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Sample records for cells reveals genes

  1. Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation.

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    Hongkai Ji

    Full Text Available The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP, global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs. We further document that a Myc core signature (MCS set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

  2. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    International Nuclear Information System (INIS)

    Vanderslice, P.; Ballinger, S.M.; Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H.

    1990-01-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  3. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    Science.gov (United States)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  4. RNA-Seq reveals spliceosome and proteasome genes as most consistent transcripts in human cancer cells.

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    Tara Macrae

    Full Text Available Accurate quantification of gene expression by qRT-PCR relies on normalization against a consistently expressed control gene. However, control genes in common use often vary greatly between samples, especially in cancer. The advent of Next Generation Sequencing technology offers the possibility to better select control genes with the least cell to cell variability in steady state transcript levels. Here we analyze the transcriptomes of 55 leukemia samples to identify the most consistent genes. This list is enriched for components of the proteasome (ex. PSMA1 and spliceosome (ex. SF3B2, and also includes the translation initiation factor EIF4H, and many heterogeneous nuclear ribonucleoprotein genes (ex. HNRNPL. We have validated the consistency of our new control genes in 1933 cancer and normal tissues using publically available RNA-seq data, and their usefulness in qRT-PCR analysis is clearly demonstrated.

  5. Cell-Specific PEAR1 Methylation Studies Reveal a Locus that Coordinates Expression of Multiple Genes

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    Benedetta Izzi

    2018-04-01

    Full Text Available Chromosomal interactions connect distant enhancers and promoters on the same chromosome, activating or repressing gene expression. PEAR1 encodes the Platelet-Endothelial Aggregation Receptor 1, a contact receptor involved in platelet function and megakaryocyte and endothelial cell proliferation. PEAR1 expression during megakaryocyte differentiation is controlled by DNA methylation at its first CpG island. We identified a PEAR1 cell-specific methylation sensitive region in endothelial cells and megakaryocytes that showed strong chromosomal interactions with ISGL20L2, RRNAD1, MRLP24, HDGF and PRCC, using available promoter capture Hi-C datasets. These genes are involved in ribosome processing, protein synthesis, cell cycle and cell proliferation. We next studied the methylation and expression profile of these five genes in Human Umbilical Vein Endothelial Cells (HUVECs and megakaryocyte precursors. While cell-specific PEAR1 methylation corresponded to variability in expression for four out of five genes, no methylation change was observed in their promoter regions across cell types. Our data suggest that PEAR1 cell-type specific methylation changes may control long distance interactions with other genes. Further studies are needed to show whether such interaction data might be relevant for the genome-wide association data that showed a role for non-coding PEAR1 variants in the same region and platelet function, platelet count and cardiovascular risk.

  6. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Hoei-Hansen, Christina E; Wirkner, Ute

    2004-01-01

    in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported......Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly...

  7. Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

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    Clive H Glover

    2006-11-01

    Full Text Available Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42 showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

  8. Transcriptome of interstitial cells of Cajal reveals unique and selective gene signatures.

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    Moon Young Lee

    Full Text Available Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC, which serve as slow-wave electrical pacemakers for gastrointestinal (GI smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.

  9. What Population Reveals about Individual Cell Identity: Single-Cell Parameter Estimation of Models of Gene Expression in Yeast.

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    Artémis Llamosi

    2016-02-01

    Full Text Available Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation. Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity.

  10. A Chinese Herbal Decoction, Danggui Buxue Tang, Stimulates Proliferation, Differentiation and Gene Expression of Cultured Osteosarcoma Cells: Genomic Approach to Reveal Specific Gene Activation

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    Roy C. Y. Choi

    2011-01-01

    Full Text Available Danggui Buxue Tang (DBT, a Chinese herbal decoction used to treat ailments in women, contains Radix Astragali (Huangqi; RA and Radix Angelicae Sinensis (Danggui; RAS. When DBT was applied onto cultured MG-63 cells, an increase of cell proliferation and differentiation of MG-63 cell were revealed: both of these effects were significantly higher in DBT than RA or RAS extract. To search for the biological markers that are specifically regulated by DBT, DNA microarray was used to reveal the gene expression profiling of DBT in MG-63 cells as compared to that of RA- or RAS-treated cells. Amongst 883 DBT-regulated genes, 403 of them are specifically regulated by DBT treatment, including CCL-2, CCL-7, CCL-8, and galectin-9. The signaling cascade of this DBT-regulated gene expression was also elucidated in cultured MG-63 cells. The current results reveal the potential usage of this herbal decoction in treating osteoporosis and suggest the uniqueness of Chinese herbal decoction that requires a well-defined formulation. The DBT-regulated genes in the culture could serve as biological responsive markers for quality assurance of the herbal preparation.

  11. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling.

    Science.gov (United States)

    Almstrup, Kristian; Hoei-Hansen, Christina E; Wirkner, Ute; Blake, Jonathon; Schwager, Christian; Ansorge, Wilhelm; Nielsen, John E; Skakkebaek, Niels E; Rajpert-De Meyts, Ewa; Leffers, Henrik

    2004-07-15

    Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly expressed in testicular CIS, including many never reported in testicular neoplasms. Expression was further verified by semiquantitative reverse transcription-PCR and in situ hybridization. Among the highest expressed genes were NANOG and POU5F1, and reverse transcription-PCR revealed possible changes in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported as unstable in cultured ESCs. The close similarity between CIS and ESCs explains the pluripotency of CIS. Moreover, the findings are consistent with an early prenatal origin of TGCTs and thus suggest that etiologic factors operating in utero are of primary importance for the incidence trends of TGCTs. Finally, some of the highly expressed genes identified in this study are promising candidates for new diagnostic markers for CIS and/or TGCTs.

  12. Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks.

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    Prasanna Vidyasekar

    Full Text Available Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated and 2542 (downregulated genes (>2 fold in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated and 444 (downregulated genes (>2 fold under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2.

  13. Gene expression profiling reveals novel regulation by bisphenol-A in estrogen receptor-α-positive human cells

    International Nuclear Information System (INIS)

    Singleton, David W.; Feng, Yuxin; Yang, Jun; Puga, Alvaro; Lee, Adrian V.; Khan, Sohaib A.

    2006-01-01

    Bisphenol-A (BPA) shows proliferative actions in uterus and mammary glands and may influence the development of male and female reproductive tracts in utero or during early postnatal life. Because of its ability to function as an estrogen receptor (ER) agonist, BPA has the potential to disrupt normal endocrine signaling through regulation of ER target genes. Some genes are regulated by both estradiol (E2) and BPA, but those exclusive to either agent have not been described. Using a yeast strain incorporating a vitellogenin A2 ERE-LacZ reporter gene into the genome, we found that BPA induced expression of the reporter in colonies transformed with the ERα expression plasmid, illustrating BPA-mediated regulation within a chromatin context. Additionally, a reporter gene transiently transfected into the endometrial cancer (Ishikawa) cell line also showed BPA activity, although at 100-fold less potency than E2. To compare global gene expression in response to BPA and E2, we used a variant of the MCF-7 breast cancer cell line stably expressing HA-tagged ERα. Cultures were treated for 3 h with an ethanol vehicle, E2 (10 -8 M), or BPA (10 -6 M), followed by isolation of RNA and microarray analysis with the human U95A probe array (Affymetrix, Santa Clara, CA, USA). More than 300 genes were changed 2-fold or more by either or both agents, with roughly half being up-regulated and half down-regulated. A number of growth- and development-related genes, such as HOXC1 and C6, Wnt5A, Frizzled, TGFβ-2, and STAT inhibitor 2, were found to be affected exclusively by BPA. We used quantitative real-time PCR to verify regulation of the HOXC6 gene, which showed decreased expression of approximately 2.5-fold by BPA. These results reveal novel effects by BPA and E2, raising interesting possibilities regarding the role of endocrine disruptors in sexual development

  14. Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

    Science.gov (United States)

    Macovei, Anca; Faè, Matteo; Biggiogera, Marco; de Sousa Araújo, Susana; Carbonera, Daniela; Balestrazzi, Alma

    2018-01-01

    The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells. PMID:29868059

  15. Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

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    Anca Macovei

    2018-05-01

    Full Text Available The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2 involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively and a control (CTRL line carrying the empty vector were investigated. Transmission electron microscopy (TEM revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL, the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells.

  16. Adventitial SCA-1+ Progenitor Cell Gene Sequencing Reveals the Mechanisms of Cell Migration in Response to Hyperlipidemia

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    Ioannis Kokkinopoulos

    2017-08-01

    Full Text Available Adventitial progenitor cells, including SCA-1+ and mesenchymal stem cells, are believed to be important in vascular remodeling. It has been shown that SCA-1+ progenitor cells are involved in neointimal hyperplasia of vein grafts, but little is known concerning their involvement in hyperlipidemia-induced atherosclerosis. We employed single-cell sequencing technology on primary adventitial mouse SCA-1+ cells from wild-type and atherosclerotic-prone (ApoE-deficient mice and found that a group of genes controlling cell migration and matrix protein degradation was highly altered. Adventitial progenitors from ApoE-deficient mice displayed an augmented migratory potential both in vitro and in vivo. This increased migratory ability was mimicked by lipid loading to SCA-1+ cells. Furthermore, we show that lipid loading increased miRNA-29b expression and induced sirtuin-1 and matrix metalloproteinase-9 levels to promote cell migration. These results provide direct evidence that blood cholesterol levels influence vascular progenitor cell function, which could be a potential target cell for treatment of vascular disease.

  17. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

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    Dan Jiang

    Full Text Available Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1 show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1, nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

  18. Targeted and genome-scale methylomics reveals gene body signatures in human cell lines

    Science.gov (United States)

    Ball, Madeleine Price; Li, Jin Billy; Gao, Yuan; Lee, Je-Hyuk; LeProust, Emily; Park, In-Hyun; Xie, Bin; Daley, George Q.; Church, George M.

    2012-01-01

    Cytosine methylation, an epigenetic modification of DNA, is a target of growing interest for developing high throughput profiling technologies. Here we introduce two new, complementary techniques for cytosine methylation profiling utilizing next generation sequencing technology: bisulfite padlock probes (BSPPs) and methyl sensitive cut counting (MSCC). In the first method, we designed a set of ~10,000 BSPPs distributed over the ENCODE pilot project regions to take advantage of existing expression and chromatin immunoprecipitation data. We observed a pattern of low promoter methylation coupled with high gene body methylation in highly expressed genes. Using the second method, MSCC, we gathered genome-scale data for 1.4 million HpaII sites and confirmed that gene body methylation in highly expressed genes is a consistent phenomenon over the entire genome. Our observations highlight the usefulness of techniques which are not inherently or intentionally biased in favor of only profiling particular subsets like CpG islands or promoter regions. PMID:19329998

  19. 5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells.

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    Krzysztof Poterlowicz

    2017-09-01

    Full Text Available Mammalian genomes contain several dozens of large (>0.5 Mbp lineage-specific gene loci harbouring functionally related genes. However, spatial chromatin folding, organization of the enhancer-promoter networks and their relevance to Topologically Associating Domains (TADs in these loci remain poorly understood. TADs are principle units of the genome folding and represents the DNA regions within which DNA interacts more frequently and less frequently across the TAD boundary. Here, we used Chromatin Conformation Capture Carbon Copy (5C technology to characterize spatial chromatin interaction network in the 3.1 Mb Epidermal Differentiation Complex (EDC locus harbouring 61 functionally related genes that show lineage-specific activation during terminal keratinocyte differentiation in the epidermis. 5C data validated by 3D-FISH demonstrate that the EDC locus is organized into several TADs showing distinct lineage-specific chromatin interaction networks based on their transcription activity and the gene-rich or gene-poor status. Correlation of the 5C results with genome-wide studies for enhancer-specific histone modifications (H3K4me1 and H3K27ac revealed that the majority of spatial chromatin interactions that involves the gene-rich TADs at the EDC locus in keratinocytes include both intra- and inter-TAD interaction networks, connecting gene promoters and enhancers. Compared to thymocytes in which the EDC locus is mostly transcriptionally inactive, these interactions were found to be keratinocyte-specific. In keratinocytes, the promoter-enhancer anchoring regions in the gene-rich transcriptionally active TADs are enriched for the binding of chromatin architectural proteins CTCF, Rad21 and chromatin remodeler Brg1. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1 in keratinocytes. Thus, spatial interactions between gene

  20. Exome sequencing of oral squamous cell carcinoma in users of Arabian snuff reveals novel candidates for driver genes.

    Science.gov (United States)

    Al-Hebshi, Nezar Noor; Li, Shiyong; Nasher, Akram Thabet; El-Setouhy, Maged; Alsanosi, Rashad; Blancato, Jan; Loffredo, Christopher

    2016-07-15

    The study sought to identify genetic aberrations driving oral squamous cell carcinoma (OSCC) development among users of shammah, an Arabian preparation of smokeless tobacco. Twenty archival OSCC samples, 15 of which with a history of shammah exposure, were whole-exome sequenced at an average depth of 127×. Somatic mutations were identified using a novel, matched controls-independent filtration algorithm. CODEX and Exomedepth coupled with a novel, Database of Genomic Variant-based filter were employed to call somatic gene-copy number variations. Significantly mutated genes were identified with Oncodrive FM and the Youn and Simon's method. Candidate driver genes were nominated based on Gene Set Enrichment Analysis. The observed mutational spectrum was similar to that reported by the TCGA project. In addition to confirming known genes of OSCC (TP53, CDKNA2, CASP8, PIK3CA, HRAS, FAT1, TP63, CCND1 and FADD) the analysis identified several candidate novel driver events including mutations of NOTCH3, CSMD3, CRB1, CLTCL1, OSMR and TRPM2, amplification of the proto-oncogenes FOSL1, RELA, TRAF6, MDM2, FRS2 and BAG1, and deletion of the recently described tumor suppressor SMARCC1. Analysis also revealed significantly altered pathways not previously implicated in OSCC including Oncostatin-M signalling pathway, AP-1 and C-MYB transcription networks and endocytosis. There was a trend for higher number of mutations, amplifications and driver events in samples with history of shammah exposure particularly those that tested EBV positive, suggesting an interaction between tobacco exposure and EBV. The work provides further evidence for the genetic heterogeneity of oral cancer and suggests shammah-associated OSCC is characterized by extensive amplification of oncogenes. © 2016 UICC.

  1. Metabolic differentiation of surface and invasive cells of yeast colony biofilms revealed by gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Maršíková, J.; Wilkinson, D.; Hlaváček, Otakar; Gilfillan, G.D.; Mizeranschi, A.; Hughes, T.; Begany, Markéta; Rešetárová, Stanislava; Váchová, Libuše; Palková, Z.

    2017-01-01

    Roč. 18, OCT 23 (2017), s. 814 ISSN 1471-2164 R&D Projects: GA MŠk(CZ) 7F14083; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Saccharomyces cerevisiae * Colony biofilms * Cell differentiation Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.729, year: 2016

  2. Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

    OpenAIRE

    Kaneko, Kumi; Ikeda, Tsubomi; Nagai, Mirai; Hori, Sayaka; Umatani, Chie; Tadano, Hiroto; Ugajin, Atsushi; Nakaoka, Takayoshi; Paul, Rajib Kumar; Fujiyuki, Tomoko; Shirai, Kenichi; Kunieda, Takekazu; Takeuchi, Hideaki; Kubo, Takeo

    2013-01-01

    The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for n...

  3. Transcriptome-wide survey of mouse CNS-derived cells reveals monoallelic expression within novel gene families.

    Directory of Open Access Journals (Sweden)

    Sierra M Li

    Full Text Available Monoallelic expression is an integral component of regulation of a number of essential genes and gene families. To probe for allele-specific expression in cells of CNS origin, we used next-generation sequencing (RNA-seq to analyze four clonal neural stem cell (NSC lines derived from Mus musculus C57BL/6 (B6×Mus musculus molossinus (JF1 adult female mice. We established a JF1 cSNP library, then ascertained transcriptome-wide expression from B6 vs. JF1 alleles in the NSC lines. Validating the assay, we found that 262 of 268 X-linked genes evaluable in at least one cell line showed monoallelic expression (at least 85% expression of the predominant allele, p-value<0.05. For autosomal genes 170 of 7,198 genes (2.4% of the total showed monoallelic expression in at least 2 evaluable cell lines. The group included eight known imprinted genes with the expected pattern of allele-specific expression. Among the other autosomal genes with monoallelic expression were five members of the glutathione transferase gene superfamily, which processes xenobiotic compounds as well as carcinogens and cancer therapeutic agents. Monoallelic expression within this superfamily thus may play a functional role in the response to diverse and potentially lethal exogenous factors, as is the case for the immunoglobulin and olfactory receptor superfamilies. Other genes and gene families showing monoallelic expression include the annexin gene family and the Thy1 gene, both linked to inflammation and cancer, as well as genes linked to alcohol dependence (Gabrg1 and epilepsy (Kcnma1. The annotated set of genes will provide a resource for investigation of mechanisms underlying certain cases of these and other major disorders.

