gene sets define common functions in a tissue group and are related to tissue specific diseases, which is supported by results from Gene Ontology and KEGG pathway analysis. Gene expression divergence is larger in certain human tissues than in the mouse homologous tissues. Conclusion The results from tissue lineage and gene expression analysis indicate that common function features of neighbor tissue groups were defined by the conservatively expressed genes and were related to tissue specific diseases, and differentially expressed genes contribute to the functional divergence of tissues. The difference of gene expression divergence in human and mouse homologous tissues reflected the organism complexity, i.e. distinct neural development levels and different body sizes. PMID:21172044

  15. Differential expression of vascular endothelial growth factor-A isoforms in the mouse uterus during early pregnancy.

    PubMed

    Walter, Lisa M; Rogers, Peter A W; Girling, Jane E

    2010-12-01

    While vascular endothelial growth factor (VEGF)-A mediates endometrial vascular remodelling during early pregnancy in mice, individual VEGF-A isoforms have not been investigated, despite their different biological properties. Using mice as a model, the expression of VEGF-A isoforms and receptors in the mouse uterus during early pregnancy was quantified. It was postulated that selected isoform expression would increase concurrent with increased endometrial endothelial cell proliferation at this time. Uteri were collected on days 1-5 of pregnancy and mRNA expression was quantified by quantitative reverse-transcription polymerase chain reaction, VEGF-A protein by Western blot and VEGF receptor (VEGFR)-2 by immunohistochemistry. The lowest expression of isomers Vegf(120) and Vegf(164) was observed on day 2 of pregnancy, increasing thereafter. Vegfr-2 mRNA expression was significantly higher on days 3-5 of pregnancy relative to days 1-2 (P<0.05). No significant changes were noted in Vegf(188), Nrp1 or Nrp2 mRNA. VEGF(188) protein expression was consistently higher than other isoforms. These data demonstrate differential regulation of VEGF-A isoforms in mouse uterus during early pregnancy. PMID:21050818

  16. Reference gene selection for gene expression studies in lily using quantitative real-time PCR.

    PubMed

    Zhang, M F; Liu, Q; Jia, G X

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is an important technology used to analyze gene-expression levels. Reference genes, which are assumed to be expressed consistently across various developmental stages and in different tissues, were selected for expression level analysis. Using digital gene expression technology, we selected nine reference genes (18S, EF, CYCOL, SAND, GAPDH, ACTIN, BHLH, TIP, and Clathrin) as candidate reference genes for further study. Using three different analysis methods (GeNorm, NormFinder, and BestKeeper), a total of 144 lily (Lilium x formolongi "Raizan 3") samples were analyzed. The samples were collected from four different tissues under various developmental stages. In addition, leaves treated with different plant hormones were collected and analyzed. The data showed that the stability of the nine reference genes differed among samples, but TIP, EF, Clathrin, and BHLH could be identified as the most stable genes overall. In addition, the relative expression level of LfFT in different lily tissues with the competence to flower was also analyzed to verify the selected reference genes. This study constitutes an important source for selecting reference genes when analyzing the expression patterns of flowering time and floral development regulation genes in lily cultivars. PMID:27173307

  17. MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

    PubMed

    Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

    2009-07-01

    Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions. PMID:19445975

  18. Apoptotic Genes are Differentially Expressed in Aged Gingival Tissue

    PubMed Central

    González, O.A.; Stromberg, A.J.; Huggins, P.M.; Gonzalez-Martinez, J.; Novak, M.J.; Ebersole, J.L.

    2011-01-01

    Cellular and molecular changes of the periodontium associated with a higher prevalence of oral diseases (e.g., chronic periodontitis) in aged populations have received little attention. Since impaired apoptosis during aging appears to be related to chronic inflammatory disorders, we hypothesized that the expression of genes associated with apoptotic processes are altered in aged healthy and periodontitis-affected gingival tissue. Ontology analysis of 88 genes related to apoptotic pathways was performed in gingival biopsies of healthy and periodontitis sites from young, adult, and aged non-human primates (Macaca mulatta), using the GeneChip® Rhesus Macaque Genome Array. Lower expression of anti-apoptotic and higher expression of pro-apoptotic genes were associated with healthy gingival tissue from young compared with aged animals. Few differences in gene expression were observed in healthy gingival tissue between adult and aged animals. Comparison between healthy and periodontitis gingival tissues showed that the up- or down-regulated apoptotic genes in diseased gingival tissue are different in adults compared with aged animals. These results suggest that apoptotic events normally occurring in gingival tissues could be reduced in aging,and unique aspects of apoptotic pathways are potentially involved in the pathophysiology of perio-dontal disease in adult vs. aged gingival tissues. PMID:21471327

  19. Polymorphic variation as a driver of differential neuropeptide gene expression.

    PubMed

    Quinn, John P; Warburton, Alix; Myers, Paul; Savage, Abigail L; Bubb, Vivien J

    2013-12-01

    The regulation of neuropeptide gene expression and their receptors in a tissue specific and stimulus inducible manner will determine in part behaviour and physiology. This can be a dynamic process resulting from short term changes in response to the environment or long term modulation imposed by epigenetically determined mechanisms established during life experiences. The latter underpins what is termed 'nature and nurture, or 'gene×environment interactions'. Dynamic gene expression of neuropeptides or their receptors is a key component of signalling in the CNS and their inappropriate regulation is therefore a predicted target underpinning psychiatric disorders and neuropathological processes. Finding the regulatory domains within our genome which have the potential to direct gene expression is a difficult challenge as 98% of our genome is non-coding and, with the exception of proximal promoter regions, such elements can be quite distant from the gene that they regulate. This review will deal with how we can find such domains by addressing both the most conserved non-exonic regions in the genome using comparative genomics and the most recent or constantly evolving DNA such as repetitive DNA or retrotransposons. We shall also explore how polymorphic changes in such domains can be associated with CNS disorders by altering the appropriate gene expression patterns which maintain normal physiology. PMID:24210140

  20. Differential expression of murine adult hemoglobins in early ontogeny

    SciTech Connect

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  1. Ectopic expression of single transcription factors directs differentiation of a medaka spermatogonial cell line.

    PubMed

    Thoma, Eva C; Wagner, Toni U; Weber, Isabell P; Herpin, Amaury; Fischer, Andreas; Schartl, Manfred

    2011-08-01

    The capability to form all cell types of the body is a unique feature of stem cells. However, many questions remain concerning the mechanisms regulating differentiation potential. The derivation of spermatogonial cell lines (SGs) from mouse and human, which can differentiate across germ-layer borders, suggested male germ cells as a potential stem cell source in addition to embryonic stem cells. Here, we present a differentiation system using an SG of the vertebrate model organism Oryzias latipes (medaka). We report differentiation of this cell line into 4 different ectodermal and mesodermal somatic cell types. In addition to differentiation into adipocytes by retinoic acid treatment, we demonstrate for the first time that directed differentiation of an SG can be induced by ectopic expression of single transcription factors, completely independent of culture conditions. Transient transfection with mitf-m, a transcription factor that has been shown to induce differentiation into melanocytes in medaka embryonic stem cells, resulted in the formation of the same cell type in spermatogonia. Similarly, the formation of neuron-like cells and matrix-depositing osteoblasts was induced by ectopic expression of mash1 and cbfa1, respectively. Interestingly, we found that the expression of all mentioned fate-inducing transcription factors leads to recapitulation of the temporal pattern of marker gene expression known from in vivo studies. PMID:21090990

  2. 17-β-estradiol up-regulates apolipoprotein genes expression during osteoblast differentiation in vitro.

    PubMed

    Gui, Yuyan; Chu, Nan; Qiu, Xuemin; Tang, Wei; Gober, Hans-Jürgen; Li, Dajin; Wang, Ling

    2016-05-23

    Apolipoproteins are of great physiological importance and are associated with different diseases. Many independent studies of patterns of gene expression during osteoblast differentiation have been described, and some apolipoproteins have been induced during this process. 17-β-estradiol (E2) may enhance osteoblast physiological function. However, no studies have indicated whether E2 can modulate the expression of apolipoproteins during osteoblast differentiation in vitro. The aim of the current study was to observe the regulation of apolipoprotein mRNA expression by E2 during this process. Primary osteoblasts were collected from the calvaria of newborn mice and were subjected to osteoblast differentiation in vitro with serial concentrations of E2. RNA was isolated on days 0, 5, and 25 of differentiation. Real-time PCR was performed to analyze the levels of apolipoprotein mRNA. Results showed that during osteoblast differentiation all of the apolipoprotein genes were up-regulated by E2 in a dose-dependent manner. Moreover, only ApoE was strongly induced during the mineralization of cultured osteoblasts. This result suggests that ApoE might be involved in osteoblast differentiation. The hypothesis is that E2 promotes osteoblast differentiation by up-regulating ApoE gene expression, though further study is needed to confirm this hypothesis. PMID:27074899

  3. Time course differential gene expression in response to porcine circovirus type 2 subclinical infection

    PubMed Central

    Tomás, Anna; Fernandes, Lana T.; Sánchez, Armand; Segalés, Joaquim

    2009-01-01

    This study was aimed at characterizing the potential differences in gene expression in piglets inoculated with Porcine circovirus type 2 (PCV2), the essential causative agent of postweaning multisystemic wasting syndrome. Seven-day-old caesarean-derived, colostrum-deprived piglets were distributed into two groups: control (n = 8) and pigs inoculated with 105.2 TCID50 of the Burgos PCV2 isolate (n = 16). One control and three inoculated pigs were necropsied on days 1, 2, 5, and 8 post-infection (p.i.). The remaining pigs (four of each group) were sequentially bled on days 0, 7, 14, 21, and 29 p.i. (necropsy). Total RNA from the mediastinal lymph node (MLN) and lysed whole blood (LWB) samples were hybridized to Affymetrix Porcine GeneChip®. Forty-three probes were differentially expressed (DE) in MLN samples (FDR < 0.1, fold change > 2) and were distributed into three clusters: globally down-regulated genes, and up-regulated genes at early (first week p.i.) and late (day 29 p.i.) stages of infection. In LWB samples, maximal differences were observed at day 7 p.i., with 54 probes DE between control and inoculated pigs. Main Gene Ontology biological processes assigned to up-regulated genes were related to the immune response. Six common genes were found in both types of samples, all of which belonged to the interferon signaling antiviral effector pathway. Down-regulated genes were mainly related to cell adhesion and migration in MLN, and cellular organization and biogenesis in LWB. Microarray results were validated by quantitative real-time PCR. This study provides, for the first time, the characterization of the early and late molecular events taking place in response to a subclinical PCV2 infection. PMID:19825344

  4. Differential expression of human placental neurotrophic factors in preterm and term deliveries.

    PubMed

    Dhobale, Madhavi V; Pisal, Hemlata R; Mehendale, Savita S; Joshi, Sadhana R

    2013-12-01

    Neurotrophic factors such as brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are involved in development of the placenta and fetal brain. A series of human and animal studies in our department have shown that micronutrients (folic acid, vitamin B12) and omega 3 fatty acids like DHA are all interlinked in the one carbon cycle. Any alterations in one carbon components will lead to changes in methylation patterns that further affect the gene expression at critical periods of development resulting in complications during pregnancy. This may further contribute to risk for neurodevelopmental disorders in children born preterm. Therefore this study for the first time examines the mRNA levels from preterm and term placentae. A total number of 38 women delivering preterm (<37 weeks gestation) and 37 women delivering at term (=>37 weeks gestation) were recruited. The mRNA levels of BDNF and NGF were analyzed by real time quantitative polymerase chain reaction. Our results indicate that BDNF and NGF mRNA levels were lower in preterm group as compared to term group. There was a positive association of placental BDNF and NGF mRNA levels with cord plasma BDNF and NGF levels. The differential expression of BDNF and NGF gene in preterm placentae may also alter the vascular development in preterm deliveries. Our data suggests that the reduced mRNA levels of BDNF and NGF may possibly be a result of altered epigenetic mechanisms and may have an implication for altered fetal programming in children born preterm. PMID:24076518

  5. Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation.