  4. Model-based deconvolution of cell cycle time-series data reveals gene expression details at high resolution.

    Directory of Open Access Journals (Sweden)

    Dan Siegal-Gaskins

    2009-08-01

    Full Text Available In both prokaryotic and eukaryotic cells, gene expression is regulated across the cell cycle to ensure "just-in-time" assembly of select cellular structures and molecular machines. However, present in all time-series gene expression measurements is variability that arises from both systematic error in the cell synchrony process and variance in the timing of cell division at the level of the single cell. Thus, gene or protein expression data collected from a population of synchronized cells is an inaccurate measure of what occurs in the average single-cell across a cell cycle. Here, we present a general computational method to extract "single-cell"-like information from population-level time-series expression data. This method removes the effects of 1 variance in growth rate and 2 variance in the physiological and developmental state of the cell. Moreover, this method represents an advance in the deconvolution of molecular expression data in its flexibility, minimal assumptions, and the use of a cross-validation analysis to determine the appropriate level of regularization. Applying our deconvolution algorithm to cell cycle gene expression data from the dimorphic bacterium Caulobacter crescentus, we recovered critical features of cell cycle regulation in essential genes, including ctrA and ftsZ, that were obscured in population-based measurements. In doing so, we highlight the problem with using population data alone to decipher cellular regulatory mechanisms and demonstrate how our deconvolution algorithm can be applied to produce a more realistic picture of temporal regulation in a cell.

  5. Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

    International Nuclear Information System (INIS)

    Pascal, Laura E; Liu, Alvin Y; Vêncio, Ricardo ZN; Page, Laura S; Liebeskind, Emily S; Shadle, Christina P; Troisch, Pamela; Marzolf, Bruz; True, Lawrence D; Hood, Leroy E

    2009-01-01

    Prostate cancer cells in primary tumors have been typed CD10 - /CD13 - /CD24 hi /CD26 + /CD38 lo /CD44 - /CD104 - . This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure. CD26 + cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells. The G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines. Genes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types

  6. Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with risk of clear cell ovarian cancer

    NARCIS (Netherlands)

    Hampras, S.S.; Sucheston-Campbell, L.E.; Cannioto, R.; Chang-Claude, J.; Modugno, F.; Dork, T.; Hillemanns, P.; Preus, L.; Knutson, K.L.; Wallace, P.K.; Hong, C.C.; Friel, G.; Davis, W.; Nesline, M.; Pearce, C.L.; Kelemen, L.E.; Goodman, M.T.; Bandera, E.V.; Terry, K.L.; Schoof, N.; Eng, K.H.; Clay, A.; Singh, P.K.; Joseph, J.M.; Aben, K.K.H.; Anton-Culver, H.; Antonenkova, N.; Baker, H.; Bean, Y.; Beckmann, M.W.; Bisogna, M.; Bjorge, L.; Bogdanova, N.; Brinton, L.A.; Brooks-Wilson, A.; Bruinsma, F.; Butzow, R.; Campbell, I.G.; Carty, K.; Cook, L.S.; Cramer, D.W; Cybulski, C.; Dansonka-Mieszkowska, A.; Dennis, J.; Despierre, E.; Dicks, E.; Doherty, J.A.; Bois, A. du; Durst, M.; Easton, D.; Eccles, D.; Edwards, R.P.; Ekici, A.B.; Fasching, P.A.; Fridley, B.L.; Gao, Y.T.; Gentry-Maharaj, A.; Giles, G.G.; Glasspool, R.; Gronwald, J.; Harrington, P.; Harter, P.; Hasmad, H.N.; Hein, A.; Heitz, F.; Hildebrandt, M.A.T.; Hogdall, C.; Hogdall, E.; Hosono, S.; Iversen, E.S.; Jakubowska, A.; Jensen, A.; Ji, B.T.; Karlan, B.Y.; Kellar, M.; Kelley, J.L.; Kiemeney, L.A.L.M.; Klapdor, R.; Kolomeyevskaya, N.; Krakstad, C.; Kjaer, S.K.; Kruszka, B.; Kupryjanczyk, J.; Lambrechts, D.; Lambrechts, S.; Le, N.D.; Lee, A.W.; Lele, S.; Leminen, A.; Lester, J.; Levine, D.A.; Liang, D.; Lissowska, J.; Liu, S.; Lu, K.; Lubinski, J.; Lundvall, L.; Massuger, L.F.A.G.; Matsuo, K.; McGuire, V.; et al.,

    2016-01-01

    BACKGROUND: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer. METHODS: In a population of 15,596 epithelial ovarian cancer (EOC) cases and

  7. Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with risk of clear cell ovarian cancer

    DEFF Research Database (Denmark)

    Hampras, Shalaka S; Sucheston-Campbell, Lara E; Cannioto, Rikki

    2016-01-01

    BACKGROUND: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer. METHODS: In a population of 15,596 epithelial ovarian cancer (EOC) cases a...

  8. Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

    Directory of Open Access Journals (Sweden)

    Ingo Ahrens

    Full Text Available Endothelial progenitor cells (EPCs can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP, PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15 or pro-angiogenic (galectin-3 properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP was the most up-regulated gene.

  9. Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression.

    Science.gov (United States)

    Crocker, Amanda; Guan, Xiao-Juan; Murphy, Coleen T; Murthy, Mala

    2016-05-17

    Learning and memory formation in Drosophila rely on a network of neurons in the mushroom bodies (MBs). Whereas numerous studies have delineated roles for individual cell types within this network in aspects of learning or memory, whether or not these cells can also be distinguished by the genes they express remains unresolved. In addition, the changes in gene expression that accompany long-term memory formation within the MBs have not yet been studied by neuron type. Here, we address both issues by performing RNA sequencing on single cell types (harvested via patch pipets) within the MB. We discover that the expression of genes that encode cell surface receptors is sufficient to identify cell types and that a subset of these genes, required for sensory transduction in peripheral sensory neurons, is not only expressed within individual neurons of the MB in the central brain, but is also critical for memory formation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells.

    Science.gov (United States)

    Balestrini, Raffaella; Gómez-Ariza, Jorge; Lanfranco, Luisa; Bonfante, Paola

    2007-09-01

    The establishment of a symbiotic interaction between plant roots and arbuscular mycorrhizal (AM) fungi requires both partners to undergo significant morphological and physiological modifications which eventually lead to reciprocal beneficial effects. Extensive changes in gene expression profiles recently have been described in transcriptomic studies that have analyzed the whole mycorrhizal root. However, because root colonization by AM fungi involves different cell types, a cell-specific gene expression pattern is likely to occur. We have applied the laser microdissection (LMD) technology to investigate expression profiles of both plant and fungal genes in Lycopersicon esculentum roots colonized by Glomus mosseae. A protocol to harvest arbuscule-containing cells from paraffin sections of mycorrhizal roots has been developed using a Leica AS LMD system. RNA of satisfactory quantity and quality has been extracted for molecular analysis. Transcripts for plant phosphate transporters (LePTs), selected as molecular markers for a functional symbiosis, have been detected by reverse-transcriptase polymerase chain reaction assays and associated to distinct cell types, leading to novel insights into the distribution of LePT mRNAs. In fact, the transcripts of the five phosphate transporters (PTs) have been detected contemporaneously in the same arbusculated cell population, unlike from the neighboring noncolonized cells. In addition, fungal H(+)ATPase (GmHA5) and phosphate transporter (GmosPT) mRNAs were found exclusively in arbusculated cells. The discovery that five plant and one fungal PT genes are consistently expressed inside the arbusculated cells provides a new scenario for plant-fungus nutrient exchanges.

  11. Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

    Science.gov (United States)

    Grandy, Rodrigo A; Whitfield, Troy W; Wu, Hai; Fitzgerald, Mark P; VanOudenhove, Jennifer J; Zaidi, Sayyed K; Montecino, Martin A; Lian, Jane B; van Wijnen, André J; Stein, Janet L; Stein, Gary S

    2016-02-15

    Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Gene Expression Profile Reveals Abnormalities of Multiple Signaling Pathways in Mesenchymal Stem Cell Derived from Patients with Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Yu Tang

    2012-01-01

    Full Text Available We aimed to compare bone-marrow-derived mesenchymal stem cells (BMMSCs between systemic lupus erythematosus (SLE and normal controls by means of cDNA microarray, immunohistochemistry, immunofluorescence, and immunoblotting. Our results showed there were a total of 1, 905 genes which were differentially expressed by BMMSCs derived from SLE patients, of which, 652 genes were upregulated and 1, 253 were downregulated. Gene ontology (GO analysis showed that the majority of these genes were related to cell cycle and protein binding. Pathway analysis exhibited that differentially regulated signal pathways involved actin cytoskeleton, focal adhesion, tight junction, and TGF-β pathway. The high protein level of BMP-5 and low expression of Id-1 indicated that there might be dysregulation in BMP/TGF-β signaling pathway. The expression of Id-1 in SLE BMMSCs was reversely correlated with serum TNF-α levels. The protein level of cyclin E decreased in the cell cycling regulation pathway. Moreover, the MAPK signaling pathway was activated in BMMSCs from SLE patients via phosphorylation of ERK1/2 and SAPK/JNK. The actin distribution pattern of BMMSCs from SLE patients was also found disordered. Our results suggested that there were distinguished differences of BMMSCs between SLE patients and normal controls.

  13. Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls.

    Science.gov (United States)

    MacMillan, Colleen P; Birke, Hannah; Chuah, Aaron; Brill, Elizabeth; Tsuji, Yukiko; Ralph, John; Dennis, Elizabeth S; Llewellyn, Danny; Pettolino, Filomena A

    2017-07-18

    Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a 'typical' lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre. Transcriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue's lacking many of the gene transcripts normally associated with lignin biosynthesis. Our study in cotton confirmed some features of the currently accepted gene regulatory cascade for 'typical' plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in

  14. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

    Science.gov (United States)

    Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

    1997-11-01

    Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

  15. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection.

    Science.gov (United States)

    Chung, Chee Yeun; Seo, Hyemyung; Sonntag, Kai Christian; Brooks, Andrew; Lin, Ling; Isacson, Ole

    2005-07-01

    Molecular differences between dopamine (DA) neurons may explain why the mesostriatal DA neurons in the A9 region preferentially degenerate in Parkinson's disease (PD) and toxic models, whereas the adjacent A10 region mesolimbic and mesocortical DA neurons are relatively spared. To characterize innate physiological differences between A9 and A10 DA neurons, we determined gene expression profiles in these neurons in the adult mouse by laser capture microdissection, microarray analysis and real-time PCR. We found 42 genes relatively elevated in A9 DA neurons, whereas 61 genes were elevated in A10 DA neurons [> 2-fold; false discovery rate (FDR) neurotoxic or protective biochemical pathways. Three A9-elevated molecules [G-protein coupled inwardly rectifying K channel 2 (GIRK2), adenine nucleotide translocator 2 (ANT-2) and the growth factor IGF-1] and three A10-elevated peptides (GRP, CGRP and PACAP) were further examined in both alpha-synuclein overexpressing PC12 (PC12-alphaSyn) cells and rat primary ventral mesencephalic (VM) cultures exposed to MPP+ neurotoxicity. GIRK2-positive DA neurons were more vulnerable to MPP+ toxicity and overexpression of GIRK2 increased the vulnerability of PC12-alphaSyn cells to the toxin. Blocking of ANT decreased vulnerability to MPP+ in both cell culture systems. Exposing cells to IGF-1, GRP and PACAP decreased vulnerability of both cell types to MPP+, whereas CGRP protected PC12-alphaSyn cells but not primary VM DA neurons. These results indicate that certain differentially expressed molecules in A9 and A10 DA neurons may play key roles in their relative vulnerability to toxins and PD.

  16. Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

    International Nuclear Information System (INIS)

    Nagao, Kenji; Ohta, Takayuki; Hinohara, Atsushi; Tahara, Tomoyuki; Hagiwara, Tetsuya; Maeda, Yoshitake; Yoneya, Takashi; Sohma, Yoshiaki; Heike, Toshio; Nakahata, Tatsutoshi; Inagaki, Yoshimasa; Nishikawa, Mitsuo

    2008-01-01

    The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis

  17. Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation.

    Science.gov (United States)

    Pang, Lisa Y; Gatenby, Emma L; Kamida, Ayako; Whitelaw, Bruce A; Hupp, Ted R; Argyle, David J

    2014-01-01

    Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

  18. Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation.

    Directory of Open Access Journals (Sweden)

    Lisa Y Pang

    Full Text Available Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs, which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05, and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

  19. Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

    Science.gov (United States)

    Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

    2011-12-01

    Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

  20. Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    Directory of Open Access Journals (Sweden)

    De Marzo Angelo M

    2011-06-01

    Full Text Available Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.

  1. Comparative Analysis of WUSCHEL-Related Homeobox Genes Revealed Their Parent-of-Origin and Cell Type-Specific Expression Pattern During Early Embryogenesis in Tobacco

    Directory of Open Access Journals (Sweden)

    Xuemei Zhou

    2018-03-01

    Full Text Available WUSCHEL-related homeobox (WOX gene is a plant-specific clade of homeobox transcription factors. Increasing evidences reveal that WOXs play critical roles in early embryogenesis, which involves zygote development, initiation of zygote division, and apical or basal cell lineage establishment. However, how WOXs regulate these developmental events remains largely unknown, and even detailed expression pattern in gametes and early proembryos is not yet available. Here, 13 WOX family genes were identified in Nicotiana tabacum genome. Comparative analysis of 13 WOX family genes with their homologs in Arabidopsis thaliana reveals relatively conserved expression pattern of WUS and WOX5 in shoot/root apical meristem. Whereas variations were also found, e.g., lacking homolog of WOX8 (a marker for suspensor cell in tobacco genome and the expression of WOX2/WOX9 in both apical cell and basal cell. Transient transcriptional activity analysis revealed that WOXs in WUS clade have repressive activities for their target's transcription, whereas WOXs in ancient and intermediate clade have activation activities, giving a molecular basis for the phylogenetic classification of tobacco WOXs into three major clades. Expression pattern analysis revealed that some WOXs (e.g., WOX 13a expressed in both male and female gametes and some WOXs (e.g., WOX 11 and WOX 13b displayed the characteristics of parent-of-origin genes. Interestingly, some WOXs (e.g., WOX2 and WOX9, which are essential for early embryo patterning, were de novo transcribed in zygote, indicating relevant mechanism for embryo pattern formation is only established in zygote right after fertilization and not carried in by gametes. We also found that most WOXs displayed a stage-specific and cell type-specific expression pattern. Taken together, this work provides a detailed landscape of WOXs in tobacco during fertilization and early embryogenesis, which will facilitate the understanding of their specific roles

  2. Transcriptional profiling of primary endometrial epithelial cells following acute HIV-1 exposure reveals gene signatures related to innate immunity.

    Science.gov (United States)

    Zahoor, Muhammad Atif; Woods, Matthew William; Dizzell, Sara; Nazli, Aisha; Mueller, Kristen M; Nguyen, Philip V; Verschoor, Chris P; Kaushic, Charu

    2018-04-01

    Genital epithelial cells (GECs) line the mucosal surface of the female genital tract (FGT) and are the first cells that interface with both commensal microbiota and sexually transmitted pathogens. Despite the protective barrier formed by GECs, the FGT is a major site of HIV-1 infection. This highlights the importance of studying the interaction of HIV-1 and GECs. Using microarray analysis, we characterized the transcriptional profile of primary endometrial GECs grown in the presence or absence of physiological levels of E2 (10 -9  mol/L) or P4 (10 -7  mol/L) following acute exposure to HIV-1 for 6 hours. Acute exposure of primary endometrial GECs to HIV-1 resulted in the expression of genes related to inflammation, plasminogen activation, adhesion and diapedesis and interferon response. Interestingly, exposure to HIV-1 in the presence of E2 and P4 resulted in differential transcriptional profiles, suggesting that the response of primary endometrial GECs to HIV-1 exposure is modulated by female sex hormones. The gene expression signature of endometrial GECs indicates that the response of these cells may be key to determining host susceptibility to HIV-1 and that sex hormones modulate these interactions. This study allows us to explore possible mechanisms that explain the hormone-mediated fluctuation of HIV-1 susceptibility in women. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Hydrogen Peroxide-induced Cell Death in Arabidopsis : Transcriptional and Mutant Analysis Reveals a Role of an Oxoglutarate-dependent Dioxygenase Gene in the Cell Death Process

    NARCIS (Netherlands)

    Gechev, Tsanko S.; Minkov, Ivan N.; Hille, Jacques

    2005-01-01

    Hydrogen peroxide is a major regulator of plant programmed cell death (PCD) but little is known about the downstream genes from the H2O2-signaling network that mediate the cell death. To address this question, a novel system for studying H2O2-induced programmed cell death in Arabidopsis thaliana was

  4. Suicide gene reveals the myocardial neovascularization role of mesenchymal stem cells overexpressing CXCR4 (MSC(CXCR4.