    PubMed

    Mazemondet, Orianne; Hubner, Rayk; Frahm, Jana; Koczan, Dirk; Bader, Benjamin M; Weiss, Dieter G; Uhrmacher, Adelinde M; Frech, Moritz J; Rolfs, Arndt; Luo, Jiankai

    2011-12-01

    ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation. PMID:21805133

  6. Validation of a quantitative 12-multigene expression assay (Oncotype DX® Colon Cancer Assay) in Korean patients with stage II colon cancer: implication of ethnic differences contributing to differences in gene expression

    PubMed Central

    Jeong, Duck Hyoun; Kim, Woo Ram; Min, Byung Soh; Kim, Young Wan; Song, Mi Kyung; Kim, Nam Kyu

    2015-01-01

    Purpose To evaluate the Recurrence Score® of the quantitative 12-multigene expression assay and to determine risk groups based on the continuous Recurrence Score® in Korean patients. Method A total of 95 patients with pathological T3N0 tumors and mismatch repair-proficient tumors were enrolled. The Recurrence Score® was used to classify risk groups (low risk, <30; intermediate risk, 30–40; high risk, ≥41). Results Fifty-four patients (56.8%) were aged over 70 years. There were 49 men (51.6%) and 56 cases of right-sided colon cancer (58.9%). Eight cases (8.4%) had well-differentiated tumors, and 86 cases (90.5%) showed moderate differentiation. Only one case (1.1%) had a poorly differentiated tumor. Three patients (3.2%) had lymphovascular invasion. Sixty-one patients were identified as low risk (64.2%) and 34 patients as intermediate risk (35.8%). There were no high-risk patients. Although not significant, the 3-year recurrence risk increased with the Recurrence Score®. Conclusion Distribution patterns of risk groups based on the Recurrence Score®, particularly the absence of a high-risk group, were different from the prior validation studies. These findings suggest that ethnic differences between Koreans and Western patients are potential contributing factors for different gene expressions in the quantitative 12-multigene expression assay. PMID:26719709

  7. Differential Effects of Language Attrition in the Domains of Verb Placement and Object Expression

    ERIC Educational Resources Information Center

    Flores, Cristina

    2012-01-01

    This study investigates the differential effects of language attrition in two diverse linguistic domains: verb placement and object expression. Linguistic phenomena at the syntax--discourse interface, such as object expression, have been shown to be more vulnerable to attrition than narrow syntax properties, such as verb placement. This study aims…

  8. Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

    PubMed

    Shahrabi-Farahani, Shokoufeh; Wang, Lili; Zwaans, Bernadette M M; Santana, Jeans M; Shimizu, Akio; Takashima, Seiji; Kreuter, Michael; Coultas, Leigh; D'Amore, Patricia A; Arbeit, Jeffrey M; Akslen, Lars A; Bielenberg, Diane R

    2014-07-01

    Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity. PMID:24791743

  9. Differential Language Functioning of Monolinguals and Bilinguals on Positive-Negative Emotional Expression

    ERIC Educational Resources Information Center

    Kheirzadeh, Shiela; Hajiabed, Mohammadreza

    2016-01-01

    The present interdisciplinary research investigates the differential emotional expression between Persian monolinguals and Persian-English bilinguals. In other words, the article was an attempt to answer the questions whether bilinguals and monolinguals differ in the expression of positive and negative emotions elicited through sad and happy…

  10. Induced myelomonocytic differentiation in leukemia cells is accompanied by noncanonical transcription factor expression

    PubMed Central

    Jensen, Holly A.; Yourish, Harmony B.; Bunaciu, Rodica P.; Varner, Jeffrey D.; Yen, Andrew

    2015-01-01

    Transcription factors that drive non-neoplastic myelomonocytic differentiation are well characterized but have not been systematically analyzed in the leukemic context. We investigated widely used, patient-derived myeloid leukemia cell lines with proclivity for differentiation into granulocytes by retinoic acid (RA) and/or monocytes by 1,25-dihyrdroxyvitamin D3 (D3). Using K562 (FAB M1), HL60 (FAB M2), RA-resistant HL60 sublines, NB4 (FAB M3), and U937 (FAB M5), we correlated nuclear transcription factor expression to immunophenotype, G1/G0 cell cycle arrest and functional inducible oxidative metabolism. We found that myelomonocytic transcription factors are aberrantly expressed in these cell lines. Monocytic-lineage factor EGR1 was not induced by D3 (the monocytic inducer) but instead by RA (the granulocytic inducer) in lineage bipotent myeloblastic HL60. In promyelocytic NB4 cells, EGR1 levels were increased by D3, while Gfi-1 expression (which promotes the granulocytic lineage) was upregulated during D3-induced monocytic differentiation in HL60, and by RA treatment in monocytic U937 cells. Furthermore, RARα and VDR expression were not strongly correlated to differentiation. In response to different differentiation inducers, U937 exhibited the most distinct transcription factor expression profile, while similarly mature NB4 and HL60 were better coupled. Overall, the differentiation induction agents RA and D3 elicited cell-specific responses across these common FAB M1-M5 cell lines. PMID:26566473

  11. A Genome-Wide Screen Indicates Correlation between Differentiation and Expression of Metabolism Related Genes

    PubMed Central

    Shende, Akhilesh; Singh, Anupama; Meena, Anil; Ghosal, Ritika; Ranganathan, Madhav; Bandyopadhyay, Amitabha

    2013-01-01

    Differentiated tissues may be considered as materials with distinct properties. The differentiation program of a given tissue ensures that it acquires material properties commensurate with its function. It may be hypothesized that some of these properties are acquired through production of tissue-specific metabolites synthesized by metabolic enzymes. To establish correlation between metabolism and organogenesis we have carried out a genome-wide expression study of metabolism related genes by RNA in-situ hybridization. 23% of the metabolism related genes studied are expressed in a tissue-restricted but not tissue-exclusive manner. We have conducted the screen on whole mount chicken (Gallus gallus) embryos from four distinct developmental stages to correlate dynamic changes in expression patterns of metabolic enzymes with spatio-temporally unique developmental events. Our data strongly suggests that unique combinations of metabolism related genes, and not specific metabolic pathways, are upregulated during differentiation. Further, expression of metabolism related genes in well established signaling centers that regulate different aspects of morphogenesis indicates developmental roles of some of the metabolism related genes. The database of tissue-restricted expression patterns of metabolism related genes, generated in this study, should serve as a resource for systematic identification of these genes with tissue-specific functions during development. Finally, comprehensive understanding of differentiation is not possible unless the downstream genes of a differentiation cascade are identified. We propose, metabolic enzymes constitute a significant portion of these downstream target genes. Thus our study should help elucidate different aspects of tissue differentiation. PMID:23717462

  12. Murine and Human Spermatids Are Characterized by Numerous, Newly Synthesized and Differentially Expressed Transcription Factors and Bromodomain-Containing Proteins.

    PubMed

    Klaus, Elisabeth Sabine; Gonzalez, Nicola Helena; Bergmann, Martin; Bartkuhn, Marek; Weidner, Wolfgang; Kliesch, Sabine; Rathke, Christina

    2016-07-01

    Much of spermatid differentiation takes place in the absence of active transcription, but in the early phase, large amounts of mRNA are synthesized, translationally repressed, and stored. Most nucleosomal histones are then degraded, and chromatin is repackaged by protamines. For both transcription and the histone-to-protamine transition in differentiating spermatids, chromatin must be opened. This raises the question of whether two different processes exist. It is conceivable that for initiation of the histone-to-protamine transition, the already accessible, actively transcribed chromatin regions are utilized or vice versa. We analyzed the enrichment of different canonical TATA-box-binding, protein-associated factors and their variants in murine spermatids, diverse bromodomain-containing proteins, and components of the Polycomb repressive complexes PRC1 and PRC2 using quantitative PCR. We compared the enrichment of corresponding proteins in human and murine spermatids and analyzed the time frame of postmeiotic transcription and expression of histones, transition proteins, and protamines in human and murine spermatids using immunohistology. We correlated the expression of different transcription factors and bromodomain-containing proteins and the pattern of acetylated histones to active transcription and to the histone-to-protamine transition in both human and murine spermatids. Our findings suggest that differentiating spermatids use both common and specific features to open chromatin first for transcription and subsequently for histone-to-protamine transition. PMID:27170439

  13. Changes in expression and secretion patterns of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway molecules during murine neural stem/progenitor cell differentiation in vitro☆

    PubMed Central

    Lu, Jiang; Lu, Kehuan; Li, Dongsheng

    2012-01-01

    In the present study, we investigated the dynamic expression of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway related factors in the process of in vitro hippocampal neural stem/progenitor cell differentiation from embryonic Sprague-Dawley rats or embryonic Kunming species mice, using fluorescent quantitative reverse transcription-PCR and western blot analyses. Results demonstrated that the dynamic expression of fibroblast growth factor 8 was similar to fibroblast growth factor receptor 1 expression but not to other fibroblast growth factor receptors. Enzyme-linked immunosorbent assay demonstrated that fibroblast growth factor 8 and Sonic Hedgehog signaling pathway protein factors were secreted by neural cells into the intercellular niche. Our experimental findings indicate that fibroblast growth factor 8 and Sonic Hedgehog expression may be related to the differentiation of neural stem/progenitor cells. PMID:25624789

  14. Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

    PubMed

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

    2016-08-01

    Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. PMID:27233607

  15. Identification and Differential Expression of microRNAs in Ovaries of Laying and Broody Geese (Anser cygnoides) by Solexa Sequencing

    PubMed Central

    Xu, Qi; Zhang, Yang; Chen, Yang; Tong, Yi-Yu; Rong, Guang-Hui; Huang, Zheng-Yang; Zhao, Rong-Xue; Zhao, Wen-Ming; Wu, Xin-sheng; Chang, Guo- Bin; Chen, Guo-Hong

    2014-01-01

    Background Recent functional studies have demonstrated that the microRNAs (miRNAs) play critical roles in ovarian gonadal development, steroidogenesis, apoptosis, and ovulation in mammals. However, little is known about the involvement of miRNAs in the ovarian function of fowl. The goose (Anas cygnoides) is a commercially important food that is cultivated widely in China but the goose industry has been hampered by high broodiness and poor egg laying performance, which are influenced by ovarian function. Methodology/Principal Findings In this study, the miRNA transcriptomes of ovaries from laying and broody geese were profiled using Solexa deep sequencing and bioinformatics was used to determine differential expression of the miRNAs. As a result, 11,350,396 and 9,890,887 clean reads were obtained in laying and broodiness goose, respectively, and 1,328 conserved known miRNAs and 22 novel potential miRNA candidates were identified. A total of 353 conserved microRNAs were significantly differentially expressed between laying and broody ovaries. Compared with miRNA expression in the laying ovary, 127 miRNAs were up-regulated and 126 miRNAs were down-regulated in the ovary of broody birds. A subset of the differentially expressed miRNAs (G-miR-320, G-miR-202, G-miR-146, and G-miR-143*) were validated using real-time quantitative PCR. In addition, 130,458 annotated mRNA transcripts were identified as putative target genes. Gene ontology annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that the differentially expressed miRNAs are involved in ovarian function, including hormone secretion, reproduction processes and so on. Conclusions The present study provides the first global miRNA transcriptome data in A. cygnoides and identifies novel and known miRNAs that are differentially expressed between the ovaries of laying and broody geese. These findings contribute to our understanding of the functional involvement of miRNAs in the broody

  16. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

    PubMed

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

    2016-05-20

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  17. Quantitative Expression Profile of Distinct Functional Regions in the Adult Mouse Brain

    PubMed Central

    Nagano, Mamoru; Uno, Kenichiro D.; Tsujino, Kaori; Hanashima, Carina; Shigeyoshi, Yasufumi; Ueda, Hiroki R.