    Directory of Open Access Journals (Sweden)

    Jialiang Liang

    Full Text Available BACKGROUND: Our previous studies indicated that MSC(CXCR4 improved cardiac function after myocardial infarction (MI. This study was aimed to investigate the specific role of MSC(CXCR4 in neovascularization of infarcted myocardium using a suicide gene approach. METHODS: MSCs were transduced with either lentivirus-null vector/GFP (MSC(Null as control or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSC(Null or MSC(CXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging. RESULTS: The expression of VEGF-A and HIF-1α was significantly higher in MSC(CXCR4 as compared to MSC(Null under hypoxia. Additionally, MSC(CXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSC(CXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor. In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSC(CXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSC(CXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function. CONCLUSION: The

  5. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

    Science.gov (United States)

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

  6. Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin.

    Science.gov (United States)

    Shu, Benshui; Zhang, Jingjing; Sethuraman, Veeran; Cui, Gaofeng; Yi, Xin; Zhong, Guohua

    2017-10-16

    As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

  7. Expression atlas and comparative coexpression network analyses reveal important genes involved in the formation of lignified cell wall in Brachypodium distachyon.

    Science.gov (United States)

    Sibout, Richard; Proost, Sebastian; Hansen, Bjoern Oest; Vaid, Neha; Giorgi, Federico M; Ho-Yue-Kuang, Severine; Legée, Frédéric; Cézart, Laurent; Bouchabké-Coussa, Oumaya; Soulhat, Camille; Provart, Nicholas; Pasha, Asher; Le Bris, Philippe; Roujol, David; Hofte, Herman; Jamet, Elisabeth; Lapierre, Catherine; Persson, Staffan; Mutwil, Marek

    2017-08-01

    While Brachypodium distachyon (Brachypodium) is an emerging model for grasses, no expression atlas or gene coexpression network is available. Such tools are of high importance to provide insights into the function of Brachypodium genes. We present a detailed Brachypodium expression atlas, capturing gene expression in its major organs at different developmental stages. The data were integrated into a large-scale coexpression database ( www.gene2function.de), enabling identification of duplicated pathways and conserved processes across 10 plant species, thus allowing genome-wide inference of gene function. We highlight the importance of the atlas and the platform through the identification of duplicated cell wall modules, and show that a lignin biosynthesis module is conserved across angiosperms. We identified and functionally characterised a putative ferulate 5-hydroxylase gene through overexpression of it in Brachypodium, which resulted in an increase in lignin syringyl units and reduced lignin content of mature stems, and led to improved saccharification of the stem biomass. Our Brachypodium expression atlas thus provides a powerful resource to reveal functionally related genes, which may advance our understanding of important biological processes in grasses. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  8. Genome-wide analysis of gene expression during adipogenesis in human adipose-derived stromal cells reveals novel patterns of gene expression during adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Melvin Anyasi Ambele

    2016-05-01

    Full Text Available We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified genes that were up-regulated only on days 1 and 7 or days 14 and 21 that could serve as potential early and late-stage differentiation markers.

  9. Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

    Science.gov (United States)

    Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

    2010-04-29

    Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

  10. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    Directory of Open Access Journals (Sweden)

    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  11. Stem cell heterogeneity revealed

    DEFF Research Database (Denmark)

    Andersen, Marianne S; Jensen, Kim B

    2016-01-01

    The skin forms a protective, water-impermeable barrier consisting of heavily crosslinked epithelial cells. However, the specific role of stem cells in sustaining this barrier remains a contentious issue. A detailed analysis of the interfollicular epidermis now proposes a model for how a composite...... of cells with different properties are involved in its maintenance....

  12. Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation

    Directory of Open Access Journals (Sweden)

    Papa Maria

    2011-01-01

    Full Text Available Abstract Background Estrogen receptors alpha (ERα and beta (ERβ are transcription factors (TFs that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC. The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results Expression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions Results indicate that the

  13. Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation.

    Science.gov (United States)

    Petersen, Maja Borup Kjær; Azad, Ajuna; Ingvorsen, Camilla; Hess, Katja; Hansson, Mattias; Grapin-Botton, Anne; Honoré, Christian

    2017-10-10

    The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level.

    Directory of Open Access Journals (Sweden)

    Andrew R Dalby

    Full Text Available Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC.

  15. Dynamic Response Genes in CD4+ T Cells Reveal a Network of Interactive Proteins that Classifies Disease Activity in Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Sandra Hellberg

    2016-09-01

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory disease of the CNS and has a varying disease course as well as variable response to treatment. Biomarkers may therefore aid personalized treatment. We tested whether in vitro activation of MS patient-derived CD4+ T cells could reveal potential biomarkers. The dynamic gene expression response to activation was dysregulated in patient-derived CD4+ T cells. By integrating our findings with genome-wide association studies, we constructed a highly connected MS gene module, disclosing cell activation and chemotaxis as central components. Changes in several module genes were associated with differences in protein levels, which were measurable in cerebrospinal fluid and were used to classify patients from control individuals. In addition, these measurements could predict disease activity after 2 years and distinguish low and high responders to treatment in two additional, independent cohorts. While further validation is needed in larger cohorts prior to clinical implementation, we have uncovered a set of potentially promising biomarkers.

  16. Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

    Directory of Open Access Journals (Sweden)

    João A Paredes

    Full Text Available Loss of thymidine kinase 2 (TK2 causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA depletion syndrome (MDS in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

  17. Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

    Science.gov (United States)

    Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

    2013-01-01

    Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

  18. Cancer gene profiling in non-small cell lung cancers reveals activating mutations in JAK2 and JAK3 with therapeutic implications

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    Shuyu D. Li

    2017-10-01

    Full Text Available Abstract Background Next-generation sequencing (NGS of cancer gene panels are widely applied to enable personalized cancer therapy and to identify novel oncogenic mutations. Methods We performed targeted NGS on 932 clinical cases of non-small-cell lung cancers (NSCLCs using the Ion AmpliSeq™ Cancer Hotspot panel v2 assay. Results Actionable mutations were identified in 65% of the cases with available targeted therapeutic options, including 26% of the patients with mutations in National Comprehensive Cancer Network (NCCN guideline genes. Most notably, we discovered JAK2 p.V617F somatic mutation, a hallmark of myeloproliferative neoplasms, in 1% (9/932 of the NSCLCs. Analysis of cancer cell line pharmacogenomic data showed that a high level of JAK2 expression in a panel of NSCLC cell lines is correlated with increased sensitivity to a selective JAK2 inhibitor. Further analysis of TCGA genomic data revealed JAK2 gain or loss due to genetic alterations in NSCLC clinical samples are associated with significantly elevated or reduced PD-L1 expression, suggesting that the activating JAK2 p.V617F mutation could confer sensitivity to both JAK inhibitors and anti-PD1 immunotherapy. We also detected JAK3 germline activating mutations in 6.7% (62/932 of the patients who may benefit from anti-PD1 treatment, in light of recent findings that JAK3 mutations upregulate PD-L1 expression. Conclusion Taken together, this study demonstrated the clinical utility of targeted NGS with a focused hotspot cancer gene panel in NSCLCs and identified activating mutations in JAK2 and JAK3 with clinical implications inferred through integrative analysis of cancer genetic, genomic, and pharmacogenomic data. The potential of JAK2 and JAK3 mutations as response markers for the targeted therapy against JAK kinases or anti-PD1 immunotherapy warrants further investigation.

  19. Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    DEFF Research Database (Denmark)

    Milani, Lili; Lundmark, Anders; Nordlund, Jessica

    2008-01-01

    To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2, 529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood...

  20. Cognitive assessment of mice strains heterozygous for cell-adhesion genes reveals strain-specific alterations in timing.

    Science.gov (United States)

    Gallistel, C R; Tucci, Valter; Nolan, Patrick M; Schachner, Melitta; Jakovcevski, Igor; Kheifets, Aaron; Barboza, Luendro

    2014-03-05

    We used a fully automated system for the behavioural measurement of physiologically meaningful properties of basic mechanisms of cognition to test two strains of heterozygous mutant mice, Bfc (batface) and L1, and their wild-type littermate controls. Both of the target genes are involved in the establishment and maintenance of synapses. We find that the Bfc heterozygotes show reduced precision in their representation of interval duration, whereas the L1 heterozygotes show increased precision. These effects are functionally specific, because many other measures made on the same mice are unaffected, namely: the accuracy of matching temporal investment ratios to income ratios in a matching protocol, the rate of instrumental and classical conditioning, the latency to initiate a cued instrumental response, the trials on task and the impulsivity in a switch paradigm, the accuracy with which mice adjust timed switches to changes in the temporal constraints, the days to acquisition, and mean onset time and onset variability in the circadian anticipation of food availability.

  1. Genomic Analyses Reveal Global Functional Alterations That Promote Tumor Growth and Novel Tumor Suppressor Genes in Natural Killer-Cell Malignancies

    DEFF Research Database (Denmark)

    Kucuk, Can; Iqbal, Javeed; J. deLeeuw, Ronald

    in cell proliferation, growth and energy metabolic processes important for the neoplastic cells. In deleted regions, genes showing decreased expression included transcription factors or repressors (e.g. SP4, PRDM1, NCOR1 and ZNF10), tumor suppressors or negative regulators of the cell cycle (e.g. CDKN2C...

  2. Integrated microRNA and gene expression profiling reveals the crucial miRNAs in curcumin anti-lung cancer cell invasion.

    Science.gov (United States)

    Zhan, Jian-Wei; Jiao, De-Min; Wang, Yi; Song, Jia; Wu, Jin-Hong; Wu, Li-Jun; Chen, Qing-Yong; Ma, Sheng-Lin

    2017-09-01

    Curcumin (diferuloylmethane) has chemopreventive and therapeutic properties against many types of tumors, both in vitro and in vivo. Previous reports have shown that curcumin exhibits anti-invasive activities, but the mechanisms remain largely unclear. In this study, both microRNA (miRNA) and messenger RNA (mRNA) expression profiles were used to characterize the anti-metastasis mechanisms of curcumin in human non-small cell lung cancer A549 cell line. Microarray analysis revealed that 36 miRNAs were differentially expressed between the curcumin-treated and control groups. miR-330-5p exhibited maximum upregulation, while miR-25-5p exhibited maximum downregulation in the curcumin treatment group. mRNA expression profiles and functional analysis indicated that 226 differentially expressed mRNAs belonged to different functional categories. Significant pathway analysis showed that mitogen-activated protein kinase, transforming growth factor-β, and Wnt signaling pathways were significantly downregulated. At the same time, axon guidance, glioma, and ErbB tyrosine kinase receptor signaling pathways were significantly upregulated. We constructed a miRNA gene network that contributed to the curcumin inhibition of metastasis in lung cancer cells. let-7a-3p, miR-1262, miR-499a-5p, miR-1276, miR-331-5p, and miR-330-5p were identified as key microRNA regulators in the network. Finally, using miR-330-5p as an example, we confirmed the role of miR-330-5p in mediating the anti-migration effect of curcumin, suggesting the importance of miRNAs in the regulation of curcumin biological activity. Our findings provide new insights into the anti-metastasis mechanism of curcumin in lung cancer. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  3. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain

    Science.gov (United States)

    Nguyen, AnhThu; Rauch, Tibor A.; Pfeifer, Gerd P.; Hu, Valerie W.

    2010-01-01

    Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.—Nguyen, A., Rauch, T. A., Pfeifer, G. P., Hu, V. W. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain. PMID:20375269

  4. Transcriptome Analysis of Porcine PBMCs Reveals the Immune Cascade Response and Gene Ontology Terms Related to Cell Death and Fibrosis in the Progression of Liver Failure

    Directory of Open Access Journals (Sweden)

    YiMin Zhang

    2018-01-01

    Full Text Available Background. The key gene sets involved in the progression of acute liver failure (ALF, which has a high mortality rate, remain unclear. This study aims to gain a deeper understanding of the transcriptional response of peripheral blood mononuclear cells (PBMCs following ALF. Methods. ALF was induced by D-galactosamine (D-gal in a porcine model. PBMCs were separated at time zero (baseline group, 36 h (failure group, and 60 h (dying group after D-gal injection. Transcriptional profiling was performed using RNA sequencing and analysed using DAVID bioinformatics resources. Results. Compared with the baseline group, 816 and 1,845 differentially expressed genes (DEGs were identified in the failure and dying groups, respectively. A total of five and two gene ontology (GO term clusters were enriched in 107 GO terms in the failure group and 154 GO terms in the dying group. These GO clusters were primarily immune-related, including genes regulating the inflammasome complex and toll-like receptor signalling pathways. Specifically, GO terms related to cell death, including apoptosis, pyroptosis, and autophagy, and those related to fibrosis, coagulation dysfunction, and hepatic encephalopathy were enriched. Seven Kyoto Encyclopedia of Genes and Genomes (KEGG pathways, cytokine-cytokine receptor interaction, hematopoietic cell lineage, lysosome, rheumatoid arthritis, malaria, and phagosome and pertussis pathways were mapped for DEGs in the failure group. All of these seven KEGG pathways were involved in the 19 KEGG pathways mapped in the dying group. Conclusion. We found that the dramatic PBMC transcriptome changes triggered by ALF progression was predominantly related to immune responses. The enriched GO terms related to cell death, fibrosis, and so on, as indicated by PBMC transcriptome analysis, seem to be useful in elucidating potential key gene sets in the progression of ALF. A better understanding of these gene sets might be of preventive or

  5. A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

    Science.gov (United States)

    Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

    2015-09-01

    Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Medroxyprogesterone acetate-treated human, primary endometrial epithelial cells reveal unique gene expression signature linked to innate immunity and HIV-1 susceptibility.

    Science.gov (United States)

    Woods, Matthew W; Zahoor, Muhammad Atif; Dizzell, Sara; Verschoor, Chris P; Kaushic, Charu

    2018-01-01

    Medroxyprogesterone acetate (MPA), a progestin-based hormonal contraceptive designed to mimic progesterone, has been linked to increased human immunodeficiency virus (HIV-1) susceptibility. Genital epithelial cells (GECs) form the mucosal lining of the female genital tract (FGT) and provide the first line of protection against HIV-1. The impact of endogenous sex hormones or MPA on the gene expression profile of GECs has not been comprehensively documented. Using microarray analysis, we characterized the transcriptional profile of primary endometrial epithelial cells grown in physiological levels of E2, P4, and MPA. Each hormone treatment altered the gene expression profile of GECs in a unique manner. Interestingly, although MPA is a progestogen, the gene expression profile induced by it was distinct from P4. MPA increased gene expression of genes related to inflammation and cholesterol synthesis linked to innate immunity and HIV-1 susceptibility. The analysis of gene expression profiles provides insights into the effects of sex hormones and MPA on GECs and allows us to posit possible mechanisms of the MPA-mediated increase in HIV-1 acquisition. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

    NARCIS (Netherlands)

    Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.

    2006-01-01

    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  8. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

    NARCIS (Netherlands)

    Boerboom, A.M.J.F.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.

    2006-01-01

    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  9. Comprehensive identification of genes driven by ERV9-LTRs reveals TNFRSF10B as a re-activatable mediator of testicular cancer cell death

    Science.gov (United States)

    Beyer, U; Krönung, S K; Leha, A; Walter, L; Dobbelstein, M

    2016-01-01

    The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression. PMID:26024393

  10. Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities.

    Science.gov (United States)

    Gorgoglione, Bartolomeo; Wang, Tiehui; Secombes, Christopher J; Holland, Jason W

    2013-07-16

    The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate/inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease.

  11. Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities

    Science.gov (United States)

    2013-01-01

    The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate / inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell / antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease. PMID:23865616

  12. Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering

    Directory of Open Access Journals (Sweden)

    Mehanathan eMuthamilarasan

    2015-11-01

    Full Text Available Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl, callose (Gsl and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2 and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization.

  13. Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering.