    2011-01-01

    The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems. PMID:21858037

  18. Quantitative single-cell gene expression measurements of multiple genes in response to hypoxia treatment.

    PubMed

    Zeng, Jia; Wang, Jiangxin; Gao, Weimin; Mohammadreza, Aida; Kelbauskas, Laimonas; Zhang, Weiwen; Johnson, Roger H; Meldrum, Deirdre R

    2011-07-01

    Cell-to-cell heterogeneity in gene transcription plays a central role in a variety of vital cell processes. To quantify gene expression heterogeneity patterns among cells and to determine their biological significance, methods to measure gene expression levels at the single-cell level are highly needed. We report an experimental technique based on the DNA-intercalating fluorescent dye SYBR green for quantitative expression level analysis of up to ten selected genes in single mammalian cells. The method features a two-step procedure consisting of a step to isolate RNA from a single mammalian cell, synthesize cDNA from it, and a qPCR step. We applied the method to cell populations exposed to hypoxia, quantifying expression levels of seven different genes spanning a wide dynamic range of expression in randomly picked single cells. In the experiment, 72 single Barrett's esophageal epithelial (CP-A) cells, 36 grown under normal physiological conditions (controls) and 36 exposed to hypoxia for 30 min, were randomly collected and used for measuring the expression levels of 28S rRNA, PRKAA1, GAPDH, Angptl4, MT3, PTGES, and VEGFA genes. The results demonstrate that the method is sensitive enough to measure alterations in gene expression at the single-cell level, clearly showing heterogeneity within a cell population. We present technical details of the method development and implementation, and experimental results obtained by use of the procedure. We expect the advantages of this technique will facilitate further developments and advances in the field of single-cell gene expression profiling on a nanotechnological scale, and eventually as a tool for future point-of-care medical applications. PMID:21614642

  19. Heterosis and differential gene expression in hybrids and parents in Bombyx mori by digital gene expression profiling

    PubMed Central

    Wang, Hua; Fang, Yan; Wang, Lipeng; Zhu, Wenjuan; Ji, Haipeng; Wang, Haiying; Xu, Shiqing; Sima, Yanghu

    2015-01-01

    Heterosis is a concern to all breeders, but the mechanism of heterosis remains unknown. In F1 organisms, genetic material is inherited from the two parents and theoretically, heterosis might be caused by differences in gene expression or modification. Differential gene expression was analyzed in hybrids and parents in Bombyx mori. The results showed that there were significant changes in gene expression in the fat body involving biological regulation, cellular and metabolic processes. Consistent trends in expression patterns covering different hybrid combinations were seen in 74 genes. Moreover, these differential gene expression patterns included overdominance, dominance, and additive effects. By correlating these patterns with economic traits, a potential relationship was found. Differential gene expression was seen in different cross combinations and in different sexes. In addition, a regulatory mechanism involving metabolism and ErbB signaling pathways was also found, suggesting that such a network might also be related to heterosis in Bombyx mori. Together, our data provide a comprehensive overview and useful resource for transcriptional analysis of heterosis of Bombyx mori. PMID:25736158

  20. Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF

    SciTech Connect

    Chen Xiaoli; Matsumoto, Hideko; Hinck, Cynthia S.; Al-Hasani, Hadi; St-Denis, Jean-Francois; Whiteheart, Sidney W.; Cushman, Samuel W. . E-mail: sam_cushman@nih.gov

    2005-07-22

    In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by {approx}50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.

  1. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

    PubMed

    Akiyama, Nobuko; Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Demizu, Yosuke; Yasuda, Hisataka; Yagi, Shintaro; Wu, Guoying; Matsumoto, Mitsuru; Sakamoto, Reiko; Yoshida, Nobuaki; Penninger, Josef M; Kobayashi, Yasuhiro; Inoue, Jun-Ichiro; Akiyama, Taishin

    2016-07-25

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs. PMID:27401343

  2. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation. PMID:26378917

  3. Lead Poisoning Disturbs Oligodendrocytes Differentiation Involved in Decreased Expression of NCX3 Inducing Intracellular Calcium Overload

    PubMed Central

    Ma, Teng; Wu, Xiyan; Cai, Qiyan; Wang, Yun; Xiao, Lan; Tian, Yanping; Li, Hongli

    2015-01-01

    Lead (Pb) poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs) differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs) differentiation via decreasing the expression of Olig 2, CNPase proteins in vitro. Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3) mRNA expression, one of the major means of calcium (Ca2+) extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP) expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload. PMID:26287169

  4. Effect of low-expression gene filtering on detection of differentially expressed genes in RNA-seq data

    PubMed Central

    Sha, Ying; Phan, John H.; Wang, May D.

    2016-01-01

    We compare methods for filtering RNA-seq lowexpression genes and investigate the effect of filtering on detection of differentially expressed genes (DEGs). Although RNA-seq technology has improved the dynamic range of gene expression quantification, low-expression genes may be indistinguishable from sampling noise. The presence of noisy, low-expression genes can decrease the sensitivity of detecting DEGs. Thus, identification and filtering of these low-expression genes may improve DEG detection sensitivity. Using the SEQC benchmark dataset, we investigate the effect of different filtering methods on DEG detection sensitivity. Moreover, we investigate the effect of RNA-seq pipelines on optimal filtering thresholds. Results indicate that the filtering threshold that maximizes the total number of DEGs closely corresponds to the threshold that maximizes DEG detection sensitivity. Transcriptome reference annotation, expression quantification method, and DEG detection method are statistically significant RNA-seq pipeline factors that affect the optimal filtering threshold. PMID:26737772

  5. Differential expression of ribosomal proteins in myelodysplastic syndromes.

    PubMed

    Rinker, Elizabeth B; Dueber, Julie C; Qualtieri, Julianne; Tedesco, Jason; Erdogan, Begum; Bosompem, Amma; Kim, Annette S

    2016-02-01

    Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed. PMID:26408650

  6. Reliable reference miRNAs for quantitative gene expression analysis of stress responses in Caenorhabditis elegans

    PubMed Central

    2014-01-01

    Background Quantitative real-time PCR (qPCR) has become the “gold standard” for measuring expression levels of individual miRNAs. However, little is known about the validity of reference miRNAs, the improper use of which can result in misleading interpretation of data. Results Here we undertook a systematic approach to identify highly stable miRNAs in different stress conditions such as low oxygen (hypoxia), UV-stress and high temperature (heat-stress) in the nematode Caenorhabditis elegans. We conducted genome-wide RNA-seq for small RNAs and selected abundant miRNAs with minimal variation of expression between the different conditions. We further validated the stable expression of a selection of those constitutively expressed candidates in the different stress conditions by SYBR Green qPCR. The selected miRNA candidates were analyzed for stability by applying the widely used geNorm logarithm. With this approach, we were able to successfully identify suitable reference miRNAs for each stress condition. Interestingly, we also found that 3 miRNAs, namely mir-2-5p, mir-46-3p and mir-47-3p, are stable in all the above-mentioned conditions suggesting that they might have general functions independent of stress. Conclusions Our analysis offers a comprehensive list of stably expressed miRNAs in different stress conditions that can be confidently used as reference miRNAs for qPCR analysis in C. elegans. PMID:24656064

  7. MicroRNA-765 regulates neural stem cell proliferation and differentiation by modulating Hes1 expression

    PubMed Central

    Li, Siou; Zhao, Weina; Xu, Qing; Yu, Yang; Yin, Changhao

    2016-01-01

    Neural stem cells (NSCs) are multipotent, self-renewing and undifferentiated cells that have the ability to differentiate to both glial and neuronal lineages. miRNAs act a key role in regulating neuronal fate and self-renewal of NSCs. In this study, we found that ectopic expression of miR-765 promoted NSCs proliferation. Moreover, miR-765 overexpression increased the ki-67 and β-tubulin-III expression inNSCs. Overexpression of miR-765 inhibited the expression of GFAP in NSCs. Furthermore, Hes1 was identified as a direct target gene of miR-765 in NSCs. Overexpression of Hes1 decreased miR-765-induced proliferation of NSCs and inhibited NSCs differentiation to neurons in miR-765-treated NSCs. These results demonstrated that miR-765 acted a crucial role in NSCs differentiation and proliferation by inhibiting Hes1 expression. PMID:27508032

  8. Differential Expression of Lipid and Carbohydrate Metabolism Genes in Upper Airway versus Diaphragm Muscle

    PubMed Central

    van Lunteren, Erik; Spiegler, Sarah; Moyer, Michelle

    2010-01-01

    Study Objectives: Contractile properties of upper airway muscles influence upper airway patency, an issue of particular importance for subjects with obstructive sleep apnea. Expression of genes related to cellular energetics is, in turn, critical for the maintenance of contractile integrity over time during repetitive activation. We tested the hypothesis that sternohyoid has lower expression of genes related to lipid and carbohydrate energetic pathways than the diaphragm. Methods: Sternohyoid and diaphragm from normal adult rats were examined with gene expression arrays. Analysis focused on genes belonging to Gene Ontology (GO) groups carbohydrate metabolism and lipid metabolism. Results: There were 433 genes with at least ± 2-fold significant differential expression between sternohyoid and diaphragm, of which 192 had sternohyoid > diaphragm and 241 had diaphragm > sternohyoid expression. Among genes with higher sternohyoid expression, there was over-representation of the GO group carbohydrate metabolism (P = 0.0053, n = 13 genes, range of differential expression 2.1- to 6.2-fold) but not lipid metabolism (P = 0.44). Conversely, among genes with higher diaphragm expression, there was over-representation of the GO group lipid metabolism (P = 0.0000065, n = 32 genes, range of differential expression 2.0- to 37.9-fold) but not carbohydrate metabolism (P = 0.23). Nineteen genes with diaphragm > sternohyoid expression were related to fatty acid metabolism (P = 0.000000058), in particular fatty acid β oxidation and biosynthesis in the mitochondria. Conclusions: Sternohyoid has much lower gene expression than diaphragm for mitochondrial enzymes that participate in fatty acid oxidation and biosynthesis. This likely contributes to the lower fatigue resistance of pharyngeal upper airway muscles compared with the diaphragm. Citation: van Lunteren E; Spiegler S; Moyer M. Differential expression of lipid and carbohydrate metabolism genes in upper airway versus diaphragm

  9. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    SciTech Connect

    Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao; Zhang, Bin; Xu, Jie; Huang, Fei; Zhao, Rui-Ni; Chen, Yong-Jin

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  10. RAGE Expression and ROS Generation in Neurons: Differentiation versus Damage

    PubMed Central

    Piras, S.; Furfaro, A. L.; Domenicotti, C.; Traverso, N.; Marinari, U. M.; Pronzato, M. A.; Nitti, M.

    2016-01-01

    RAGE is a multiligand receptor able to bind advanced glycation end-products (AGEs), amphoterin, calgranulins, and amyloid-beta peptides, identified in many tissues and cells, including neurons. RAGE stimulation induces the generation of reactive oxygen species (ROS) mainly through the activity of NADPH oxidases. In neuronal cells, RAGE-induced ROS generation is able to favor cell survival and differentiation or to induce death through the imbalance of redox state. The dual nature of RAGE signaling in neurons depends not only on the intensity of RAGE activation but also on the ability of RAGE-bearing cells to adapt to ROS generation. In this review we highlight these aspects of RAGE signaling regulation in neuronal cells. PMID:27313835

  11. Do count-based differential expression methods perform poorly when genes are expressed in only one condition?

    PubMed

    Zhou, Xiaobei; Robinson, Mark D

    2015-01-01

    A response to 'Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data' by Rapaport F, Khanin R, Liang Y, Pirun M, Krek A, Zumbo P, Mason CE, Socci ND and Betel D in Genome Biology, 2013, 14:R95. PMID:26450178

  12. Quantitative Trait Locus and Brain Expression of HLA-DPA1 Offers Evidence of Shared Immune Alterations in Psychiatric Disorders.

    PubMed

    Morgan, Ling Z; Rollins, Brandi; Sequeira, Adolfo; Byerley, William; DeLisi, Lynn E; Schatzberg, Alan F; Barchas, Jack D; Myers, Richard M; Watson, Stanley J; Akil, Huda; Bunney, William E; Vawter, Marquis P

    2016-01-01

    Genome-wide association studies of schizophrenia encompassing the major histocompatibility locus (MHC) were highly significant following genome-wide correction. This broad region implicates many genes including the MHC complex class II. Within this interval we examined the expression of two MHC II genes (HLA-DPA1 and HLA-DRB1) in brain from individual subjects with schizophrenia (SZ), bipolar disorder (BD), major depressive disorder (MDD), and controls by differential gene expression methods. A third MHC II mRNA, CD74, was studied outside of the MHC II locus, as it interacts within the same immune complex. Exon microarrays were performed in anterior cingulate cortex (ACC) in BD compared to controls, and both HLA-DPA1 and CD74 were decreased in expression in BD. The expression of HLA-DPA1 and CD74 were both reduced in hippocampus, amygdala, and dorsolateral prefrontal cortex regions in SZ and BD compared to controls by specific qPCR assay. We found several novel HLA-DPA1 mRNA variants spanning HLA-DPA1 exons 2-3-4 as suggested by exon microarrays. The intronic rs9277341 SNP was a significant cis expression quantitative trait locus (eQTL) that was associated with the total expression of HLA-DPA1 in five brain regions. A biomarker study of MHC II mRNAs was conducted in SZ, BD, MDD, and control lymphoblastic cell lines (LCL) by qPCR assay of 87 subjects. There was significantly decreased expression of HLA-DPA1 and CD74 in BD, and trends for reductions in SZ in LCLs. The discovery of multiple splicing variants in brain for HLA-DPA1 is important as the HLA-DPA1 gene is highly conserved, there are no reported splicing variants, and the functions in brain are unknown. Future work on the function and localization of MHC Class II proteins in brain will help to understand the role of alterations in neuropsychiatric disorders. The HLA-DPA1 eQTL is located within a large linkage disequilibrium block that has an irrefutable association with schizophrenia. Future tests in a

  13. Analysis of differential gene expression under low-temperature stress in Nile tilapia (Oreochromis niloticus) using digital gene expression.

    PubMed

    Yang, Changgeng; Jiang, Ming; Wen, Hua; Tian, Juan; Liu, Wei; Wu, Fan; Gou, Gengwu

    2015-06-15

    Tilapia (Oreochromis niloticus) do not survive well at low temperatures. Therefore, improvement of the low-temperature resistance has become an important issue for aquaculture development of tilapia. The objective of this study was to construct a digital gene expression tag profile to identify genes potentially related to low temperature in tilapia. In this study, tilapia was treated at 30°C to lethal temperature 10°C in decrement of 1°CD(-1). Digital gene expression analysis was performed using the Illumina technique to investigate differentially expressed genes in tilapia cultured at different temperatures (30°C, 26°C, 20°C, 16°C, and 10°C). A total of 206,861, 188,082, 185,827, 188,067, and 214,171 distinct tags were obtained by sequencing these five libraries, respectively. Compared with the 30°C library, there were 304, 407, 709, and 772 upregulated genes and 342, 793, 771, and 1466 downregulated genes in 26°C, 20°C, 16°C, and 10°C libraries, respectively. Trend analysis of these differentially expressed genes identified six statistically significant trends. Functional annotation analysis of the differentially expressed genes identified various functions associated with the response to low-temperature stress. When tilapia are subjected to low-temperature stress, expression changes were observed in genes associated with nucleic acid synthesis and metabolism, amino acid metabolism and protein synthesis, lipid and carbohydrate content and types, material transport, apoptosis, and immunity. The differentially expressed genes obtained in this study may provide useful insights to help further understand the effects of low temperature on tilapia. PMID:25617524

  14. Regulation of mineralocorticoid receptor expression during neuronal differentiation of murine embryonic stem cells

    PubMed Central

    Munier, Mathilde; Meduri, Geri; Viengchareun, Say; Leclerc, Phillipe; Le Menuet, Damien; Lombès, Marc

    2010-01-01

    Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem (ES) cell lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene Green Fluorescent Protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites) and the increase in the expression of neuronal markers (nestin, β-tubulin III, MAP2) as demonstrated by immunocytochemistry and qPCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively and correlated with MR expression. Finally, while progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by siRNA. Concluding, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases. PMID:20207834

  15. SOX2 and OCT4 mRNA-Expressing Cells, Detected by Molecular Beacons, Localize to the Center of Neurospheres during Differentiation

    PubMed Central

    Ilieva, Mirolyuba; Dufva, Martin

    2013-01-01

    Neurospheres are used as in vitro assay to measure the properties of neural stem cells. To investigate the molecular and phenotypic heterogeneity of neurospheres, molecular beacons (MBs) targeted against the stem cell markers OCT4 and SOX2 were designed, and synthesized with a 2’-O-methyl RNA backbone. OCT4 and SOX2 MBs were transfected into human embryonic mesencephalon derived cells, which spontaneously form neurospheres when grown on poly-L-ornitine/fibronectin matrix and medium complemented with bFGF. OCT4 and SOX2 gene expression were tracked in individual cell using the MBs. Quantitative image analysis every day for seven days showed that the OCT4 and SOX2 mRNA-expressing cells clustered in the centre of the neurospheres cultured in differentiation medium. By contrast, cells at the periphery of the differentiating spheres developed neurite outgrowths and expressed the tyrosine hydroxylase protein, indicating terminal differentiation. Neurospheres cultured in growth medium contained OCT4 and SOX2-positive cells distributed throughout the entire sphere, and no differentiating neurones. Gene expression of SOX2 and OCT4 mRNA detected by MBs correlated well with gene and protein expression measured by qRT-PCR and immunostaining, respectively. These experimental data support the theoretical model that stem cells cluster in the centre of neurospheres, and demonstrate the use of MBs for the spatial localization of specific gene-expressing cells within heterogeneous cell populations. PMID:24013403

  16. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    PubMed

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  17. Tetrandrine induces microRNA differential expression in human hypertrophic scar fibroblasts in vitro.

    PubMed

    Ning, P; Peng, Y; Liu, D W; Hu, Y H; Liu, Y; Liu, D M

    2016-01-01

    MicroRNAs (miRNAs) have recently been shown to play a role in normal wound healing process. miRNAs may be linked to pathologic wound healing and closely related to the formation of hypertrophic scars. This study aimed to explore the effects of tetrandrine on the miRNA expression profile in human hypertrophic scar fibroblasts (HSFs) in vitro. HSFs were randomly divided into two groups: the tetrandrine treatment group and the control group. The experimental and control groups were collected and analyzed by miRNA array after a 48-h culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the array results. The targets of differentially expressed miRNA were functionally annotated using bioinformatic approaches. miRNA microarray analysis identified 193 differentially expressed miRNAs and the expression of 186 miRNAs in the experimental group decreased while that of 7 miRNAs increased compared to the control group. The most significantly downregulated miRNA was hsa-miR-1246, and hsa-miR-27b had the highest expression level. Significant differentially expressed miRNAs were predicted to be related to several important signaling pathways related to scar wound healing. The differential miRNA expression identified in this study provides the experimental basis for further understanding the anti-fibrosis effect of tetrandrine. PMID:26909951

  18. Characterization of Differentially Expressed Genes Involved in Pathways Associated with Gastric Cancer

    PubMed Central

    Li, Hao; Yu, Beiqin; Li, Jianfang; Su, Liping; Yan, Min; Zhang, Jun; Li, Chen; Zhu, Zhenggang; Liu, Bingya

    2015-01-01

    To explore the patterns of gene expression in gastric cancer, a total of 26 paired gastric cancer and noncancerous tissues from patients were enrolled for gene expression microarray analyses. Limma methods were applied to analyze the data, and genes were considered to be significantly differentially expressed if the False Discovery Rate (FDR) value was < 0.01, P-value was <0.01 and the fold change (FC) was >2. Subsequently, Gene Ontology (GO) categories were used to analyze the main functions of the differentially expressed genes. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we found pathways significantly associated with the differential genes. Gene-Act network and co-expression network were built respectively based on the relationships among the genes, proteins and compounds in the database. 2371 mRNAs and 350 lncRNAs considered as significantly differentially expressed genes were selected for the further analysis. The GO categories, pathway analyses and the Gene-Act network showed a consistent result that up-regulated genes were responsible for tumorigenesis, migration, angiogenesis and microenvironment formation, while down-regulated genes were involved in metabolism. These results of this study provide some novel findings on coding RNAs, lncRNAs, pathways and the co-expression network in gastric cancer which will be useful to guide further investigation and target therapy for this disease. PMID:25928635

  19. MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells

    PubMed Central

    Huszar, Jessica M.; Payne, Christopher J.

    2014-01-01

    Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3. PMID:24726732

  20. MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells.

    PubMed

    Huszar, Jessica M; Payne, Christopher J

    2014-05-01

    Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3. PMID:24726732

  1. Differential gene expression induced by exposure of captive mink to fuel oil: A model for the sea otter

    USGS Publications Warehouse

    Bowen, L.; Riva, F.; Mohr, C.; Aldridge, B.; Schwartz, J.; Miles, A.K.; Stott, J.L.