    Science.gov (United States)

    Muthamilarasan, Mehanathan; Khan, Yusuf; Jaishankar, Jananee; Shweta, Shweta; Lata, Charu; Prasad, Manoj

    2015-01-01

    Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si) is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2, and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization.

  14. High-throughput sequencing reveals key genes and immune homeostatic pathways activated in myeloid dendritic cells by Porphyromonas gingivalis 381 and its fimbrial mutants.

    Science.gov (United States)

    Arjunan, P; El-Awady, A; Dannebaum, R O; Kunde-Ramamoorthy, G; Cutler, C W

    2016-02-01

    The human microbiome consists of highly diverse microbial communities that colonize our skin and mucosal surfaces, aiding in maintenance of immune homeostasis. The keystone pathogen Porphyromonas gingivalis induces a dysbiosis and disrupts immune homeostasis through as yet unclear mechanisms. The fimbrial adhesins of P. gingivalis facilitate biofilm formation, invasion of and dissemination by blood dendritic cells; hence, fimbriae may be key factors in disruption of immune homeostasis. In this study we employed RNA-sequencing transcriptome profiling to identify differentially expressed genes (DEGs) in human monocyte-derived dendritic cells (MoDCs) in response to in vitro infection/exposure by Pg381 or its isogenic mutant strains that solely express minor-Mfa1 fimbriae (DPG3), major-FimA fimbriae (MFI) or are deficient in both fimbriae (MFB) relative to uninfected control. Our results yielded a total of 479 DEGs that were at least two-fold upregulated and downregulated in MoDCs significantly (P ≤ 0.05) by all four strains and certain DEGs that were strain-specific. Interestingly, the gene ontology biological and functional analysis shows that the upregulated genes in DPG3-induced MoDCs were more significant than other strains and associated with inflammation, immune response, anti-apoptosis, cell proliferation, and other homeostatic functions. Both transcriptome and quantitative polymerase chain reaction results show that DPG3, which solely expresses Mfa1, increased ZNF366, CD209, LOX1, IDO1, IL-10, CCL2, SOCS3, STAT3 and FOXO1 gene expression. In conclusion, we have identified key DC-mediated immune homeostatic pathways that could contribute to dysbiosis in periodontal infection with P. gingivalis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles.

    Science.gov (United States)

    Gadala-Maria, Daniel; Yaari, Gur; Uduman, Mohamed; Kleinstein, Steven H

    2015-02-24

    Individual variation in germline and expressed B-cell immunoglobulin (Ig) repertoires has been associated with aging, disease susceptibility, and differential response to infection and vaccination. Repertoire properties can now be studied at large-scale through next-generation sequencing of rearranged Ig genes. Accurate analysis of these repertoire-sequencing (Rep-Seq) data requires identifying the germline variable (V), diversity (D), and joining (J) gene segments used by each Ig sequence. Current V(D)J assignment methods work by aligning sequences to a database of known germline V(D)J segment alleles. However, existing databases are likely to be incomplete and novel polymorphisms are hard to differentiate from the frequent occurrence of somatic hypermutations in Ig sequences. Here we develop a Tool for Ig Genotype Elucidation via Rep-Seq (TIgGER). TIgGER analyzes mutation patterns in Rep-Seq data to identify novel V segment alleles, and also constructs a personalized germline database containing the specific set of alleles carried by a subject. This information is then used to improve the initial V segment assignments from existing tools, like IMGT/HighV-QUEST. The application of TIgGER to Rep-Seq data from seven subjects identified 11 novel V segment alleles, including at least one in every subject examined. These novel alleles constituted 13% of the total number of unique alleles in these subjects, and impacted 3% of V(D)J segment assignments. These results reinforce the highly polymorphic nature of human Ig V genes, and suggest that many novel alleles remain to be discovered. The integration of TIgGER into Rep-Seq processing pipelines will increase the accuracy of V segment assignments, thus improving B-cell repertoire analyses.

  16. Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes.

    Science.gov (United States)

    Camilios-Neto, Doumit; Bonato, Paloma; Wassem, Roseli; Tadra-Sfeir, Michelle Z; Brusamarello-Santos, Liziane C C; Valdameri, Glaucio; Donatti, Lucélia; Faoro, Helisson; Weiss, Vinicius A; Chubatsu, Leda S; Pedrosa, Fábio O; Souza, Emanuel M

    2014-05-16

    The rapid growth of the world's population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB. We performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes. PGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.

  17. Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries

    DEFF Research Database (Denmark)

    Jakobsen, Janus Schou; Waage, Johannes; Rapin, Nicolas

    2013-01-01

    quantified the genome-wide binding patterns of two key hepatocyte transcription factors, CEBPA and CEBPB (also known as C/EBPalpha and C/EBPbeta), at multiple time points during the highly dynamic process of liver regeneration elicited by partial hepatectomy in mouse. Combining these profiles with RNA...... polymerase II binding data, we find three temporal classes of transcription factor binding to be associated with distinct sets of regulated genes involved in the acute phase response, metabolic/homeostatic functions, or cell cycle progression. Moreover, we demonstrate a previously unrecognized early phase......IP experiments involving a panel of central transcription factors and/or by comparison to external ChIP-seq data. Our quantitative investigation not only provides in vivo evidence for the involvement of many new factors in liver regeneration but also points to similarities in the circuitries regulating self...

  18. CRISPR loci reveal networks of gene exchange in archaea

    Directory of Open Access Journals (Sweden)

    Brodt Avital

    2011-12-01

    Full Text Available Abstract Background CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past "infection history" of the organism. Results Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. Conclusions CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense. Open peer review This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten

  19. CRISPR loci reveal networks of gene exchange in archaea.

    Science.gov (United States)

    Brodt, Avital; Lurie-Weinberger, Mor N; Gophna, Uri

    2011-12-21

    CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past "infection history" of the organism. Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense. This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten).

  20. Meta-Analysis of Transcriptome Data Related to Hippocampus Biopsies and iPSC-Derived Neuronal Cells from Alzheimer's Disease Patients Reveals an Association with FOXA1 and FOXA2 Gene Regulatory Networks.

    Science.gov (United States)

    Wruck, Wasco; Schröter, Friederike; Adjaye, James

    2016-01-01

    Although the incidence of Alzheimer's disease (AD) is continuously increasing in the aging population worldwide, effective therapies are not available. The interplay between causative genetic and environmental factors is partially understood. Meta-analyses have been performed on aspects such as polymorphisms, cytokines, and cognitive training. Here, we propose a meta-analysis approach based on hierarchical clustering analysis of a reliable training set of hippocampus biopsies, which is condensed to a gene expression signature. This gene expression signature was applied to various test sets of brain biopsies and iPSC-derived neuronal cell models to demonstrate its ability to distinguish AD samples from control. Thus, our identified AD-gene signature may form the basis for determination of biomarkers that are urgently needed to overcome current diagnostic shortfalls. Intriguingly, the well-described AD-related genes APP and APOE are not within the signature because their gene expression profiles show a lower correlation to the disease phenotype than genes from the signature. This is in line with the differing characteristics of the disease as early-/late-onset or with/without genetic predisposition. To investigate the gene signature's systemic role(s), signaling pathways, gene ontologies, and transcription factors were analyzed which revealed over-representation of response to stress, regulation of cellular metabolic processes, and reactive oxygen species. Additionally, our results clearly point to an important role of FOXA1 and FOXA2 gene regulatory networks in the etiology of AD. This finding is in corroboration with the recently reported major role of the dopaminergic system in the development of AD and its regulation by FOXA1 and FOXA2.

  1. Cloning of the gene encoding the δ subunit of the human T-cell receptor reveals its physical organization within the α-subunit locus and its involvement in chromosome translocations in T-cell malignancy

    International Nuclear Information System (INIS)

    Isobe, M.; Russo, G.; Haluska, F.G.; Croce, C.M.

    1988-01-01

    By taking advantage of chromosomal walking techniques, the authors have obtained clones that encompass the T-cell receptor (TCR) δ-chain gene. They analyzed clones spanning the entire J α region extending 115 kilobases 5' of the TCR α-chain constant region and have shown that the TCR δ-chain gene is located over 80 kilobases 5' of C α . TCR δ-chain gene is rearranged in the γ/δ-expressing T-cell line Peer and is deleted in α/β-expressing T-cell lines. Sequence analysis of portions of this genomic region demonstrates its identity with previously described cDNA clones corresponding to the C δ and J δ segments. Furthermore, they have analyzed a t(8;14)-(q24;q11) chromosome translocation from a T-cell leukemia and have shown that the J δ segment is rearranged in cells deriving from this tumor and probably directly involved in the translocation. Thus, the newly clones TCR δ chain is implicated in the genesis of chromosome translocations in T-cell malignancies carrying cytogenetic abnormalities of band 14q11

  2. 'Big bang' of B-cell development revealed.

    Science.gov (United States)

    Murre, Cornelis

    2018-01-15

    Earlier studies have identified transcription factors that specify B-cell fate, but the underlying mechanisms remain to be revealed. Two new studies by Miyai and colleagues (pp. 112-126) and Li and colleagues (pp. 96-111) in this issue of Genes & Development provide new and unprecedented insights into the genetic and epigenetic mechanisms that establish B-cell identity. © 2018 Murre; Published by Cold Spring Harbor Laboratory Press.

  3. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  4. Comparative mapping reveals similar linkage of functional genes to ...

    Indian Academy of Sciences (India)

    genes between O. sativa and B. napus may have consistent function and control similar traits, which may be ..... acea chromosomes reveals islands of conserved organization. ... 1998 Conserved structure and function of the Arabidopsis flow-.

  5. Revealing gene action for production characteristics by inbreeding ...

    African Journals Online (AJOL)

    Revealing gene action for production characteristics by inbreeding, based on a long-term selection ... The gene action involved in the expression of production characters was investigated, using the effect of the theoretical inbreeding ..... and predicted selection responses for growth, fat and lean traits in mice. J. Anim. Sci.

  6. Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B-Treated Cervical Cancer HeLa Cells.

    Science.gov (United States)

    Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

    2017-09-01

    Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H 2 O 2 -induced cell death is via neutralization of reactive oxygen species.

  7. Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B–Treated Cervical Cancer HeLa Cells

    Science.gov (United States)

    Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

    2016-01-01

    Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H2O2–induced cell death is via neutralization of reactive oxygen species. PMID:27458249

  8. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

    Science.gov (United States)

    Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

  9. A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT

    DEFF Research Database (Denmark)

    Lafferty-Whyte, K; Cairney, C J; Will, M B

    2009-01-01

    Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular le......TERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues....

  10. American Society of Gene & Cell Therapy

    Science.gov (United States)

    ... Gene & Cell Therapy Defined Gene therapy and cell therapy are overlapping fields of biomedical research that aim to repair the direct cause of genetic diseases. Read More Gene & Cell Therapy FAQ's Read the most common questions raised by ...

  11. Novel algorithms reveal streptococcal transcriptomes and clues about undefined genes.

    Science.gov (United States)

    Ryan, Patricia A; Kirk, Brian W; Euler, Chad W; Schuch, Raymond; Fischetti, Vincent A

    2007-07-01

    Bacteria-host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

  12. mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Gordon M Cann

    Full Text Available Circulating tumor cells (CTC mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

  13. mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.

    Science.gov (United States)

    Cann, Gordon M; Gulzar, Zulfiqar G; Cooper, Samantha; Li, Robin; Luo, Shujun; Tat, Mai; Stuart, Sarah; Schroth, Gary; Srinivas, Sandhya; Ronaghi, Mostafa; Brooks, James D; Talasaz, Amirali H

    2012-01-01

    Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

  14. Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2001-01-01

    Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

  15. Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Bolt, Gert; Hansen, Jens J

    2015-01-01

    orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII...... FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce...

  16. Reveal genes functionally associated with ACADS by a network study.

    Science.gov (United States)

    Chen, Yulong; Su, Zhiguang

    2015-09-15

    Establishing a systematic network is aimed at finding essential human gene-gene/gene-disease pathway by means of network inter-connecting patterns and functional annotation analysis. In the present study, we have analyzed functional gene interactions of short-chain acyl-coenzyme A dehydrogenase gene (ACADS). ACADS plays a vital role in free fatty acid β-oxidation and regulates energy homeostasis. Modules of highly inter-connected genes in disease-specific ACADS network are derived by integrating gene function and protein interaction data. Among the 8 genes in ACADS web retrieved from both STRING and GeneMANIA, ACADS is effectively conjoined with 4 genes including HAHDA, HADHB, ECHS1 and ACAT1. The functional analysis is done via ontological briefing and candidate disease identification. We observed that the highly efficient-interlinked genes connected with ACADS are HAHDA, HADHB, ECHS1 and ACAT1. Interestingly, the ontological aspect of genes in the ACADS network reveals that ACADS, HAHDA and HADHB play equally vital roles in fatty acid metabolism. The gene ACAT1 together with ACADS indulges in ketone metabolism. Our computational gene web analysis also predicts potential candidate disease recognition, thus indicating the involvement of ACADS, HAHDA, HADHB, ECHS1 and ACAT1 not only with lipid metabolism but also with infant death syndrome, skeletal myopathy, acute hepatic encephalopathy, Reye-like syndrome, episodic ketosis, and metabolic acidosis. The current study presents a comprehensible layout of ACADS network, its functional strategies and candidate disease approach associated with ACADS network. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Computational integration of homolog and pathway gene module expression reveals general stemness signatures.

    Directory of Open Access Journals (Sweden)

    Martina Koeva

    Full Text Available The stemness hypothesis states that all stem cells use common mechanisms to regulate self-renewal and multi-lineage potential. However, gene expression meta-analyses at the single gene level have failed to identify a significant number of genes selectively expressed by a broad range of stem cell types. We hypothesized that stemness may be regulated by modules of homologs. While the expression of any single gene within a module may vary from one stem cell type to the next, it is possible that the expression of the module as a whole is required so that the expression of different, yet functionally-synonymous, homologs is needed in different stem cells. Thus, we developed a computational method to test for stem cell-specific gene expression patterns from a comprehensive collection of 49 murine datasets covering 12 different stem cell types. We identified 40 individual genes and 224 stemness modules with reproducible and specific up-regulation across multiple stem cell types. The stemness modules included families regulating chromatin remodeling, DNA repair, and Wnt signaling. Strikingly, the majority of modules represent evolutionarily related homologs. Moreover, a score based on the discovered modules could accurately distinguish stem cell-like populations from other cell types in both normal and cancer tissues. This scoring system revealed that both mouse and human metastatic populations exhibit higher stemness indices than non-metastatic populations, providing further evidence for a stem cell-driven component underlying the transformation to metastatic disease.

  18. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6 causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Directory of Open Access Journals (Sweden)

    Takao Sasado

    Full Text Available Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68. CPSF6 is a component of the Cleavage Factor Im complex (CFIm which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  19. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Science.gov (United States)

    Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  20. Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes

    Directory of Open Access Journals (Sweden)

    Paules Richard S

    2007-11-01

    biological processes affected by IR- and/or UV- induced DNA damage. Conclusion EPIG competed with CLICK and performed better than CAST in extracting patterns from simulated data. EPIG extracted more biological informative patterns and co-expressed genes from both C. elegans and IR/UV-treated human fibroblasts. Using Gene Ontology analysis of the genes in the patterns extracted by EPIG, several key biological categories related to p53-dependent cell cycle control were revealed from the IR/UV data. Among them were mitotic cell cycle, DNA replication, DNA repair, cell cycle checkpoint, and G0-like status transition. EPIG can be applied to data sets from a variety of experimental designs.