    2007-01-01

    Free-ranging sea otters are subject to hydrocarbon exposure from a variety of sources, both natural and anthropogenic. Effects of direct exposure to unrefined crude oil, such as that associated with the Exxon Valdez oil spill, are readily apparent. However, the impact of subtle but pathophysiologically relevant concentrations of crude oil on sea otters is difficult to assess. The present study was directed at developing a model for assessing the impact of low concentrations of fuel oil on sea otters. Quantitative PCR was used to identify differential gene expression in American mink that were exposed to low concentrations of bunker C fuel oil. A total of 23 genes, representing 10 different physiological systems, were analyzed for perturbation. Six genes with immunological relevance were differentially expressed in oil-fed mink. Interleukin-18 (IL-18), IL-10, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and complement cytolysis inhibitor (CLI) were down-regulated while IL-2 was up-regulated. Expression of two additional genes was affected; heat shock protein 70 (HSP70) was up-regulated and thyroid hormone receptor (THR) was down-regulated. While the significance of each perturbation is not immediately evident, we identified differential expression of genes that would be consistent with the presence of immune system-modifying and endocrine-disrupting compounds in fuel oil. Application of this approach to identify effects of petroleum contamination on sea otters should be possible following expansion of this mink model to identify a greater number of affected genes in peripheral blood leukocytes. ?? 2007 Ecohealth Journal Consortium.

  2. Microarray analysis of differentially expressed genes in ovarian and fallopian tube epithelium from risk-reducing salpingo-oophorectomies.

    PubMed

    Veskimäe, Kristina; Staff, Synnöve; Tabaro, Francesco; Nykter, Matti; Isola, Jorma; Mäenpää, Johanna

    2015-05-01

    Mutations in the BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Ovarian cancer risk can be decreased by risk-reducing salpingo-oophorectomy (RRSO). Studies on RRSO material have altered the paradigm of serous ovarian cancer pathogenesis. The purpose of this study was to identify candidate genes possibly involved in the pathogenesis of serous ovarian cancer by carrying out a microarray analysis of differentially expressed genes in BRCA1/2- mutation positive ovarian and fallopian tube epithelium derived from RRSO surgery. Freshly frozen ovarian and fallopian tube samples from nine BRCA1/2 mutation carriers scheduled for RRSO were prospectively collected together with five mutation-negative control patients undergoing salpingo-oophorectomy for benign indications. Microarray analysis of genome-wide gene expression was performed on ovarian and fallopian tube samples from the BRCA1/2 and control patients. The validation of microarray data was performed by quantitative real-time polymerase chain reaction (qRT-PCR) in selected cases of RRSO samples and also in high grade serous carcinoma samples collected from patients with a BRCA phenotype. From 22,733 genes, 454 transcripts were identified that were differentially expressed in BRCA1/2 mutation carriers when compared with controls, pooling all ovarian and fallopian tube samples together. Of these, 299 genes were statistically significantly downregulated and 155 genes upregulated. Differentially expressed genes in BRCA1/2 samples reported here might be involved in serous ovarian carcinogenesis and provide interesting targets for further studies. PMID:25706666

  3. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells*S⃞

    PubMed Central

    Prokhorova, Tatyana A.; Rigbolt, Kristoffer T. G.; Johansen, Pia T.; Henningsen, Jeanette; Kratchmarova, Irina; Kassem, Moustapha; Blagoev, Blagoy

    2009-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase ζ (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations. PMID:19151416

  4. Differential expression of vascular endothelial growth factor in human fetal skeletal site-specific tissues: Mandible versus femur.

    PubMed

    Marini, Mirca; Bertolai, Roberto; Ambrosini, Stefano; Sarchielli, Erica; Vannelli, Gabriella Barbara; Sgambati, Eleonora

    2015-04-01

    Vascular endothelial growth factor (VEGF) is a well-known mediator that signals through pathways in angiogenesis and osteogenesis. Angiogenesis and bone formation are coupled during either skeletal development or bone remodeling and repair occurring in postnatal life. In this study, we examined for the first time the expression of VEGF in human fetal mandibular and femoral bone in comparison with the respective adult tissues. Similarly to other craniofacial bones, but at variance with the axial and appendicular skeleton, during development mandible does not arise from mesoderm but neural crest cells of the neuroectoderm germ layer, and undergoes intramembranous instead of endochondral ossification. By quantitative real-time PCR technique, we could show that VEGF gene expression levels were significantly higher in fetal than in adult samples, especially in femoral tissue. Western blotting analysis confirmed higher protein expression of VEGF in the fetal femur respect to the mandible. Moreover, immunohistochemistry revealed that in both fetal tissues VEGF expression was mainly localized in pre- and osteoblasts. Differential expression of VEGF in femoral and mandibular bone tissues could be related to their different structure, function and development during organogenesis. PMID:25769656

  5. Quantitative analysis of gene expression in preimplantation mouse embryos using green fluorescent protein reporter.

    PubMed

    Medvedev, Serguei Yuri; Tokunaga, Tomoyuki; Schultz, Richard M; Furukawa, Tsutomu; Nagai, Takashi; Yamaguchi, Manabu; Hosoe, Misa; Yakovlev, Alexander F; Takahashi, Seiya; Izaike, Yoshiaki

    2002-07-01

    We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available. PMID:12080029

  6. Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells

    SciTech Connect

    Du Liping; Neis, Mark M.; Ladd, Patricia A.; Yost, Garold S.; Keeney, Diane S. . E-mail: diane.keeney@vanderbilt.edu

    2006-06-01

    Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.

  7. Characterization and Improvement of RNA-Seq Precision in Quantitative Transcript Expression Profiling

    SciTech Connect

    Labaj, Pawel P.; Leparc, German G.; Linggi, Bryan E.; Markillie, Lye Meng; Wiley, H. S.; Kreil, David P.

    2011-07-01

    Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large scale RNA-Seq data sets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. Results: We report on a comprehensive study of target coverage and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive target coverage of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, less than 30% of all transcripts could be quantified reliably with a relative error < 20%. Based on established tools, we then introduce a new approach for mapping and analyzing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision.

  8. Differential expression of TRPM7 in rat hepatoma and embryonic and adult hepatocytes.

    PubMed

    Lam, D Hung; Grant, Caroline E; Hill, Ceredwyn E

    2012-04-01

    TRPM7 channels are implicated in cellular survival, proliferation, and differentiation. However, a profile of TRPM7 activity in a specific cell type has not been determined from embryonic to terminally differentiated state. Here, we characterized TRPM7 expression in a spectrum of rat liver cells at different developmental stages. Using the whole-cell patch clamp technique, TRPM7-like Na(+) currents were identified in RLC-18 cells, a differentiated, proliferating hepatocellular line derived from day 17 embryonic rat liver. Currents were outwardly rectifying, enhanced in divalent-free solutions, and inhibited by intracellular Mg(2+). Reverse transcription - polymerase chain reaction (RT-PCR) revealed that RLC-18 cells express both TRPM6 and TRPM7. However, mean currents were reduced almost 80% by 1 mmol/L 2-aminoethoxyphenylborate (2-APB) and were abolished in RLC-18 cells heterologously expressing a dominant negative TRPM7 construct, suggesting that TRPM7 is the major current carrier in these cells. Functional comparison showed that relative to terminally differentiated adult rat hepatocytes, currents were 1.8 and 3.9 times higher in, respectively, RLC-18 and WIF-B cells, a rat hepatoma - human fibroblast cross. Our results demonstrate that plasma membrane TRPM7 channels are more highly expressed in proliferating cells as compared with terminally differentiated and nondividing rat hepatocytes and suggest that downregulation of this channel is associated with hepatocellular differentiation. PMID:22429021

  9. Detecting differential protein expression in large-scale population proteomics

    SciTech Connect

    Ryu, Soyoung; Qian, Weijun; Camp, David G.; Smith, Richard D.; Tompkins, Ronald G.; Davis, Ronald W.; Xiao, Wenzhong

    2014-06-17

    Mass spectrometry-based high-throughput quantitative proteomics shows great potential in clinical biomarker studies, identifying and quantifying thousands of proteins in biological samples. However, methods are needed to appropriately handle issues/challenges unique to mass spectrometry data in order to detect as many biomarker proteins as possible. One issue is that different mass spectrometry experiments generate quite different total numbers of quantified peptides, which can result in more missing peptide abundances in an experiment with a smaller total number of quantified peptides. Another issue is that the quantification of peptides is sometimes absent, especially for less abundant peptides and such missing values contain the information about the peptide abundance. Here, we propose a Significance Analysis for Large-scale Proteomics Studies (SALPS) that handles missing peptide intensity values caused by the two mechanisms mentioned above. Our model has a robust performance in both simulated data and proteomics data from a large clinical study. Because varying patients’ sample qualities and deviating instrument performances are not avoidable for clinical studies performed over the course of several years, we believe that our approach will be useful to analyze large-scale clinical proteomics data.

  10. Differential Expression of MicroRNAs in Tissues and Plasma Co-exists as a Biomarker for Pancreatic Cancer

    PubMed Central

    Ali, Shadan; Dubaybo, Hala; Brand, Randall E; Sarkar, Fazlul H

    2016-01-01

    Objective Pancreatic cancer (PC) is a lethal disease with disappointing results from current treatment modalities, suggesting that novel therapeutic strategies are urgently needed. Since microRNAs (miRNAs) are important player in biology, the clinical utility of miRNAs for designing novel therapeutics is an active area of research. The objective of the present study was to examine differentially expressed miRNAs between normal and tumor tissues, and in plasma samples obtained from PC patients, chronic pancreatitis (CP) patients and healthy subjects (HC). Material and methods The miRNA expression profiling using formalin-fixed paraffin embedded (FFPE) tissues from normal and tumor specimens was accomplished using miRBase version 19 (LC Sciences, Houston, TX, USA). Quantitative real-time PCR (qRT-PCR) was subsequently performed in individual samples for 7 selected miRNAs. In addition, qRT-PCR was also performed for assessing the expression of 8 selected miRNAs in plasma samples. Results A significant difference in the expressions of miR-21, miR-205, miR-155, miR-31, miR-203, miR-214 and miR-129-2 were found in tumor tissue samples. Lower expression of miR-214 was found to be associated with better overall survival. We also observed differential expression of 8 miRNAs in plasma samples of CP and PC patients compared to HC. Interestingly, over expression of miR-21, and miR-31 was noted in both tumor tissues and in the plasma. Conclusion We found deregulated expression of miRNAs that could distinguish normal from PC in two different types of samples (tissues and plasma). Interestingly, lower expression of miR-214 was found to be associated with better overall survival. Although not statistically significant, we also observed higher expression of let-7a and lower expression of miR-508 to be associated with overall better survival. We conclude that our study nicely lays the foundation for detailed future investigations for assessing the role of these miRNAs in the

  11. Differential gene expression in migratory streams of cortical interneurons

    PubMed Central

    Antypa, Mary; Faux, Clare; Eichele, Gregor; Parnavelas, John G; Andrews, William D

    2011-01-01

    Cortical interneurons originate in the ganglionic eminences of the subpallium and migrate into the cortex in well-defined tangential streams. At the start of corticogenesis, two streams of migrating neurons are evident: a superficial one at the level of the preplate (PPL), and a deeper one at the level of the intermediate zone (IZ). Currently, little is known about the signalling mechanisms that regulate interneuron migration, and almost nothing is known about the molecules that may be involved in their choice of migratory stream. Here, we performed a microarray analysis, comparing the changes in gene expression between cells migrating in the PPL and those migrating in the IZ at embryonic day 13.5. This analysis identified genes, many of them novel, that were upregulated in one of the two streams. Moreover, polymerase chain reaction, in situ hybridization experiments and immunohistochemistry showed the expression of these genes in interneurons migrating within the PPL or IZ, suggesting that they play a role in their migration and choice of stream. PMID:22103416

  12. KLF4 Expression Correlates with the Degree of Differentiation in Colorectal Cancer

    PubMed Central

    Hu, Ruolei; Zuo, Yan; Zuo, Li; Liu, Chao; Zhang, Sumei; Wu, Qiang; Zhou, Qing; Gui, Shuyu; Wei, Wei

    2011-01-01

    Background/Aims Krüppel-like factor 4 (KLF4) is an epithelial-specific transcription factor primarily expressed in the gastrointestinal tract that mediates growth arrest in the colonic epithelium. We tried to find whether KLF4 expression is associated with the progression and differentiation of colorectal cancer. Methods We detected KLF4 expression in 109 colorectal specimens (40 normal appearing mucosa, 7 adenomas, and 62 carcinomas) by immunohistochemistry using a tissue microarray. Western blot and RT-PCR analyses were also performed. Results The upregulation of KLF4 expression in carcinoma tissue was statistically significant (p<0.05) when compared to normal appearing mucosa. The negative and weak positive staining rates in normal appearing mucosa, adenoma, and carcinoma were 42.5%, 71.4%, and 82.3%, respectively, indicating a decreased degree of KLF4 expression over the course of progressive transformation of normal cells into malignant derivatives. KLF4 protein levels showed no correlation with sex, age, or metastatic state (p>0.05), while KLF4 protein expression correlated with the diagnostic stage (p<0.05). Furthermore, strong KLF4 staining was detected in 22.9% (11/48) and 0% (0/14) of well/moderately and poorly differentiated colorectal cancers, respectively. Our results clearly indicate that KLF4 protein expression significantly correlates with the degree of differentiation in colorectal cancers (p<0.05). KLF4 expression in RKO cells is also upregulated by butyrate, an inducer of differentiation. Conclusions Downregulation of KLF4 expression may lead to more poorly differentiated tumors. PMID:21814594

  13. Differential expression of genes in milk of dairy cattle during lactation.

    PubMed

    Yang, Jie; Jiang, Jicai; Liu, Xuan; Wang, Haifei; Guo, Gang; Zhang, Qin; Jiang, Li

    2016-04-01

    The milk fat globule (MFG) is one of the most representative of mammary gland tissues and can be utilized to study gene expression of lactating cows during lactation. In this study, RNA-seq technology was employed to detect differential expression of genes in MFGs at day 10 and day 70 after calving between two groups of cows with extremely high (H group) and low (L group) 305-day milk yield, milk fat yield and milk protein yield. In total, 1232, 81, 429 and 178 significantly differentially expressed genes (false discovery rate q < 0.05) were detected between H10 and L10, H70 and L70, H10 and H70, and L10 and L70 respectively. Gene Ontology enrichment and pathway analysis revealed that these differentially expressed genes were enriched in biological processes involved in mammary gland development, protein and lipid metabolism process, signal transduction, cellular process, differentiation and immune function. Among these differentially expressed genes, 178 (H10 vs. L10), 4 (H70 vs. L70), 68 (H10 vs. H70) and 22 (L10 vs. L70) were found to be located within previously reported QTL regions for milk production traits. Based on these results, some promising candidate genes for milk production traits in dairy cattle were suggested. PMID:26692495

  14. Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation.

    PubMed

    Dakir, E L Habib; Feigenbaum, Lionel; Linnoila, R Ilona

    2008-12-01

    Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. PMID:18701433

  15. Microarray Analyses of Gene Expression during Chondrocyte Differentiation Identifies Novel Regulators of Hypertrophy

    PubMed Central

    James, Claudine G.; Appleton, C. Thomas G.; Ulici, Veronica; Underhill, T. Michael; Beier, Frank

    2005-01-01

    Ordered chondrocyte differentiation and maturation is required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. We used Affymetrix microarrays to examine temporal gene expression patterns during chondrogenic differentiation in a mouse micromass culture system. Robust normalization of the data identified 3300 differentially expressed probe sets, which corresponds to 1772, 481, and 249 probe sets exhibiting minimum 2-, 5-, and 10-fold changes over the time period, respectively. GeneOntology annotations for molecular function show changes in the expression of molecules involved in transcriptional regulation and signal transduction among others. The expression of identified markers was confirmed by RT-PCR, and cluster analysis revealed groups of coexpressed transcripts. One gene that was up-regulated at later stages of chondrocyte differentiation was Rgs2. Overexpression of Rgs2 in the chondrogenic cell line ATDC5 resulted in accelerated hypertrophic differentiation, thus providing functional validation of microarray data. Collectively, these analyses provide novel information on the temporal expression of molecules regulating endochondral bone development. PMID:16135533

  16. GOGOT: a method for the identification of differentially expressed fragments from cDNA-AFLP data

    PubMed Central

    2007-01-01

    Background One-dimensional (1-D) electrophoretic data obtained using the cDNA-AFLP method have attracted great interest for the identification of differentially expressed transcript-derived fragments (TDFs). However, high-throughput analysis of the cDNA-AFLP data is currently limited by the need for labor-intensive visual evaluation of multiple electropherograms. We would like to have high-throughput ways of identifying such TDFs. Results We describe a method, GOGOT, which automatically detects the differentially expressed TDFs in a set of time-course electropherograms. Analysis by GOGOT is conducted as follows: correction of fragment lengths of TDFs, alignment of identical TDFs across different electropherograms, normalization of peak heights, and identification of differentially expressed TDFs using a special statistic. The output of the analysis is a highly reduced list of differentially expressed TDFs. Visual evaluation confirmed that the peak alignment was performed perfectly for the TDFs by virtue of the correction of peak fragment lengths before alignment in step 1. The validity of the automated ranking of TDFs by the special statistic was confirmed by the visual evaluation of a third party. Conclusion GOGOT is useful for the automated detection of differentially expressed TDFs from cDNA-AFLP temporal electrophoretic data. The current algorithm may be applied to other electrophoretic data and temporal microarray data. PMID:17535446

  17. Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18.

    PubMed

    Bao, W B; Ye, L; Pan, Z Y; Zhu, J; Du, Z D; Zhu, G Q; Huang, X G; Wu, S L

    2012-10-01

    In this study, Agilent two-colour microarray-based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full-sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two-fold change minimum threshold, we found 18 genes that were differentially expressed (10 up-regulated and eight down-regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation- and immune-related pathways. Based on the genes identified in the screen and the pathway analysis results, real-time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid-related pathways), SLA-1 and SLA-3 genes (of inflammation- and immune-related pathways), as well as the differential genes FUT1, TAP1 and SLA-DQA. Subsequently, real-time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18. PMID:22497274

  18. Differential Expression of MicroRNA Species in Human Uterine Leiomyoma versus Normal Myometrium

    PubMed Central

    Marsh, Erica E.; Lin, Zhihong; Yin, Ping; Milad, Magdy; Chakravarti, Debabrata; Bulun, Serdar E.

    2009-01-01

    Objective To determine whether microRNAs are differentially expressed in human leiomyoma versus matched myometrial tissue. Design Microarray with real-time PCR validation. Setting Academic medical center Patients Premenopausal subjects (n=13), who were undergoing hysterectomies for leiomyoma-related symptoms. Interventions none Main Outcome Measure Statistically differential expression of microRNAs in leiomyoma versus myometrium. Results Forty-six miRNA species were differentially expressed in leiomyoma versus normal myometrium with p-values <0.01. Of these, 19 were overexpressed whereas 27 were downregulated in leiomyomas. The fold changes ranged from 1.2 to 11.8. These findings were confirmed using real time RT-PCR for selected miRNAs (miRNAs 21, 34a, 125b, 139 and 323). Conclusions Our findings indicate that miRNAs are differentially expressed between human leiomyoma and matched myometrium. Given this differential expression, miRNAs may play a role in the pathogenesis of uterine leiomyoma and may serve as future therapeutic targets for the treatment of these tumors. PMID:17765232

  19. Differential effects of environment on potato phenylpropanoid and carotenoid expression

    PubMed Central

    2012-01-01

    Background Plant secondary metabolites, including phenylpropanoids and carotenoids, are stress inducible, have important roles in potato physiology and influence the nutritional value of potatoes. The type and magnitude of environmental effects on tuber phytonutrients is unclear, especially under modern agricultural management that minimizes stress. Understanding factors that influence tuber secondary metabolism could facilitate production of more nutritious crops. Metabolite pools of over forty tuber phenylpropanoids and carotenoids, along with the expression of twenty structural genes, were measured in high-phenylpropanoid purple potatoes grown in environmentally diverse locations in North America (Alaska, Texas and Florida). Results Phenylpropanoids, including chlorogenic acid (CGA), were higher in samples from the northern latitudes, as was the expression of phenylpropanoid genes including phenylalanine ammonia lyase (PAL), which had over a ten-fold difference in relative abundance. Phenylpropanoid gene expression appeared coordinately regulated and was well correlated with metabolite pools, except for hydroxycinnamoyl-CoA:quinatehydroxcinnamoyl transferase (HQT; r = -0.24). In silico promoter analysis identified two cis-acting elements in the HQT promoter not found in the other phenylpropanoid genes. Anthocyanins were more abundant in Alaskan samples and correlated with flavonoid genes including DFR (r = 0.91), UFGT (r = 0.94) and F3H (r = 0.77). The most abundant anthocyanin was petunidin-3-coum-rutinoside-5-glu, which ranged from 4.7 mg g-1 in Alaska to 2.3 mg g-1 in Texas. Positive correlations between tuber sucrose and anthocyanins (r = 0.85), suggested a stimulatory effect of sucrose. Smaller variation was observed in total carotenoids, but marked differences occurred in individual carotenoids, which had over a ten-fold range. Violaxanthin, lutein or zeaxanthin were the predominant carotenoids in tubers from Alaska, Texas and Florida respectively. Unlike

  20. Gene expression analysis by quantitative real-time PCR for floral tissues.

    PubMed

    Bustamante, Mariana; Jin, Jian; Casagran, Oriol; Nolan, Tania; Riechmann, José Luis

    2014-01-01

    Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler(®) 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment. PMID:24395270

  1. Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).

    PubMed

    Zhang, F; Wang, X J; Huang, Y H; Zhao, Z G; Zhang, S S; Gong, X S; Xie, L; Kang, D M; Jing, X

    2014-08-01

    A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides. PMID:25182623

  2. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    NASA Astrophysics Data System (ADS)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  3. Quantitative analysis of miRNA expression in seven human foetal and adult organs.

    PubMed

    Tang, Yanping; Liu, Dong; Zhang, Lijie; Ingvarsson, Sigurdur; Chen, Huiping

    2011-01-01

    miRNAs have been found to repress gene expression at posttranscriptional level in cells. Studies have shown that expression of miRNAs is tissue-specific and developmental-stage-specific. The mechanism behind this could be explained by miRNA pathways. In this study, totally 54 miRNAs were analysed in 7 matched human foetal and adult organs (brain, colon, heart, kidney, liver, lung and spleen) using real-time PCR. Quantitative analysis showed that a big proportion of the 54 miRNAs have higher general expression in the organs of the foetal period than the adult period, with the exception of the heart. The miRNA gene promoter methylation level in the adult stages was higher than in the foetal stages. Moreover, there is a high general expression level of several miRNAs in both stages of brain, kidney, liver, lung and spleen, but not seen in colon and heart. Our results indicate that the miRNAs may play a bigger role in the foetal stage than the adult stage of brain, colon, kidney, liver, lung and spleen. The majority of the miRNAs analysed may play an important role in the growth and development of brain, kidney, liver, lung and spleen. However, a minority of the miRNAs may be functional in colon and heart. PMID:22194897

  4. The use of relative quantitative RT-PCR for expression analysis in azalea flower color sports.

    PubMed

    De Keyser, E; De Riek, J; Van Bockstaele, E

    2003-01-01

    The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all 'Hellmut Vogel' flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed. PMID:24757769

  5. Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp

    NASA Technical Reports Server (NTRS)

    Peterson, K. J.; Irvine, S. Q.; Cameron, R. A.; Davidson, E. H.