  1. Systems-level analysis of risk genes reveals the modular nature of schizophrenia.

    Science.gov (United States)

    Liu, Jiewei; Li, Ming; Luo, Xiong-Jian; Su, Bing

    2018-05-19

    Schizophrenia (SCZ) is a complex mental disorder with high heritability. Genetic studies (especially recent genome-wide association studies) have identified many risk genes for schizophrenia. However, the physical interactions among the proteins encoded by schizophrenia risk genes remain elusive and it is not known whether the identified risk genes converge on common molecular networks or pathways. Here we systematically investigated the network characteristics of schizophrenia risk genes using the high-confidence protein-protein interactions (PPI) from the human interactome. We found that schizophrenia risk genes encode a densely interconnected PPI network (P = 4.15 × 10 -31 ). Compared with the background genes, the schizophrenia risk genes in the interactome have significantly higher degree (P = 5.39 × 10 -11 ), closeness centrality (P = 7.56 × 10 -11 ), betweeness centrality (P = 1.29 × 10 -11 ), clustering coefficient (P = 2.22 × 10 -2 ), and shorter average shortest path length (P = 7.56 × 10 -11 ). Based on the densely interconnected PPI network, we identified 48 hub genes and 4 modules formed by highly interconnected schizophrenia genes. We showed that the proteins encoded by schizophrenia hub genes have significantly more direct physical interactions. Gene ontology (GO) analysis revealed that cell adhesion, cell cycle, immune system response, and GABR-receptor complex categories were enriched in the modules formed by highly interconnected schizophrenia risk genes. Our study reveals that schizophrenia risk genes encode a densely interconnected molecular network and demonstrates the modular nature of schizophrenia. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process

    International Nuclear Information System (INIS)

    Chandran, Uma R; Ma, Changqing; Dhir, Rajiv; Bisceglia, Michelle; Lyons-Weiler, Maureen; Liang, Wenjing; Michalopoulos, George; Becich, Michael; Monzon, Federico A

    2007-01-01

    Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer

  3. Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

    Directory of Open Access Journals (Sweden)

    Kouji Satoh

    Full Text Available Rice (Oryza sativa L. is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE genes, 33K annotated non-expressed (ANE genes, and 5.5K non-annotated expressed (NAE genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

  4. Comprehensive regional and temporal gene expression profiling of the rat brain during the first 24 h after experimental stroke identifies dynamic ischemia-induced gene expression patterns, and reveals a biphasic activation of genes in surviving tissue

    DEFF Research Database (Denmark)

    Rickhag, Karl Mattias; Wieloch, Tadeusz; Gidö, Gunilla

    2006-01-01

    middle cerebral artery occlusion in the rat. K-means cluster analysis revealed two distinct biphasic gene expression patterns that contained 44 genes (including 18 immediate early genes), involved in cell signaling and plasticity (i.e. MAP2K7, Sprouty2, Irs-2, Homer1, GPRC5B, Grasp). The first gene...

  5. Genome-wide analysis of gene expression in primate taste buds reveals links to diverse processes.

    Directory of Open Access Journals (Sweden)

    Peter Hevezi

    Full Text Available Efforts to unravel the mechanisms underlying taste sensation (gustation have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM procured fungiform (FG and circumvallate (CV taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology.

  6. Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function

    Directory of Open Access Journals (Sweden)

    Antommattei Frances M

    2008-10-01

    Full Text Available Abstract Background Geobacter species are δ-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production. Recent studies in other bacteria have demonstrated that signaling pathways homologous to the paradigm established for Escherichia coli chemotaxis can regulate type IV pili-dependent motility, the synthesis of flagella and type IV pili, the production of extracellular matrix material, and biofilm formation. The classification of these pathways by comparative genomics improves the ability to understand how Geobacter thrives in natural environments and better their use in microbial fuel cells. Results The genomes of G. sulfurreducens, G. metallireducens, and G. uraniireducens contain multiple (~70 homologs of chemotaxis genes arranged in several major clusters (six, seven, and seven, respectively. Unlike the single gene cluster of E. coli, the Geobacter clusters are not all located near the flagellar genes. The probable functions of some Geobacter clusters are assignable by homology to known pathways; others appear to be unique to the Geobacter sp. and contain genes of unknown function. We identified large numbers of methyl-accepting chemotaxis protein (MCP homologs that have diverse sensing domain architectures and generate a potential for sensing a great variety of environmental signals. We discuss mechanisms for class-specific segregation of the MCPs in the cell membrane, which serve to maintain pathway specificity and diminish crosstalk. Finally, the regulation of gene expression in Geobacter differs from E. coli. The sequences of predicted promoter elements suggest that the alternative sigma factors

  7. Genes but not genomes reveal bacterial domestication of Lactococcus lactis.

    Directory of Open Access Journals (Sweden)

    Delphine Passerini

    Full Text Available BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE. METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable

  8. Listening to the Noise: Random Fluctuations Reveal Gene Network Parameters

    Science.gov (United States)

    Munsky, Brian; Trinh, Brooke; Khammash, Mustafa

    2010-03-01

    The cellular environment is abuzz with noise originating from the inherent random motion of reacting molecules in the living cell. In this noisy environment, clonal cell populations exhibit cell-to-cell variability that can manifest significant prototypical differences. Noise induced stochastic fluctuations in cellular constituents can be measured and their statistics quantified using flow cytometry, single molecule fluorescence in situ hybridization, time lapse fluorescence microscopy and other single cell and single molecule measurement techniques. We show that these random fluctuations carry within them valuable information about the underlying genetic network. Far from being a nuisance, the ever-present cellular noise acts as a rich source of excitation that, when processed through a gene network, carries its distinctive fingerprint that encodes a wealth of information about that network. We demonstrate that in some cases the analysis of these random fluctuations enables the full identification of network parameters, including those that may otherwise be difficult to measure. We use theoretical investigations to establish experimental guidelines for the identification of gene regulatory networks, and we apply these guideline to experimentally identify predictive models for different regulatory mechanisms in bacteria and yeast.

  9. Heart morphogenesis gene regulatory networks revealed by temporal expression analysis.

    Science.gov (United States)

    Hill, Jonathon T; Demarest, Bradley; Gorsi, Bushra; Smith, Megan; Yost, H Joseph

    2017-10-01

    During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, Tbx5 and nkx2-5 , and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation. © 2017. Published by The Company of Biologists Ltd.

  10. Gene Delivery for Metastatic Prostate Cancer Cells

    National Research Council Canada - National Science Library

    Pang, Shen

    2001-01-01

    .... Enhanced by the bystander effect, the specific expression of the DTA gene causes significant cell death in prostate cancer cell cultures, with very low background cell eradication in control cell lines...

  11. Genomic analysis of primordial dwarfism reveals novel disease genes.

    Science.gov (United States)

    Shaheen, Ranad; Faqeih, Eissa; Ansari, Shinu; Abdel-Salam, Ghada; Al-Hassnan, Zuhair N; Al-Shidi, Tarfa; Alomar, Rana; Sogaty, Sameera; Alkuraya, Fowzan S

    2014-02-01

    Primordial dwarfism (PD) is a disease in which severely impaired fetal growth persists throughout postnatal development and results in stunted adult size. The condition is highly heterogeneous clinically, but the use of certain phenotypic aspects such as head circumference and facial appearance has proven helpful in defining clinical subgroups. In this study, we present the results of clinical and genomic characterization of 16 new patients in whom a broad definition of PD was used (e.g., 3M syndrome was included). We report a novel PD syndrome with distinct facies in two unrelated patients, each with a different homozygous truncating mutation in CRIPT. Our analysis also reveals, in addition to mutations in known PD disease genes, the first instance of biallelic truncating BRCA2 mutation causing PD with normal bone marrow analysis. In addition, we have identified a novel locus for Seckel syndrome based on a consanguineous multiplex family and identified a homozygous truncating mutation in DNA2 as the likely cause. An additional novel PD disease candidate gene XRCC4 was identified by autozygome/exome analysis, and the knockout mouse phenotype is highly compatible with PD. Thus, we add a number of novel genes to the growing list of PD-linked genes, including one which we show to be linked to a novel PD syndrome with a distinct facial appearance. PD is extremely heterogeneous genetically and clinically, and genomic tools are often required to reach a molecular diagnosis.

  12. Novel candidate genes important for asthma and hypertension comorbidity revealed from associative gene networks.

    Science.gov (United States)

    Saik, Olga V; Demenkov, Pavel S; Ivanisenko, Timofey V; Bragina, Elena Yu; Freidin, Maxim B; Goncharova, Irina A; Dosenko, Victor E; Zolotareva, Olga I; Hofestaedt, Ralf; Lavrik, Inna N; Rogaev, Evgeny I; Ivanisenko, Vladimir A

    2018-02-13

    Hypertension and bronchial asthma are a major issue for people's health. As of 2014, approximately one billion adults, or ~ 22% of the world population, have had hypertension. As of 2011, 235-330 million people globally have been affected by asthma and approximately 250,000-345,000 people have died each year from the disease. The development of the effective treatment therapies against these diseases is complicated by their comorbidity features. This is often a major problem in diagnosis and their treatment. Hence, in this study the bioinformatical methodology for the analysis of the comorbidity of these two diseases have been developed. As such, the search for candidate genes related to the comorbid conditions of asthma and hypertension can help in elucidating the molecular mechanisms underlying the comorbid condition of these two diseases, and can also be useful for genotyping and identifying new drug targets. Using ANDSystem, the reconstruction and analysis of gene networks associated with asthma and hypertension was carried out. The gene network of asthma included 755 genes/proteins and 62,603 interactions, while the gene network of hypertension - 713 genes/proteins and 45,479 interactions. Two hundred and five genes/proteins and 9638 interactions were shared between asthma and hypertension. An approach for ranking genes implicated in the comorbid condition of two diseases was proposed. The approach is based on nine criteria for ranking genes by their importance, including standard methods of gene prioritization (Endeavor, ToppGene) as well as original criteria that take into account the characteristics of an associative gene network and the presence of known polymorphisms in the analysed genes. According to the proposed approach, the genes IL10, TLR4, and CAT had the highest priority in the development of comorbidity of these two diseases. Additionally, it was revealed that the list of top genes is enriched with apoptotic genes and genes involved in

  13. A PCR-aided transcript titration assay revealing very low expression of a gene at band 3p21 in 33 cells lines derived from all types of lung cancer

    NARCIS (Netherlands)

    Kok, K; Buchhagen, D L; Carritt, B; Buys, C H; van den Berg, Anke

    1993-01-01

    We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of

  14. Gaussian graphical modeling reveals specific lipid correlations in glioblastoma cells

    Science.gov (United States)

    Mueller, Nikola S.; Krumsiek, Jan; Theis, Fabian J.; Böhm, Christian; Meyer-Bäse, Anke

    2011-06-01

    Advances in high-throughput measurements of biological specimens necessitate the development of biologically driven computational techniques. To understand the molecular level of many human diseases, such as cancer, lipid quantifications have been shown to offer an excellent opportunity to reveal disease-specific regulations. The data analysis of the cell lipidome, however, remains a challenging task and cannot be accomplished solely based on intuitive reasoning. We have developed a method to identify a lipid correlation network which is entirely disease-specific. A powerful method to correlate experimentally measured lipid levels across the various samples is a Gaussian Graphical Model (GGM), which is based on partial correlation coefficients. In contrast to regular Pearson correlations, partial correlations aim to identify only direct correlations while eliminating indirect associations. Conventional GGM calculations on the entire dataset can, however, not provide information on whether a correlation is truly disease-specific with respect to the disease samples and not a correlation of control samples. Thus, we implemented a novel differential GGM approach unraveling only the disease-specific correlations, and applied it to the lipidome of immortal Glioblastoma tumor cells. A large set of lipid species were measured by mass spectrometry in order to evaluate lipid remodeling as a result to a combination of perturbation of cells inducing programmed cell death, while the other perturbations served solely as biological controls. With the differential GGM, we were able to reveal Glioblastoma-specific lipid correlations to advance biomedical research on novel gene therapies.

  15. Multilocus analysis reveals three candidate genes for Chinese migraine susceptibility.

    Science.gov (United States)

    An, X-K; Fang, J; Yu, Z-Z; Lin, Q; Lu, C-X; Qu, H-L; Ma, Q-L

    2017-08-01

    Several genome-wide association studies (GWASs) in Caucasian populations have identified 12 loci that are significantly associated with migraine. More evidence suggests that serotonin receptors are also involved in migraine pathophysiology. In the present study, a case-control study was conducted in a cohort of 581 migraine cases and 533 ethnically matched controls among a Chinese population. Eighteen polymorphisms from serotonin receptors and GWASs were selected, and genotyping was performed using a Sequenom MALDI-TOF mass spectrometry iPLEX platform. The genotypic and allelic distributions of MEF2D rs2274316 and ASTN2 rs6478241 were significantly different between migraine patients and controls. Univariate and multivariate analysis revealed significant associations of polymorphisms in the MEF2D and ASTN2 genes with migraine susceptibility. MEF2D, PRDM16 and ASTN2 were also found to be associated with migraine without aura (MO) and migraine with family history. And, MEF2D and ASTN2 also served as genetic risk factors for the migraine without family history. The generalized multifactor dimensionality reduction analysis identified that MEF2D and HTR2E constituted the two-factor interaction model. Our study suggests that the MEF2D, PRDM16 and ASTN2 genes from GWAS are associated with migraine susceptibility, especially MO, among Chinese patients. It appears that there is no association with serotonin receptor related genes. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Characterization of the avian Trojan gene family reveals contrasting evolutionary constraints.

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    Petar Petrov

    Full Text Available "Trojan" is a leukocyte-specific, cell surface protein originally identified in the chicken. Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges. We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules.

  17. Characterization of the avian Trojan gene family reveals contrasting evolutionary constraints.

    Science.gov (United States)

    Petrov, Petar; Syrjänen, Riikka; Smith, Jacqueline; Gutowska, Maria Weronika; Uchida, Tatsuya; Vainio, Olli; Burt, David W

    2015-01-01

    "Trojan" is a leukocyte-specific, cell surface protein originally identified in the chicken. Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges. We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules.

  18. Microarray gene expression profiling and analysis in renal cell carcinoma

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    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  19. Relaxation rates of gene expression kinetics reveal the feedback signs of autoregulatory gene networks

    Science.gov (United States)

    Jia, Chen; Qian, Hong; Chen, Min; Zhang, Michael Q.

    2018-03-01

    The transient response to a stimulus and subsequent recovery to a steady state are the fundamental characteristics of a living organism. Here we study the relaxation kinetics of autoregulatory gene networks based on the chemical master equation model of single-cell stochastic gene expression with nonlinear feedback regulation. We report a novel relation between the rate of relaxation, characterized by the spectral gap of the Markov model, and the feedback sign of the underlying gene circuit. When a network has no feedback, the relaxation rate is exactly the decaying rate of the protein. We further show that positive feedback always slows down the relaxation kinetics while negative feedback always speeds it up. Numerical simulations demonstrate that this relation provides a possible method to infer the feedback topology of autoregulatory gene networks by using time-series data of gene expression.

  20. Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

    Directory of Open Access Journals (Sweden)

    Eddy Edward M

    2007-07-01

    Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

  1. Single-cell RNA-Seq reveals cell heterogeneity and hierarchy within mouse mammary epithelia.

    Science.gov (United States)

    Sun, Heng; Miao, Zhengqiang; Zhang, Xin; Chan, Un In; Su, Sek Man; Guo, Sen; Wong, Chris Koon Ho; Xu, Xiaoling; Deng, Chu-Xia

    2018-04-17

    The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5+ cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5+ cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Genetic recombination at the human RH locus: A family study of the red-cell Evans phenotype reveals a transfer of exons 2-6 from the RHD to the RHCE gene

    Energy Technology Data Exchange (ETDEWEB)

    Huang, C.H.; Chen, Y.; Reid, M. [Lindsley F. Kimball Research Inst., New York, NY (United States); Ghosh, S.

    1996-10-01

    The human RH locus appears to consist of two structural genes, D and CE, which map on the short arm p34-36 of chromosome 1 and specify a most complex system of blood-group genetic polymorphisms. Here we describe a family study of the Evans (also known as {open_quotes}D..{open_quotes}) phenotype, a codominant trait associated with both qualitative and quantitative changes in D-antigen expression. A cataract-causing mutation was also inherited in this family and was apparently cotransmitted with Evans, suggesting a chromosomal linkage of these two otherwise unrelated traits. Southern blot analysis and allele-specific PCR showed the linkage of Evans with a SphI RFLP marker and the presence of a hybrid gene in the RH locus. To delineate the pattern of gene expression, the composition and structure of Rh-polypeptide transcripts were characterized by reverse transcriptase-PCR and nucleotide sequencing. This resulted in the identification of a novel Rh transcript expressed only in the Evans-positive erythroid cells. Sequence analysis showed that the transcript maintained a normal open reading frame but occurred as a CE-D-CE composite in which exons 2-6 of the CE gene were replaced by the homologous counterpart of the D gene. This hybrid gene was predicted to encode a CE-D-CE fusion protein whose surface expression correlates with the Evans phenotype. The mode and consequence of such a recombination event suggest the occurrence, in the RH locus, of a segmental DNA transfer via the mechanism of gene conversion. 31 refs., 6 figs., 1 tab.

  3. Memory functions reveal structural properties of gene regulatory networks

    Science.gov (United States)

    Perez-Carrasco, Ruben

    2018-01-01

    Gene regulatory networks (GRNs) control cellular function and decision making during tissue development and homeostasis. Mathematical tools based on dynamical systems theory are often used to model these networks, but the size and complexity of these models mean that their behaviour is not always intuitive and the underlying mechanisms can be difficult to decipher. For this reason, methods that simplify and aid exploration of complex networks are necessary. To this end we develop a broadly applicable form of the Zwanzig-Mori projection. By first converting a thermodynamic state ensemble model of gene regulation into mass action reactions we derive a general method that produces a set of time evolution equations for a subset of components of a network. The influence of the rest of the network, the bulk, is captured by memory functions that describe how the subnetwork reacts to its own past state via components in the bulk. These memory functions provide probes of near-steady state dynamics, revealing information not easily accessible otherwise. We illustrate the method on a simple cross-repressive transcriptional motif to show that memory functions not only simplify the analysis of the subnetwork but also have a natural interpretation. We then apply the approach to a GRN from the vertebrate neural tube, a well characterised developmental transcriptional network composed of four interacting transcription factors. The memory functions reveal the function of specific links within the neural tube network and identify features of the regulatory structure that specifically increase the robustness of the network to initial conditions. Taken together, the study provides evidence that Zwanzig-Mori projections offer powerful and effective tools for simplifying and exploring the behaviour of GRNs. PMID:29470492

  4. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  5. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    International Nuclear Information System (INIS)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-01-01

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  6. Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Ohkawa, Mayumi [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Ohno, Yoshiya [Laboratory of Immunobiology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe-shi, Hyogo 650-8530 (Japan); Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan); Tanaka, Toshiyuki [Laboratory of Immunobiology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe-shi, Hyogo 650-8530 (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8502 (Japan); Seki, Masayuki; Enomoto, Takemi [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Yagi, Hideki [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan); Hashimoto, Yoshiyuki [Tohoku University, Sendai (Japan); Masuko, Takashi, E-mail: masuko@phar.kindai.ac.jp [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan)

    2011-03-25

    Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{sup -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

  7. Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

    International Nuclear Information System (INIS)

    Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo; Tanaka, Toshiyuki; Saya, Hideyuki; Seki, Masayuki; Enomoto, Takemi; Yagi, Hideki; Hashimoto, Yoshiyuki; Masuko, Takashi

    2011-01-01

    Highlights: → We established LAT1 amino-acid transporter-disrupted DT40 cells. → LAT1-disrupted cells showed slow growth and lost the oncogenicity. → siRNA and mAb inhibited human tumor growth in vitro and in vivo. → LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1 -/- ) cell clones, derived from a heterozygous LAT1 +/- clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1 -/- DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1 -/- cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1 -/- DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1 -/- DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1 +/- DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

  8. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2014-05-01

    Full Text Available Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF, we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I and Atf4 and Hspa5 (for Up-II. Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.

  9. Integrated bioinformatics analysis reveals key candidate genes and pathways in breast cancer.

    Science.gov (United States)

    Wang, Yuzhi; Zhang, Yi; Huang, Qian; Li, Chengwen

    2018-04-19

    Breast cancer (BC) is the leading malignancy in women worldwide, yet relatively little is known about the genes and signaling pathways involved in BC tumorigenesis and progression. The present study aimed to elucidate potential key candidate genes and pathways in BC. Five gene expression profile data sets (GSE22035, GSE3744, GSE5764, GSE21422 and GSE26910) were downloaded from the Gene Expression Omnibus (GEO) database, which included data from 113 tumorous and 38 adjacent non‑tumorous tissue samples. Differentially expressed genes (DEGs) were identified using t‑tests in the limma R package. These DEGs were subsequently investigated by pathway enrichment analysis and a protein‑protein interaction (PPI) network was constructed. The most significant module from the PPI network was selected for pathway enrichment analysis. In total, 227 DEGs were identified, of which 82 were upregulated and 145 were downregulated. Pathway enrichment analysis results revealed that the upregulated DEGs were mainly enriched in 'cell division', the 'proteinaceous extracellular matrix (ECM)', 'ECM structural constituents' and 'ECM‑receptor interaction', whereas downregulated genes were mainly enriched in 'response to drugs', 'extracellular space', 'transcriptional activator activity' and the 'peroxisome proliferator‑activated receptor signaling pathway'. The PPI network contained 174 nodes and 1,257 edges. DNA topoisomerase 2‑a, baculoviral inhibitor of apoptosis repeat‑containing protein 5, cyclin‑dependent kinase 1, G2/mitotic‑specific cyclin‑B1 and kinetochore protein NDC80 homolog were identified as the top 5 hub genes. Furthermore, the genes in the most significant module were predominantly involved in 'mitotic nuclear division', 'mid‑body', 'protein binding' and 'cell cycle'. In conclusion, the DEGs, relative pathways and hub genes identified in the present study may aid in understanding of the molecular mechanisms underlying BC progression and provide

  10. DNA entropy reveals a significant difference in complexity between housekeeping and tissue specific gene promoters.

    Science.gov (United States)

    Thomas, David; Finan, Chris; Newport, Melanie J; Jones, Susan

    2015-10-01

    The complexity of DNA can be quantified using estimates of entropy. Variation in DNA complexity is expected between the promoters of genes with different transcriptional mechanisms; namely housekeeping (HK) and tissue specific (TS). The former are transcribed constitutively to maintain general cellular functions, and the latter are transcribed in restricted tissue and cells types for specific molecular events. It is known that promoter features in the human genome are related to tissue specificity, but this has been difficult to quantify on a genomic scale. If entropy effectively quantifies DNA complexity, calculating the entropies of HK and TS gene promoters as profiles may reveal significant differences. Entropy profiles were calculated for a total dataset of 12,003 human gene promoters and for 501 housekeeping (HK) and 587 tissue specific (TS) human gene promoters. The mean profiles show the TS promoters have a significantly lower entropy (pentropy distributions for the 3 datasets show that promoter entropies could be used to identify novel HK genes. Functional features comprise DNA sequence patterns that are non-random and hence they have lower entropies. The lower entropy of TS gene promoters can be explained by a higher density of positive and negative regulatory elements, required for genes with complex spatial and temporary expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Hierarchical clustering of breast cancer methylomes revealed differentially methylated and expressed breast cancer genes.

    Directory of Open Access Journals (Sweden)

    I-Hsuan Lin

    Full Text Available Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs and the hypomethylation of the megabase-sized partially methylated domains (PMDs are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation.

  12. An Integrated Cell Purification and Genomics Strategy Reveals Multiple Regulators of Pancreas Development

    Science.gov (United States)

    Benitez, Cecil M.; Qu, Kun; Sugiyama, Takuya; Pauerstein, Philip T.; Liu, Yinghua; Tsai, Jennifer; Gu, Xueying; Ghodasara, Amar; Arda, H. Efsun; Zhang, Jiajing; Dekker, Joseph D.; Tucker, Haley O.; Chang, Howard Y.; Kim, Seung K.

    2014-01-01

    The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus. PMID:25330008

  13. An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

    Directory of Open Access Journals (Sweden)

    Cecil M Benitez

    2014-10-01

    Full Text Available The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

  14. Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

    Science.gov (United States)

    Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

    2018-05-03

    Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

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    Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

    2017-08-01

    Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

  16. Patterns of expression of cell wall related genes in sugarcane

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    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  17. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

    International Nuclear Information System (INIS)

    Teutschbein, Janka; Haydn, Johannes M; Samans, Birgit; Krause, Michael; Eilers, Martin; Schartl, Manfred; Meierjohann, Svenja

    2010-01-01

    Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development

  18. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

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    Krause Michael

    2010-07-01

    Full Text Available Abstract Background Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1, early growth response 1 (Egr1, osteopontin (Opn, insulin-like growth factor binding protein 3 (Igfbp3, dual-specificity phosphatase 4 (Dusp4, and tumor-associated antigen L6 (Taal6. Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute

  19. Characterization of transformation related genes in oral cancer cells.

    Science.gov (United States)

    Chang, D D; Park, N H; Denny, C T; Nelson, S F; Pe, M

    1998-04-16

    A cDNA representational difference analysis (cDNA-RDA) and an arrayed filter technique were used to characterize transformation-related genes in oral cancer. From an initial comparison of normal oral epithelial cells and a human papilloma virus (HPV)-immortalized oral epithelial cell line, we obtained 384 differentially expressed gene fragments and arrayed them on a filter. Two hundred and twelve redundant clones were identified by three rounds of back hybridization. Sequence analysis of the remaining clones revealed 99 unique clones corresponding to 69 genes. The expression of these transformation related gene fragments in three nontumorigenic HPV-immortalized oral epithelial cell lines and three oral cancer cell lines were simultaneously monitored using a cDNA array hybridization. Although there was a considerable cell line-to-cell line variability in the expression of these clones, a reliable prediction of their expression could be made from the cDNA array hybridization. Our study demonstrates the utility of combining cDNA-RDA and arrayed filters in high-throughput gene expression difference analysis. The differentially expressed genes identified in this study should be informative in studying oral epithelial cell carcinogenesis.

  20. Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times

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    Rao Nagesha AS

    2009-09-01

    Full Text Available Abstract Background Gene expression profiling of spontaneous tumors in the dog offers a unique translational opportunity to identify prognostic biomarkers and signaling pathways that are common to both canine and human. Osteosarcoma (OS accounts for approximately 80% of all malignant bone tumors in the dog. Canine OS are highly comparable with their human counterpart with respect to histology, high metastatic rate and poor long-term survival. This study investigates the prognostic gene profile among thirty-two primary canine OS using canine specific cDNA microarrays representing 20,313 genes to identify genes and cellular signaling pathways associated with survival. This, the first report of its kind in dogs with OS, also demonstrates the advantages of cross-species comparison with human OS. Results The 32 tumors were classified into two prognostic groups based on survival time (ST. They were defined as short survivors (dogs with poor prognosis: surviving fewer than 6 months and long survivors (dogs with better prognosis: surviving 6 months or longer. Fifty-one transcripts were found to be differentially expressed, with common upregulation of these genes in the short survivors. The overexpressed genes in short survivors are associated with possible roles in proliferation, drug resistance or metastasis. Several deregulated pathways identified in the present study, including Wnt signaling, Integrin signaling and Chemokine/cytokine signaling are comparable to the pathway analysis conducted on human OS gene profiles, emphasizing the value of the dog as an excellent model for humans. Conclusion A molecular-based method for discrimination of outcome for short and long survivors is useful for future prognostic stratification at initial diagnosis, where genes and pathways associated with cell cycle/proliferation, drug resistance and metastasis could be potential targets for diagnosis and therapy. The similarities between human and canine OS makes the

  1. Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis.

    Science.gov (United States)

    Zhao, Lihua; He, Jiangman; Cai, Hanyang; Lin, Haiyan; Li, Yanqiang; Liu, Renyi; Yang, Zhenbiao; Qin, Yuan

    2014-11-01

    Megasporogenesis is essential for female fertility, and requires the accomplishment of meiosis and the formation of functional megaspores. The inaccessibility and low abundance of female meiocytes make it particularly difficult to elucidate the molecular basis underlying megasporogenesis. We used high-throughput tag-sequencing analysis to identify genes expressed in female meiocytes (FMs) by comparing gene expression profiles from wild-type ovules undergoing megasporogenesis with those from the spl mutant ovules, which lack megasporogenesis. A total of 862 genes were identified as FMs, with levels that are consistently reduced in spl ovules in two biological replicates. Fluorescence-assisted cell sorting followed by RNA-seq analysis of DMC1:GFP-labeled female meiocytes confirmed that 90% of the FMs are indeed detected in the female meiocyte protoplast profiling. We performed reverse genetic analysis of 120 candidate genes and identified four FM genes with a function in female meiosis progression in Arabidopsis. We further revealed that KLU, a putative cytochrome P450 monooxygenase, is involved in chromosome pairing during female meiosis, most likely by affecting the normal expression pattern of DMC1 in ovules during female meiosis. Our studies provide valuable information for functional genomic analyses of plant germline development as well as insights into meiosis. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  2. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

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    Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

    2004-05-18

    Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

  4. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

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    Anton V Borovjagin

    Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

  5. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

    Science.gov (United States)

    Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M

    2012-11-15

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

  6. Interdependence of cell growth and gene expression: origins and consequences.

    Science.gov (United States)

    Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

    2010-11-19

    In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

  7. Gene array analysis of PD-1H overexpressing monocytes reveals a pro-inflammatory profile

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    Preeti Bharaj

    2018-02-01

    Full Text Available We have previously reported that overexpression of Programmed Death -1 Homolog (PD-1H in human monocytes leads to activation and spontaneous secretion of multiple pro inflammatory cytokines. Here we evaluate changes in monocytes gene expression after enforced PD-1H expression by gene array. The results show that there are significant alterations in 51 potential candidate genes that relate to immune response, cell adhesion and metabolism. Genes corresponding to pro-inflammatory cytokines showed the highest upregulation, 7, 3.2, 3.0, 5.8, 4.4 and 3.1 fold upregulation of TNF-α, IL-1 β, IFN-α, γ, λ and IL-27 relative to vector control. The data are in agreement with cytometric bead array analysis showing induction of proinflammatory cytokines, IL-6, IL-1β and TNF-α by PD-1H. Other genes related to inflammation, include transglutaminase 2 (TG2, NF-κB (p65 and p50 and toll like receptors (TLR 3 and 4 were upregulated 5, 4.5 and 2.5 fold, respectively. Gene set enrichment analysis (GSEA also revealed that signaling pathways related to inflammatory response, such as NFκB, AT1R, PYK2, MAPK, RELA, TNFR1, MTOR and proteasomal degradation, were significantly upregulated in response to PD-1H overexpression. We validated the results utilizing a standard inflammatory sepsis model in humanized BLT mice, finding that PD-1H expression was highly correlated with proinflammatory cytokine production. We therefore conclude that PD-1H functions to enhance monocyte activation and the induction of a pro-inflammatory gene expression profile.

  8. Functional gene polymorphism to reveal species history: the case of the CRTISO gene in cultivated carrots.

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    Vanessa Soufflet-Freslon

    Full Text Available Carrot is a vegetable cultivated worldwide for the consumption of its root. Historical data indicate that root colour has been differentially selected over time and according to geographical areas. Root pigmentation depends on the relative proportion of different carotenoids for the white, yellow, orange and red types but only internally for the purple one. The genetic control for root carotenoid content might be partially associated with carotenoid biosynthetic genes. Carotenoid isomerase (CRTISO has emerged as a regulatory step in the carotenoid biosynthesis pathway and could be a good candidate to show how a metabolic pathway gene reflects a species genetic history.In this study, the nucleotide polymorphism and the linkage disequilibrium among the complete CRTISO sequence, and the deviation from neutral expectation were analysed by considering population subdivision revealed with 17 microsatellite markers. A sample of 39 accessions, which represented different geographical origins and root colours, was used. Cultivated carrot was divided into two genetic groups: one from Middle East and Asia (Eastern group, and another one mainly from Europe (Western group. The Western and Eastern genetic groups were suggested to be differentially affected by selection: a signature of balancing selection was detected within the first group whereas the second one showed no selection. A focus on orange-rooted carrots revealed that cultivars cultivated in Asia were mainly assigned to the Western group but showed CRTISO haplotypes common to Eastern carrots.The carotenoid pathway CRTISO gene data proved to be complementary to neutral markers in order to bring critical insight in the cultivated carrot history. We confirmed the occurrence of two migration events since domestication. Our results showed a European background in material from Japan and Central Asia. While confirming the introduction of European carrots in Japanese resources, the history of Central Asia

  9. Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.