    2000-01-01

    A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

  6. Consensus Genome-Wide Expression Quantitative Trait Loci and Their Relationship with Human Complex Trait Disease.

    PubMed

    Yu, Chen-Hsin; Pal, Lipika R; Moult, John

    2016-07-01

    Most of the risk loci identified from genome-wide association (GWA) studies do not provide direct information on the biological basis of a disease or on the underlying mechanisms. Recent expression quantitative trait locus (eQTL) association studies have provided information on genetic factors associated with gene expression variation. These eQTLs might contribute to phenotype diversity and disease susceptibility, but interpretation is handicapped by low reproducibility of the expression results. To address this issue, we have generated a set of consensus eQTLs by integrating publicly available data for specific human populations and cell types. Overall, we find over 4000 genes that are involved in high-confidence eQTL relationships. To elucidate the role that eQTLs play in human common diseases, we matched the high-confidence eQTLs to a set of 335 disease risk loci identified from the Wellcome Trust Case Control Consortium GWA study and follow-up studies for 7 human complex trait diseases-bipolar disorder (BD), coronary artery disease (CAD), Crohn's disease (CD), hypertension (HT), rheumatoid arthritis (RA), type 1 diabetes (T1D), and type 2 diabetes (T2D). The results show that the data are consistent with ∼50% of these disease loci arising from an underlying expression change mechanism. PMID:27428252

  7. Quantitative expression profile of hepatobiliary transporters in sandwich cultured rat and human hepatocytes.

    PubMed

    Li, Na; Bi, Yi-An; Duignan, David B; Lai, Yurong

    2009-01-01

    As sandwich cultured (SC) hepatocytes can repolarize to form bile canalicular networks, allowing active excretion of compounds in a vectorial manner, the model has been widely used for assessing the transporter related complexity of ADME/tox issues. A lack of quantitative information on transporter expression during cell culture has made in vitro to in vivo extrapolation of hepatobiliary transport difficult. In the present study, using our newly developed LC-MS/MS absolute quantitative methods, we determined the quantitative expression profile of three biliary transporters in SC rat and human hepatocytes. A significant shift of hepatobiliary transporter proteins was observed both in human and rat sandwich cultures. A decrease of BSEP/Bsep protein and an increase of BCRP/Bcrp protein were detected in both rat and human hepatocytes over time in culture. Interestingly, Mrp2 in rat hepatocytes was significantly diminished, while MRP2 constantly increased in human hepatocytes during the cell culture. Consequently, the interspecies difference between rat and human in absolute amount of MRP2/Mrp2 was minimized over time in culture. Following the sandwich culture, the species difference of hepatobiliary transporter protein between human and rat at day 5 post SC was diminished (MRP2/Mrp2), identical (BSEP/Bsep) or reversed (BCRP/Bcrp), compared to the in vivo situation. In addition, the absolute protein amount of BCRP/Bcrp or MRP2/Mrp2 was proportionally correlated with the intrinsic biliary clearance estimated in various lots of SC rat and human hepatocytes. The results revealed that absolute protein amount is a key determinant for hepatobiliary clearance and could provide fundamental support on extrapolation of biliary secretion from in vitro to in vivo. PMID:19545175

  8. Differential expression of several drug transporter genes in HepG2 and Huh-7 cell lines

    PubMed Central

    Louisa, Melva; Suyatna, Frans D.; Wanandi, Septelia Inawati; Asih, Puji Budi Setia; Syafruddin, Din

    2016-01-01

    Background: Cell culture techniques have many advantages for investigation of drug transport to target organ like liver. HepG2 and Huh-7 are two cell lines available from hepatoma that can be used as a model for hepatic drug transport. The present study is aimed to analyze the expression level of several drug transporter genes in two hepatoma cell lines, HepG2 and Huh-7 and their response to inhibitors. Materials and Methods: This is an in vitro study using HepG2 and Huh-7 cells. The expression level of the following drug transporter genes was quantified: P-glycoprotein/multidrug resistance protein 1, Organic Anionic Transporter Protein 1B1 (OATP1B1) and Organic Cationic Transporter-1 (OCT1). Ribonucleic acid was extracted from the cells using Tripure isolation reagent, then gene expression level of the transporters is quantified using Applied Biosystems quantitative reverse transcriptase polymerase chain reaction. Verapamil (P-glycoprotein inhibitor), nelfinavir (OATP1B1 inhibitor), quinidine (OCT1 inhibitor) were used to differentiate the inhibitory properties of these agents to the transporter expressions in HepG2 and Huh-7 cells. Results: Huh-7 shows a higher level of P-glycoprotein, OATP1B1 and OCT1 expressions compared with those of HepG2. Verapamil reduces the expressions of P-glycoprotein in HepG2 and Huh-7; nelfinavir reduces the expression of OATP1B1 in HepG2 and Huh-7; while quinidine reduces the OCT1 gene expressions in HepG2, but not in Huh-7 cells. Conclusion: This study indicates that HepG2 might be a more suitable in vitro model than Huh-7 to study drug transport in hepatocytes involving drug transporters. PMID:27376043

  9. Satb1 and Satb2 regulate embryonic stem cell differentiation and Nanog expression

    PubMed Central

    Savarese, Fabio; Dávila, Amparo; Nechanitzky, Robert; De La Rosa-Velazquez, Inti; Pereira, Carlos F.; Engelke, Rudolf; Takahashi, Keiko; Jenuwein, Thomas; Kohwi-Shigematsu, Terumi; Fisher, Amanda G.; Grosschedl, Rudolf

    2009-01-01

    Satb1 and the closely related Satb2 proteins regulate gene expression and higher-order chromatin structure of multigene clusters in vivo. In examining the role of Satb proteins in murine embryonic stem (ES) cells, we find that Satb1−/− cells display an impaired differentiation potential and augmented expression of the pluripotency determinants Nanog, Klf4, and Tbx3. Metastable states of self-renewal and differentiation competence have been attributed to heterogeneity of ES cells in the expression of Nanog. Satb1−/− cultures have a higher proportion of Nanoghigh cells, and an increased potential to reprogram human B lymphocytes in cell fusion experiments. Moreover, Satb1-deficient ES cells show an increased expression of Satb2, and we find that forced Satb2 expression in wild-type ES cells antagonizes differentiation-associated silencing of Nanog and enhances the induction of NANOG in cell fusions with human B lymphocytes. An antagonistic function of Satb1 and Satb2 is also supported by the almost normal differentiation potential of Satb1−/−Satb2−/− ES cells. Taken together with the finding that both Satb1 and Satb2 bind the Nanog locus in vivo, our data suggest that the balance of Satb1 and Satb2 contributes to the plasticity of Nanog expression and ES cell pluripotency. PMID:19933152

  10. Meta-Analysis of Differential Connectivity in Gene Co-Expression Networks in Multiple Sclerosis

    PubMed Central

    Creanza, Teresa Maria; Liguori, Maria; Liuni, Sabino; Nuzziello, Nicoletta; Ancona, Nicola

    2016-01-01

    Differential gene expression analyses to investigate multiple sclerosis (MS) molecular pathogenesis cannot detect genes harboring genetic and/or epigenetic modifications that change the gene functions without affecting their expression. Differential co-expression network approaches may capture changes in functional interactions resulting from these alterations. We re-analyzed 595 mRNA arrays from publicly available datasets by studying changes in gene co-expression networks in MS and in response to interferon (IFN)-β treatment. Interestingly, MS networks show a reduced connectivity relative to the healthy condition, and the treatment activates the transcription of genes and increases their connectivity in MS patients. Importantly, the analysis of changes in gene connectivity in MS patients provides new evidence of association for genes already implicated in MS by single-nucleotide polymorphism studies and that do not show differential expression. This is the case of amiloride-sensitive cation channel 1 neuronal (ACCN1) that shows a reduced number of interacting partners in MS networks, and it is known for its role in synaptic transmission and central nervous system (CNS) development. Furthermore, our study confirms a deregulation of the vitamin D system: among the transcription factors that potentially regulate the deregulated genes, we find TCF3 and SP1 that are both involved in vitamin D3-induced p27Kip1 expression. Unveiling differential network properties allows us to gain systems-level insights into disease mechanisms and may suggest putative targets for the treatment. PMID:27314336

  11. Meta-Analysis of Differential Connectivity in Gene Co-Expression Networks in Multiple Sclerosis.

    PubMed

    Creanza, Teresa Maria; Liguori, Maria; Liuni, Sabino; Nuzziello, Nicoletta; Ancona, Nicola

    2016-01-01

    Differential gene expression analyses to investigate multiple sclerosis (MS) molecular pathogenesis cannot detect genes harboring genetic and/or epigenetic modifications that change the gene functions without affecting their expression. Differential co-expression network approaches may capture changes in functional interactions resulting from these alterations. We re-analyzed 595 mRNA arrays from publicly available datasets by studying changes in gene co-expression networks in MS and in response to interferon (IFN)-β treatment. Interestingly, MS networks show a reduced connectivity relative to the healthy condition, and the treatment activates the transcription of genes and increases their connectivity in MS patients. Importantly, the analysis of changes in gene connectivity in MS patients provides new evidence of association for genes already implicated in MS by single-nucleotide polymorphism studies and that do not show differential expression. This is the case of amiloride-sensitive cation channel 1 neuronal (ACCN1) that shows a reduced number of interacting partners in MS networks, and it is known for its role in synaptic transmission and central nervous system (CNS) development. Furthermore, our study confirms a deregulation of the vitamin D system: among the transcription factors that potentially regulate the deregulated genes, we find TCF3 and SP1 that are both involved in vitamin D3-induced p27Kip1 expression. Unveiling differential network properties allows us to gain systems-level insights into disease mechanisms and may suggest putative targets for the treatment. PMID:27314336

  12. Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPβ.

    PubMed

    Domizi, Pablo; Aoyama, Chieko; Banchio, Claudia

    2014-04-01

    Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation. PMID:24440820

  13. Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes.

    PubMed

    Houston, B; Seawright, E; Jefferies, D; Hoogland, E; Lester, D; Whitehead, C; Farquharson, C

    1999-01-11

    Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes. PMID:9990301

  14. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma

    PubMed Central

    Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M.; Davies, Anthony J.; Weetman, Malcolm; Garden, Oliver A.; Masters, John R.; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  15. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

    PubMed

    Giuliano, Antonio; Swift, Rebecca; Arthurs, Callum; Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M; Davies, Anthony J; Weetman, Malcolm; Garden, Oliver A; Masters, John R; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  16. Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

    PubMed

    Santana, Flávia A; Nunes, Francis M F; Vieira, Carlos U; Machado, Maria Alice M S; Kerr, Warwick E; Silva, Wilson A; Bonetti, Ana Maria

    2006-03-01

    We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona. PMID:16532208

  17. Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display.