    Science.gov (United States)

    Tarazanova, Mariya; Beerthuyzen, Marke; Siezen, Roland; Fernandez-Gutierrez, Marcela M; de Jong, Anne; van der Meulen, Sjoerd; Kok, Jan; Bachmann, Herwig

    2016-01-01

    Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

  10. Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

    Science.gov (United States)

    Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

    2009-01-01

    OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

  11. Gene Profiling in Patients with Systemic Sclerosis Reveals the Presence of Oncogenic Gene Signatures

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    Marzia Dolcino

    2018-03-01

    Full Text Available Systemic sclerosis (SSc is a rare connective tissue disease characterized by three pathogenetic hallmarks: vasculopathy, dysregulation of the immune system, and fibrosis. A particular feature of SSc is the increased frequency of some types of malignancies, namely breast, lung, and hematological malignancies. Moreover, SSc may also be a paraneoplastic disease, again indicating a strong link between cancer and scleroderma. The reason of this association is still unknown; therefore, we aimed at investigating whether particular genetic or epigenetic factors may play a role in promoting cancer development in patients with SSc and whether some features are shared by the two conditions. We therefore performed a gene expression profiling of peripheral blood mononuclear cells (PBMCs derived from patients with limited and diffuse SSc, showing that the various classes of genes potentially linked to the pathogenesis of SSc (such as apoptosis, endothelial cell activation, extracellular matrix remodeling, immune response, and inflammation include genes that directly participate in the development of malignancies or that are involved in pathways known to be associated with carcinogenesis. The transcriptional analysis was then complemented by a complex network analysis of modulated genes which further confirmed the presence of signaling pathways associated with carcinogenesis. Since epigenetic mechanisms, such as microRNAs (miRNAs, are believed to play a central role in the pathogenesis of SSc, we also evaluated whether specific cancer-related miRNAs could be deregulated in the serum of SSc patients. We focused our attention on miRNAs already found upregulated in SSc such as miR-21-5p, miR-92a-3p, and on miR-155-5p, miR 126-3p and miR-16-5p known to be deregulated in malignancies associated to SSc, i.e., breast, lung, and hematological malignancies. miR-21-5p, miR-92a-3p, miR-155-5p, and miR-16-5p expression was significantly higher in SSc sera compared to

  12. Phylogenetic analysis of ferlin genes reveals ancient eukaryotic origins

    Directory of Open Access Journals (Sweden)

    Lek Monkol

    2010-07-01

    Full Text Available Abstract Background The ferlin gene family possesses a rare and identifying feature consisting of multiple tandem C2 domains and a C-terminal transmembrane domain. Much currently remains unknown about the fundamental function of this gene family, however, mutations in its two most well-characterised members, dysferlin and otoferlin, have been implicated in human disease. The availability of genome sequences from a wide range of species makes it possible to explore the evolution of the ferlin family, providing contextual insight into characteristic features that define the ferlin gene family in its present form in humans. Results Ferlin genes were detected from all species of representative phyla, with two ferlin subgroups partitioned within the ferlin phylogenetic tree based on the presence or absence of a DysF domain. Invertebrates generally possessed two ferlin genes (one with DysF and one without, with six ferlin genes in most vertebrates (three DysF, three non-DysF. Expansion of the ferlin gene family is evident between the divergence of lamprey (jawless vertebrates and shark (cartilaginous fish. Common to almost all ferlins is an N-terminal C2-FerI-C2 sandwich, a FerB motif, and two C-terminal C2 domains (C2E and C2F adjacent to the transmembrane domain. Preservation of these structural elements throughout eukaryotic evolution suggests a fundamental role of these motifs for ferlin function. In contrast, DysF, C2DE, and FerA are optional, giving rise to subtle differences in domain topologies of ferlin genes. Despite conservation of multiple C2 domains in all ferlins, the C-terminal C2 domains (C2E and C2F displayed higher sequence conservation and greater conservation of putative calcium binding residues across paralogs and orthologs. Interestingly, the two most studied non-mammalian ferlins (Fer-1 and Misfire in model organisms C. elegans and D. melanogaster, present as outgroups in the phylogenetic analysis, with results suggesting

  13. Identification of astrocytoma associated genes including cell surface markers

    International Nuclear Information System (INIS)

    Boon, Kathy; Edwards, Jennifer B; Eberhart, Charles G; Riggins, Gregory J

    2004-01-01

    Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide. We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [1]. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie [2], and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors. A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions. This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes

  14. Systems Nutrigenomics Reveals Brain Gene Networks Linking Metabolic and Brain Disorders.

    Science.gov (United States)

    Meng, Qingying; Ying, Zhe; Noble, Emily; Zhao, Yuqi; Agrawal, Rahul; Mikhail, Andrew; Zhuang, Yumei; Tyagi, Ethika; Zhang, Qing; Lee, Jae-Hyung; Morselli, Marco; Orozco, Luz; Guo, Weilong; Kilts, Tina M; Zhu, Jun; Zhang, Bin; Pellegrini, Matteo; Xiao, Xinshu; Young, Marian F; Gomez-Pinilla, Fernando; Yang, Xia

    2016-05-01

    Nutrition plays a significant role in the increasing prevalence of metabolic and brain disorders. Here we employ systems nutrigenomics to scrutinize the genomic bases of nutrient-host interaction underlying disease predisposition or therapeutic potential. We conducted transcriptome and epigenome sequencing of hypothalamus (metabolic control) and hippocampus (cognitive processing) from a rodent model of fructose consumption, and identified significant reprogramming of DNA methylation, transcript abundance, alternative splicing, and gene networks governing cell metabolism, cell communication, inflammation, and neuronal signaling. These signals converged with genetic causal risks of metabolic, neurological, and psychiatric disorders revealed in humans. Gene network modeling uncovered the extracellular matrix genes Bgn and Fmod as main orchestrators of the effects of fructose, as validated using two knockout mouse models. We further demonstrate that an omega-3 fatty acid, DHA, reverses the genomic and network perturbations elicited by fructose, providing molecular support for nutritional interventions to counteract diet-induced metabolic and brain disorders. Our integrative approach complementing rodent and human studies supports the applicability of nutrigenomics principles to predict disease susceptibility and to guide personalized medicine. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

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    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  16. Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

    Directory of Open Access Journals (Sweden)

    Dmitri Papatsenko

    2015-08-01

    Full Text Available Analyses of gene expression in single mouse embryonic stem cells (mESCs cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM. In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

  17. Whole genome transcript profiling of drug induced steatosis in rats reveals a gene signature predictive of outcome.

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    Nishika Sahini

    Full Text Available Drug induced steatosis (DIS is characterised by excess triglyceride accumulation in the form of lipid droplets (LD in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014. Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1. Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h and findings from rat hepatocyte studies (2 h, 8 h, 24 h identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21. Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.

  18. Genetic diversity and gene flow revealed by microsatellite DNA ...

    African Journals Online (AJOL)

    Dacryodes edulis is a multipurpose tree integrated in the cropping system of Central African region still dominated by subsistence agriculture. Some populations grown are wild which can provide information on the domestication process, and could also represent a potential source of gene flow. Leaves samples for DNA ...

  19. Comparative genome analysis of PHB gene family reveals deep evolutionary origins and diverse gene function.

    Science.gov (United States)

    Di, Chao; Xu, Wenying; Su, Zhen; Yuan, Joshua S

    2010-10-07

    PHB (Prohibitin) gene family is involved in a variety of functions important for different biological processes. PHB genes are ubiquitously present in divergent species from prokaryotes to eukaryotes. Human PHB genes have been found to be associated with various diseases. Recent studies by our group and others have shown diverse function of PHB genes in plants for development, senescence, defence, and others. Despite the importance of the PHB gene family, no comprehensive gene family analysis has been carried to evaluate the relatedness of PHB genes across different species. In order to better guide the gene function analysis and understand the evolution of the PHB gene family, we therefore carried out the comparative genome analysis of the PHB genes across different kingdoms. The relatedness, motif distribution, and intron/exon distribution all indicated that PHB genes is a relatively conserved gene family. The PHB genes can be classified into 5 classes and each class have a very deep evolutionary origin. The PHB genes within the class maintained the same motif patterns during the evolution. With Arabidopsis as the model species, we found that PHB gene intron/exon structure and domains are also conserved during the evolution. Despite being a conserved gene family, various gene duplication events led to the expansion of the PHB genes. Both segmental and tandem gene duplication were involved in Arabidopsis PHB gene family expansion. However, segmental duplication is predominant in Arabidopsis. Moreover, most of the duplicated genes experienced neofunctionalization. The results highlighted that PHB genes might be involved in important functions so that the duplicated genes are under the evolutionary pressure to derive new function. PHB gene family is a conserved gene family and accounts for diverse but important biological functions based on the similar molecular mechanisms. The highly diverse biological function indicated that more research needs to be carried out

  20. DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

    Directory of Open Access Journals (Sweden)

    Nikul Patel

    2012-01-01

    Full Text Available The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1, and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

  1. Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

    Directory of Open Access Journals (Sweden)

    Henry D Priest

    Full Text Available Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

  2. Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts.

    Science.gov (United States)

    Viñuelas, José; Kaneko, Gaël; Coulon, Antoine; Vallin, Elodie; Morin, Valérie; Mejia-Pous, Camila; Kupiec, Jean-Jacques; Beslon, Guillaume; Gandrillon, Olivier

    2013-02-25

    A number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells. For this purpose, we generated isogenic chicken-cell populations expressing a fluorescent reporter integrated in one copy per clone. Although the clones differed only in the genetic locus at which the reporter was inserted, they showed markedly different fluorescence distributions, revealing different levels of stochastic gene expression. Use of chromatin-modifying agents showed that direct manipulation of chromatin dynamics had a marked effect on the extent of stochastic gene expression. To better understand the molecular mechanism involved in these phenomena, we fitted these data to a two-state model describing the opening/closing process of the chromatin. We found that the differences between clones seemed to be due mainly to the duration of the closed state, and that the agents we used mainly seem to act on the opening probability. In this study, we report biological experiments combined with computational modeling, highlighting the importance of chromatin dynamics in stochastic gene expression. This work sheds a new light on the mechanisms of gene expression in higher eukaryotic cells, and argues in favor of relatively slow dynamics with long (hours to days) periods of quiet state.

  3. Matrix factorization reveals aging-specific co-expression gene modules in the fat and muscle tissues in nonhuman primates

    Science.gov (United States)

    Wang, Yongcui; Zhao, Weiling; Zhou, Xiaobo

    2016-10-01

    Accurate identification of coherent transcriptional modules (subnetworks) in adipose and muscle tissues is important for revealing the related mechanisms and co-regulated pathways involved in the development of aging-related diseases. Here, we proposed a systematically computational approach, called ICEGM, to Identify the Co-Expression Gene Modules through a novel mathematical framework of Higher-Order Generalized Singular Value Decomposition (HO-GSVD). ICEGM was applied on the adipose, and heart and skeletal muscle tissues in old and young female African green vervet monkeys. The genes associated with the development of inflammation, cardiovascular and skeletal disorder diseases, and cancer were revealed by the ICEGM. Meanwhile, genes in the ICEGM modules were also enriched in the adipocytes, smooth muscle cells, cardiac myocytes, and immune cells. Comprehensive disease annotation and canonical pathway analysis indicated that immune cells, adipocytes, cardiomyocytes, and smooth muscle cells played a synergistic role in cardiac and physical functions in the aged monkeys by regulation of the biological processes associated with metabolism, inflammation, and atherosclerosis. In conclusion, the ICEGM provides an efficiently systematic framework for decoding the co-expression gene modules in multiple tissues. Analysis of genes in the ICEGM module yielded important insights on the cooperative role of multiple tissues in the development of diseases.

  4. A systems level approach reveals new gene regulatory modules in the developing ear

    OpenAIRE

    Chen, Jingchen; Tambalo, Monica; Barembaum, Meyer; Ranganathan, Ramya; Simões-Costa, Marcos; Bronner, Marianne E.; Streit, Andrea

    2017-01-01

    The inner ear is a complex vertebrate sense organ, yet it arises from a simple epithelium, the otic placode. Specification towards otic fate requires diverse signals and transcriptional inputs that act sequentially and/or in parallel. Using the chick embryo, we uncover novel genes in the gene regulatory network underlying otic commitment and reveal dynamic changes in gene expression. Functional analysis of selected transcription factors reveals the genetic hierarchy underlying the transition ...

  5. Inertial picobalance reveals fast mass fluctuations in mammalian cells

    Science.gov (United States)

    Martínez-Martín, David; Fläschner, Gotthold; Gaub, Benjamin; Martin, Sascha; Newton, Richard; Beerli, Corina; Mercer, Jason; Gerber, Christoph; Müller, Daniel J.

    2017-10-01

    The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

  6. Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

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    Carole Bougault

    Full Text Available Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK pathways and Smad2/3 (members of the canonical transforming growth factor (TGF-β pathways. A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how

  7. Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.

    Directory of Open Access Journals (Sweden)

    Charles W Higdon

    Full Text Available In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.

  8. Revealing new mouse epicardial cell markers through transcriptomics.

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    Lars Bochmann

    2010-06-01

    Full Text Available The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1 that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.

  9. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    Science.gov (United States)

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  10. High-frequency microrheology reveals cytoskeleton dynamics in living cells

    Science.gov (United States)

    Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

    2017-08-01

    Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

  11. Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

    Directory of Open Access Journals (Sweden)

    Bin Tang

    Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

  12. Microarray analysis of gene expression alteration in human middle ear epithelial cells induced by micro particle.

    Science.gov (United States)

    Song, Jae-Jun; Kwon, Jee Young; Park, Moo Kyun; Seo, Young Rok

    2013-10-01

    The primary aim of this study is to reveal the effect of particulate matter (PM) on the human middle ear epithelial cell (HMEEC). The HMEEC was treated with PM (300 μg/ml) for 24 h. Total RNA was extracted and used for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed by using Pathway Studio 9.0 software. For selected genes, the changes in gene expression were confirmed by real-time PCR. A total of 611 genes were regulated by PM. Among them, 366 genes were up-regulated, whereas 245 genes were down-regulated. Up-regulated genes were mainly involved in cellular processes, including reactive oxygen species generation, cell proliferation, apoptosis, cell differentiation, inflammatory response and immune response. Down-regulated genes affected several cellular processes, including cell differentiation, cell cycle, proliferation, apoptosis and cell migration. A total of 21 genes were discovered as crucial components in potential signaling networks containing 2-fold up regulated genes. Four genes, VEGFA, IL1B, CSF2 and HMOX1 were revealed as key mediator genes among the up-regulated genes. A total of 25 genes were revealed as key modulators in the signaling pathway associated with 2-fold down regulated genes. Four genes, including IGF1R, TIMP1, IL6 and FN1, were identified as the main modulator genes. We identified the differentially expressed genes in PM-treated HMEEC, whose expression profile may provide a useful clue for the understanding of environmental pathophysiology of otitis media. Our work indicates that air pollution, like PM, plays an important role in the pathogenesis of otitis media. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    OpenAIRE

    Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

  14. Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

    OpenAIRE

    Zhou Qing; Plath Kathrin; Fan Guoping; Mason Mike J; Horvath Steve

    2009-01-01

    Abstract Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we...

  15. Estrogen-Responsive Genes Overlap with Triiodothyronine-Responsive Genes in a Breast Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Nancy Bueno Figueiredo

    2014-01-01

    Full Text Available It has been well established that estrogen plays an important role in the progression and treatment of breast cancer. However, the role of triiodothyronine (T3 remains controversial. We have previously shown its capacity to stimulate the development of positive estrogen receptor breast carcinoma, induce the expression of genes (PR, TGF-alpha normally stimulated by estradiol (E2, and suppress genes (TGF-beta normally inhibited by E2. Since T3 regulates growth hormones, metabolism, and differentiation, it is important to verify its action on other genes normally induced by E2. Therefore, we used DNA microarrays to compare gene expression patterns in MCF-7 breast adenocarcinoma cells treated with E2 and T3. Several genes were modulated by both E2 and T3 in MCF-7 cells (Student’s t-test, P 2.0, pFDR < 0.05. We confirmed our microarray results by real-time PCR. Our findings reveal that certain genes in MCF-7 cells can be regulated by both E2 and T3.

  16. Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control.

    Science.gov (United States)

    Katayama, Shintaro; Skoog, Tiina; Jouhilahti, Eeva-Mari; Siitonen, H Annika; Nuutila, Kristo; Tervaniemi, Mari H; Vuola, Jyrki; Johnsson, Anna; Lönnerberg, Peter; Linnarsson, Sten; Elomaa, Outi; Kankuri, Esko; Kere, Juha

    2015-06-25

    Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted. We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.

  17. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  18. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells

    NARCIS (Netherlands)

    Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

    2017-01-01

    Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression

  19. DNA capture reveals transoceanic gene flow in endangered river sharks.

    Science.gov (United States)

    Li, Chenhong; Corrigan, Shannon; Yang, Lei; Straube, Nicolas; Harris, Mark; Hofreiter, Michael; White, William T; Naylor, Gavin J P

    2015-10-27

    For over a hundred years, the "river sharks" of the genus Glyphis were only known from the type specimens of species that had been collected in the 19th century. They were widely considered extinct until populations of Glyphis-like sharks were rediscovered in remote regions of Borneo and Northern Australia at the end of the 20th century. However, the genetic affinities between the newly discovered Glyphis-like populations and the poorly preserved, original museum-type specimens have never been established. Here, we present the first (to our knowledge) fully resolved, complete phylogeny of Glyphis that includes both archival-type specimens and modern material. We used a sensitive DNA hybridization capture method to obtain complete mitochondrial genomes from all of our samples and show that three of the five described river shark species are probably conspecific and widely distributed in Southeast Asia. Furthermore we show that there has been recent gene flow between locations that are separated by large oceanic expanses. Our data strongly suggest marine dispersal in these species, overturning the widely held notion that river sharks are restricted to freshwater. It seems that species in the genus Glyphis are euryhaline with an ecology similar to the bull shark, in which adult individuals live in the ocean while the young grow up in river habitats with reduced predation pressure. Finally, we discovered a previously unidentified species within the genus Glyphis that is deeply divergent from all other lineages, underscoring the current lack of knowledge about the biodiversity and ecology of these mysterious sharks.