    PubMed

    Biswas, Pranjal; Majumdar, Uddalak; Ghosh, Sanjay

    2016-03-01

    Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways. PMID:26858975

  18. Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display

    PubMed Central

    Biswas, Pranjal; Majumdar, Uddalak; Ghosh, Sanjay

    2015-01-01

    Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways. PMID:26858975

  19. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells

    PubMed Central

    Nawandar, Dhananjay M.; Wang, Anqi; Makielski, Kathleen; Lee, Denis; Ma, Shidong; Barlow, Elizabeth; Reusch, Jessica; Jiang, Ru; Wille, Coral K.; Greenspan, Deborah; Greenspan, John S.; Mertz, Janet E.; Hutt-Fletcher, Lindsey; Johannsen, Eric C.; Lambert, Paul F.; Kenney, Shannon C.

    2015-01-01

    Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells. PMID:26431332

  20. Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype

    PubMed Central

    Branco, Ana F.; Pereira, Susana P.; Gonzalez, Susana; Gusev, Oleg; Rizvanov, Albert A.; Oliveira, Paulo J.

    2015-01-01

    H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays. PMID:26121149

  1. Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype.

    PubMed

    Branco, Ana F; Pereira, Susana P; Gonzalez, Susana; Gusev, Oleg; Rizvanov, Albert A; Oliveira, Paulo J

    2015-01-01

    H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays. PMID:26121149

  2. Quantitation of CDH1 promoter methylation in formalin-fixed paraffin-embedded tissues of breast cancer patients using differential high resolution melting analysis

    PubMed Central

    Naghitorabi, Mojgan; Mohammadi-Asl, Javad; Sadeghi, Hamid Mir Mohammad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas; Javanmard, Shaghayegh Haghjooy

    2016-01-01

    Background: E-cadherin (CDH1) plays an important role in cell–cell adhesion of epithelial tissues. Loss of E-cadherin expression can lead to loss of tissue integrity, metastasis, and cancer progression. Also loss of E-cadherin expression might be related to aberrant promoter methylation of the CDH1 gene. Many studies have been performed on CDH1 promoter methylation, especially in breast cancer. Although most of the studies have used qualitative methods for methylation analysis, this study is designed to quantitatively investigate CDH1 promoter methylation in breast cancer and its correlation with patients’ clinicopathological features. Materials and Methods: Using differential high resolution melting analysis (D-HRMA), the methylation level of the CDH1 gene promoter was quantified in 98 breast cancer formalin-fixed paraffin-embedded (FFPE) tissues and also 10 fresh frozen normal breast tissues. Results: All samples were detected to be methylated at the CDH1 promoter region. About 74.5% of the breast cancer samples were hypermethylated with an average methylation level of around 60%, while 25.5% of the patients were methylated with the mean methylation level of about 33%, and 90% of the normal samples had a mean methylation level of about 18%. Statistical analyses represented a significant correlation between CDH1 promoter methylation and cancer progression hallmarks, such as, clinical stage, nodal involvement, tumor size, and histological grade. Conclusion: In summary, quantitation of CDH1 promoter methylation can serve as a diagnostic and prognostic tool in breast cancer. Also D-HRMA can be used as a fast and reliable method for quantitation of promoter methylation. PMID:27308263

  3. Differential nuclear scaffold/matrix attachment marks expressed genes†

    PubMed Central

    Linnemann, Amelia K.; Platts, Adrian E.; Krawetz, Stephen A.

    2009-01-01

    It is well established that nuclear architecture plays a key role in poising regions of the genome for transcription. This may be achieved using scaffold/matrix attachment regions (S/MARs) that establish loop domains. However, the relationship between changes in the physical structure of the genome as mediated by attachment to the nuclear scaffold/matrix and gene expression is not clearly understood. To define the role of S/MARs in organizing our genome and to resolve the often contradictory loci-specific studies, we have surveyed the S/MARs in HeLa S3 cells on human chromosomes 14–18 by array comparative genomic hybridization. Comparison of LIS (lithium 3,5-diiodosalicylate) extraction to identify SARs and 2 m NaCl extraction to identify MARs revealed that approximately one-half of the sites were in common. The results presented in this study suggest that SARs 5′ of a gene are associated with transcript presence whereas MARs contained within a gene are associated with silenced genes. The varied functions of the S/MARs as revealed by the different extraction methods highlights their unique functional contribution. PMID:19017725

  4. Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases.

    PubMed

    Johnson, Clayton H; Klotz, Martin G; York, J Lyndal; Kruft, Volker; McEwen, Joan E

    2002-04-01

    Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species. Evidence is presented here for two additional catalase isozymes in H. capsulatum. Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts. Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated. The transcriptional expression of the H. capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA. Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H(2)O(2)) and carbon source (glucose vs glycerol). Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress. A phylogenetic analysis of presently available catalase sequences and intron residences was done. The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes. PMID:11932457

  5. Quantitative immunohistochemical expression of c Kit in breast carcinomas is predictive of patients' outcome

    PubMed Central

    Charpin, C; Giusiano, S; Charfi, S; Secq, V; Carpentier, S; Andrac, L; Lavaut, M-N; Allasia, C; Bonnier, P; Garcia, S

    2009-01-01

    Background: c Kit (CD117) expression in tissues has been reported as a relevant target for specific therapy in some human malignancies, but has been poorly documented in breast carcinomas Methods: The prognostic significance of c Kit in a series of 924 breast carcinomas (mean follow-up, 79 months) was investigated using standardised high-throughput quantitative densitometry of immunohistochemical precipitates in tissue microarrays. Results: c Kit was expressed in 14.7% breast carcinomas (and in 42 out of 586 node-negative tumours). In univariate analysis, (log-rank test) the score of c Kit expression correlated with poor patient outcome P=0.02 and particularly in node-negative cases (P=0.002). In multivariate Cox analysis, c Kit was an indicator of metastasis independent of 25 other concomitantly evaluated markers of prognosis. Logistic regression showed that c Kit ranked 10 out of 25 (P=0.041), and was included in a 10-marker signature that allowed 79.2% of the patients to be correctly classified in the metastatic or metastasis-free categories independently of hormone receptors and HER-2 status. Interestingly, c Kit was also a significant predictor of metastasis in node-negative tumours (2 out of 25 ranking, P<0.0001) and included in a six-marker signature of prognosis, correctly classifying 88.6% of the patients (P<0.0001). Conclusion: We concluded that, as assessed by quantitative immunohistochemistry, c Kit is an independent prognostic indicator that could also potentially serve as a target for specific therapy in breast carcinomas. PMID:19513067

  6. Identification of differentially expressed genes of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) in response to Tetracapsuloides bryosalmonae (Myxozoa).

    PubMed

    Kumar, Gokhlesh; Abd-Elfattah, Ahmed; El-Matbouli, Mansour

    2015-03-01

    Tetracapsuloides bryosalmonae Canning et al., 1999 (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. We have shown previously that the development and distribution of the European strain of T. bryosalmonae differs in the kidney of brown trout (Salmo trutta) Linnaeus, 1758 and rainbow trout (Oncorhynchus mykiss) Walbaum, 1792, and that intra-luminal sporogonic stages were found in brown trout but not in rainbow trout. We have now compared transcriptomes from kidneys of brown trout and rainbow trout infected with T. bryosalmonae using suppressive subtractive hybridization (SSH). The differentially expressed transcripts produced by SSH were cloned, transformed, and tested by colony PCR. Differential expression screening of PCR products was validated using dot blot, and positive clones having different signal intensities were sequenced. Differential screening and a subsequent NCBI-BLAST analysis of expressed sequence tags revealed nine clones expressed differently between both fish species. These differentially expressed genes were validated by quantitative real-time PCR of kidney samples from both fish species at different time points of infection. Expression of anti-inflammatory (TSC22 domain family protein 3) and cell proliferation (Prothymin alpha) genes were upregulated significantly in brown trout but downregulated in rainbow trout. The expression of humoral immune response (immunoglobulin mu) and endocytic pathway (Ras-related protein Rab-11b) genes were significantly upregulated in rainbow trout but downregulated in brown trout. This study suggests that differential expression of host anti-inflammatory, humoral immune and endocytic pathway responses, cell proliferation, and cell growth processes do not inhibit the development of intra-luminal sporogonic stages of the European strain of T. bryosalmonae in brown trout but may suppress it in rainbow trout. PMID:25563603

  7. MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

    PubMed Central

    Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

    2015-01-01

    The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

  8. Integrated Analysis of Expression Profile Based on Differentially Expressed Genes in Middle Cerebral Artery Occlusion Animal Models.

    PubMed

    Zhou, Huaqiang; Qiu, Zeting; Gao, Shaowei; Chen, Qinchang; Li, Si; Tan, Wulin; Liu, Xiaochen; Wang, Zhongxing

    2016-01-01

    Stroke is one of the most common causes of death, only second to heart disease. Molecular investigations about stroke are in acute shortage nowadays. This study is intended to explore a gene expression profile after brain ischemia reperfusion. Meta-analysis, differential expression analysis, and integrated analysis were employed on an eight microarray series. We explored the functions and pathways of target genes in gene ontology (GO) enrichment analysis and constructed a protein-protein interaction network. Meta-analysis identified 360 differentially expressed genes (DEGs) for Mus musculus and 255 for Rattus norvegicus. Differential expression analysis identified 44 DEGs for Mus musculus and 21 for Rattus norvegicus. Timp1 and Lcn2 were overexpressed in both species. The cytokine-cytokine receptor interaction and chemokine signaling pathway were highly enriched for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. We have exhibited a global view of the potential molecular differences between middle cerebral artery occlusion (MCAO) animal model and sham for Mus musculus or Rattus norvegicus, including the biological process and enriched pathways in DEGs. This research helps contribute to a clearer understanding of the inflammation process and accurate identification of ischemic infarction stages, which might be transformed into a therapeutic approach. PMID:27213359

  9. Integrated Analysis of Expression Profile Based on Differentially Expressed Genes in Middle Cerebral Artery Occlusion Animal Models

    PubMed Central

    Zhou, Huaqiang; Qiu, Zeting; Gao, Shaowei; Chen, Qinchang; Li, Si; Tan, Wulin; Liu, Xiaochen; Wang, Zhongxing

    2016-01-01

    Stroke is one of the most common causes of death, only second to heart disease. Molecular investigations about stroke are in acute shortage nowadays. This study is intended to explore a gene expression profile after brain ischemia reperfusion. Meta-analysis, differential expression analysis, and integrated analysis were employed on an eight microarray series. We explored the functions and pathways of target genes in gene ontology (GO) enrichment analysis and constructed a protein-protein interaction network. Meta-analysis identified 360 differentially expressed genes (DEGs) for Mus musculus and 255 for Rattus norvegicus. Differential expression analysis identified 44 DEGs for Mus musculus and 21 for Rattus norvegicus. Timp1 and Lcn2 were overexpressed in both species. The cytokine-cytokine receptor interaction and chemokine signaling pathway were highly enriched for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. We have exhibited a global view of the potential molecular differences between middle cerebral artery occlusion (MCAO) animal model and sham for Mus musculus or Rattus norvegicus, including the biological process and enriched pathways in DEGs. This research helps contribute to a clearer understanding of the inflammation process and accurate identification of ischemic infarction stages, which might be transformed into a therapeutic approach. PMID:2721335