  20. Mel-18, a mammalian Polycomb gene, regulates angiogenic gene expression of endothelial cells.

    Science.gov (United States)

    Jung, Ji-Hye; Choi, Hyun-Jung; Maeng, Yong-Sun; Choi, Jung-Yeon; Kim, Minhyung; Kwon, Ja-Young; Park, Yong-Won; Kim, Young-Myeong; Hwang, Daehee; Kwon, Young-Guen

    2010-10-01

    Mel-18 is a mammalian homolog of Polycomb group (PcG) genes. Microarray analysis revealed that Mel-18 expression was induced during endothelial progenitor cell (EPC) differentiation and correlates with the expression of EC-specific protein markers. Overexpression of Mel-18 promoted EPC differentiation and angiogenic activity of ECs. Accordingly, silencing Mel-18 inhibited EC migration and tube formation in vitro. Gene expression profiling showed that Mel-18 regulates angiogenic genes including kinase insert domain receptor (KDR), claudin 5, and angiopoietin-like 2. Our findings demonstrate, for the first time, that Mel-18 plays a significant role in the angiogenic function of ECs by regulating endothelial gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding

    NARCIS (Netherlands)

    Goeij, de J.M.; Kluijver, de A.; Duyl, van F.C.; Vacelet, J.; Wijffels, R.H.; Goeij, de A.F.P.M.; Cleutjens, J.P.M.; Schutte, B.

    2009-01-01

    This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is

  2. Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

    Science.gov (United States)

    Ding, Mingquan; Jiang, Yurong; Cao, Yuefen; Lin, Lifeng; He, Shae; Zhou, Wei; Rong, Junkang

    2014-02-10

    Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Constraints on genome dynamics revealed from gene distribution among the Ralstonia solanacearum species.

    Directory of Open Access Journals (Sweden)

    Pierre Lefeuvre

    Full Text Available Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens.

  4. Identification of Cell Cycle-Regulated Genes by Convolutional Neural Network.

    Science.gov (United States)

    Liu, Chenglin; Cui, Peng; Huang, Tao

    2017-01-01

    The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype are analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight to the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Global gene expression analysis of the zoonotic parasite Trichinella spiralis revealed novel genes in host parasite interaction.

    Directory of Open Access Journals (Sweden)

    Xiaolei Liu

    Full Text Available BACKGROUND: Trichinellosis is a typical food-borne zoonotic disease which is epidemic worldwide and the nematode Trichinella spiralis is the main pathogen. The life cycle of T. spiralis contains three developmental stages, i.e. adult worms, new borne larva (new borne L1 larva and muscular larva (infective L1 larva. Stage-specific gene expression in the parasites has been investigated with various immunological and cDNA cloning approaches, whereas the genome-wide transcriptome and expression features of the parasite have been largely unknown. The availability of the genome sequence information of T. spiralis has made it possible to deeply dissect parasite biology in association with global gene expression and pathogenesis. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we analyzed the global gene expression patterns in the three developmental stages of T. spiralis using digital gene expression (DGE analysis. Almost 15 million sequence tags were generated with the Illumina RNA-seq technology, producing expression data for more than 9,000 genes, covering 65% of the genome. The transcriptome analysis revealed thousands of differentially expressed genes within the genome, and importantly, a panel of genes encoding functional proteins associated with parasite invasion and immuno-modulation were identified. More than 45% of the genes were found to be transcribed from both strands, indicating the importance of RNA-mediated gene regulation in the development of the parasite. Further, based on gene ontological analysis, over 3000 genes were functionally categorized and biological pathways in the three life cycle stage were elucidated. CONCLUSIONS AND SIGNIFICANCE: The global transcriptome of T. spiralis in three developmental stages has been profiled, and most gene activity in the genome was found to be developmentally regulated. Many metabolic and biological pathways have been revealed. The findings of the differential expression of several protein

  6. Repression of the albumin gene in Novikoff hepatoma cells

    International Nuclear Information System (INIS)

    Capetanaki, Y.G.; Flytzanis, C.N.; Alonso, A.

    1982-01-01

    Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [/sup 32/P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements

  7. Mass Spectrometry Reveals Changes in MHC I Antigen Presentation After Lentivector Expression of a Gene Regulation System

    Directory of Open Access Journals (Sweden)

    Roland Vogel

    2013-01-01

    Full Text Available The rapamycin-inducible gene regulation system was designed to minimize immune reactions in man and may thus be suited for gene therapy. We assessed whether this system indeed induces no immune responses. The protein components of the regulation system were produced in the human cell lines HEK 293T, D407, and HER 911 following lentiviral transfer of the corresponding genes. Stable cell lines were established, and the peptides presented by major histocompatibility complex class I (MHC I molecules on transduced and wild-type (wt cells were compared by differential mass spectrometry. In all cell lines examined, expression of the transgenes resulted in prominent changes in the repertoire of MHC I-presented self-peptides. No MHC I ligands originating from the transgenic proteins were detected. In vitro analysis of immunogenicity revealed that transduced D407 cells displayed slightly higher capacity than wt controls to promote proliferation of cytotoxic T cells. These results indicate that therapeutic manipulations within the genome of target cells may affect pathways involved in the processing of peptide antigens and their presentation by MHC I. This makes the genomic modifications visible to the immune system which may recognize these events and respond. Ultimately, the findings call attention to a possible immune risk.

  8. Cell cycle and apoptosis genes in atherosclerosis

    NARCIS (Netherlands)

    Boesten, Lianne Simone Mirjam

    2006-01-01

    The work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder occurring in the large and medium-sized arteries of the body. Although in the beginning 90s promising

  9. Gene expression profiles reveal key genes for early diagnosis and treatment of adamantinomatous craniopharyngioma.

    Science.gov (United States)

    Yang, Jun; Hou, Ziming; Wang, Changjiang; Wang, Hao; Zhang, Hongbing

    2018-04-23

    Adamantinomatous craniopharyngioma (ACP) is an aggressive brain tumor that occurs predominantly in the pediatric population. Conventional diagnosis method and standard therapy cannot treat ACPs effectively. In this paper, we aimed to identify key genes for ACP early diagnosis and treatment. Datasets GSE94349 and GSE68015 were obtained from Gene Expression Omnibus database. Consensus clustering was applied to discover the gene clusters in the expression data of GSE94349 and functional enrichment analysis was performed on gene set in each cluster. The protein-protein interaction (PPI) network was built by the Search Tool for the Retrieval of Interacting Genes, and hubs were selected. Support vector machine (SVM) model was built based on the signature genes identified from enrichment analysis and PPI network. Dataset GSE94349 was used for training and testing, and GSE68015 was used for validation. Besides, RT-qPCR analysis was performed to analyze the expression of signature genes in ACP samples compared with normal controls. Seven gene clusters were discovered in the differentially expressed genes identified from GSE94349 dataset. Enrichment analysis of each cluster identified 25 pathways that highly associated with ACP. PPI network was built and 46 hubs were determined. Twenty-five pathway-related genes that overlapped with the hubs in PPI network were used as signatures to establish the SVM diagnosis model for ACP. The prediction accuracy of SVM model for training, testing, and validation data were 94, 85, and 74%, respectively. The expression of CDH1, CCL2, ITGA2, COL8A1, COL6A2, and COL6A3 were significantly upregulated in ACP tumor samples, while CAMK2A, RIMS1, NEFL, SYT1, and STX1A were significantly downregulated, which were consistent with the differentially expressed gene analysis. SVM model is a promising classification tool for screening and early diagnosis of ACP. The ACP-related pathways and signature genes will advance our knowledge of ACP pathogenesis

  10. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

    Directory of Open Access Journals (Sweden)

    Yonglong Yu

    2016-04-01

    Full Text Available Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20 during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

  11. Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell profileration and inhibition of PPARγ signaling as potential mechanism for the negative relationships between immune response and lipid metabolism

    DEFF Research Database (Denmark)

    Moyes, Kasey M; Drackley, James K; Morin, Dawn E

    2009-01-01

    Background Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decre......Background Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare....../or metabolic responses to mastitis challenge with S. uberis O140J. Results Streptococcus uberis IMI resulted in 2,102 (1,939 annotated) differentially expressed genes (DEG). Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each), the majority of which were...

  12. The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

    Science.gov (United States)

    Fabry, S; Müller, K; Lindauer, A; Park, P B; Cornelius, T; Schmitt, R

    1995-09-01

    The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II

  13. Transcriptomics reveal several gene expression patterns in the piezophile Desulfovibrio hydrothermalis in response to hydrostatic pressure.

    Directory of Open Access Journals (Sweden)

    Amira Amrani

    Full Text Available RNA-seq was used to study the response of Desulfovibrio hydrothermalis, isolated from a deep-sea hydrothermal chimney on the East-Pacific Rise at a depth of 2,600 m, to various hydrostatic pressure growth conditions. The transcriptomic datasets obtained after growth at 26, 10 and 0.1 MPa identified only 65 differentially expressed genes that were distributed among four main categories: aromatic amino acid and glutamate metabolisms, energy metabolism, signal transduction, and unknown function. The gene expression patterns suggest that D. hydrothermalis uses at least three different adaptation mechanisms, according to a hydrostatic pressure threshold (HPt that was estimated to be above 10 MPa. Both glutamate and energy metabolism were found to play crucial roles in these mechanisms. Quantitation of the glutamate levels in cells revealed its accumulation at high hydrostatic pressure, suggesting its role as a piezolyte. ATP measurements showed that the energy metabolism of this bacterium is optimized for deep-sea life conditions. This study provides new insights into the molecular mechanisms linked to hydrostatic pressure adaptation in sulfate-reducing bacteria.

  14. Transcriptomics Reveal Several Gene Expression Patterns in the Piezophile Desulfovibrio hydrothermalis in Response to Hydrostatic Pressure

    Science.gov (United States)

    Amrani, Amira; Bergon, Aurélie; Holota, Hélène; Tamburini, Christian; Garel, Marc; Ollivier, Bernard; Imbert, Jean; Dolla, Alain; Pradel, Nathalie

    2014-01-01

    RNA-seq was used to study the response of Desulfovibrio hydrothermalis, isolated from a deep-sea hydrothermal chimney on the East-Pacific Rise at a depth of 2,600 m, to various hydrostatic pressure growth conditions. The transcriptomic datasets obtained after growth at 26, 10 and 0.1 MPa identified only 65 differentially expressed genes that were distributed among four main categories: aromatic amino acid and glutamate metabolisms, energy metabolism, signal transduction, and unknown function. The gene expression patterns suggest that D. hydrothermalis uses at least three different adaptation mechanisms, according to a hydrostatic pressure threshold (HPt) that was estimated to be above 10 MPa. Both glutamate and energy metabolism were found to play crucial roles in these mechanisms. Quantitation of the glutamate levels in cells revealed its accumulation at high hydrostatic pressure, suggesting its role as a piezolyte. ATP measurements showed that the energy metabolism of this bacterium is optimized for deep-sea life conditions. This study provides new insights into the molecular mechanisms linked to hydrostatic pressure adaptation in sulfate-reducing bacteria. PMID:25215865

  15. Opto-acoustic microscopy reveals adhesion mechanics of single cells

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  16. Genes involved in cell division in mycoplasmas

    Directory of Open Access Journals (Sweden)

    Frank Alarcón

    2007-01-01

    Full Text Available Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

  17. Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate.

    Directory of Open Access Journals (Sweden)

    Heike Hadrys

    Full Text Available Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera. We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.

  18. Prediction of epigenetically regulated genes in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  19. Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage.

    Science.gov (United States)

    Hilton, Matthew J; Tu, Xiaolin; Long, Fanxin

    2007-08-01

    Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.

  20. Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Shan Goh

    Full Text Available BACKGROUND: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development. METHODOLOGY/PRINCIPAL FINDINGS: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50. When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50 values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required. CONCLUSIONS: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

  1. Recent adaptive events in human brain revealed by meta-analysis of positively selected genes.

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    Yue Huang

    Full Text Available BACKGROUND AND OBJECTIVES: Analysis of positively-selected genes can help us understand how human evolved, especially the evolution of highly developed cognitive functions. However, previous works have reached conflicting conclusions regarding whether human neuronal genes are over-represented among genes under positive selection. METHODS AND RESULTS: We divided positively-selected genes into four groups according to the identification approaches, compiling a comprehensive list from 27 previous studies. We showed that genes that are highly expressed in the central nervous system are enriched in recent positive selection events in human history identified by intra-species genomic scan, especially in brain regions related to cognitive functions. This pattern holds when different datasets, parameters and analysis pipelines were used. Functional category enrichment analysis supported these findings, showing that synapse-related functions are enriched in genes under recent positive selection. In contrast, immune-related functions, for instance, are enriched in genes under ancient positive selection revealed by inter-species coding region comparison. We further demonstrated that most of these patterns still hold even after controlling for genomic characteristics that might bias genome-wide identification of positively-selected genes including gene length, gene density, GC composition, and intensity of negative selection. CONCLUSION: Our rigorous analysis resolved previous conflicting conclusions and revealed recent adaptation of human brain functions.

  2. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  3. New insight on FGFR3-related chondrodysplasias molecular physiopathology revealed by human chondrocyte gene expression profiling.

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    Laurent Schibler

    Full Text Available Endochondral ossification is the process by which the appendicular skeleton, facial bones, vertebrae and medial clavicles are formed and relies on the tight control of chondrocyte maturation. Fibroblast growth factor receptor (FGFR3 plays a role in bone development and maintenance and belongs to a family of proteins which differ in their ligand affinities and tissue distribution. Activating mutations of the FGFR3 gene lead to craniosynostosis and multiple types of skeletal dysplasia with varying degrees of severity: thanatophoric dysplasia (TD, achondroplasia and hypochondroplasia. Despite progress in the characterization of FGFR3-mediated regulation of cartilage development, many aspects remain unclear. The aim and the novelty of our study was to examine whole gene expression differences occurring in primary human chondrocytes isolated from normal cartilage or pathological cartilage from TD-affected fetuses, using Affymetrix technology. The phenotype of the primary cells was confirmed by the high expression of chondrocytic markers. Altered expression of genes associated with many cellular processes was observed, including cell growth and proliferation, cell cycle, cell adhesion, cell motility, metabolic pathways, signal transduction, cell cycle process and cell signaling. Most of the cell cycle process genes were down-regulated and consisted of genes involved in cell cycle progression, DNA biosynthesis, spindle dynamics and cytokinesis. About eight percent of all modulated genes were found to impact extracellular matrix (ECM structure and turnover, especially glycosaminoglycan (GAG and proteoglycan biosynthesis and sulfation. Altogether, the gene expression analyses provide new insight into the consequences of FGFR3 mutations in cell cycle regulation, onset of pre-hypertrophic differentiation and concomitant metabolism changes. Moreover, impaired motility and ECM properties may also provide clues about growth plate disorganization. These

  4. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  5. Acquisition of docetaxel resistance in breast cancer cells reveals upregulation of ABCB1 expression as a key mediator of resistance accompanied by discrete upregulation of other specific genes and pathways

    DEFF Research Database (Denmark)

    Ninel Hansen, Stine; Westergaard, David; Borg Houlberg Thomsen, Mathilde

    2015-01-01

    The microtubule-targeting taxanes are important in breast cancer therapy, but no predictive biomarkers have yet been identified with sufficient scientific evidence to allow clinical routine use. The purposes of the present study were to develop a cell-culture-based discovery platform for docetaxel...

  6. Systematic Prioritization and Integrative Analysis of Copy Number Variations in Schizophrenia Reveal Key Schizophrenia Susceptibility Genes

    Science.gov (United States)

    Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

    2014-01-01

    Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and

  7. Gene expression profiles reveal key pathways and genes associated with neuropathic pain in patients with spinal cord injury.

    Science.gov (United States)

    He, Xijing; Fan, Liying; Wu, Zhongheng; He, Jiaxuan; Cheng, Bin

    2017-04-01

    Previous gene expression profiling studies of neuropathic pain (NP) following spinal cord injury (SCI) have predominantly been performed in animal models. The present study aimed to investigate gene alterations in patients with spinal cord injury and to further examine the mechanisms underlying NP following SCI. The GSE69901 gene expression profile was downloaded from the public Gene Expression Omnibus database. Samples of peripheral blood mononuclear cells (PBMCs) derived from 12 patients with intractable NP and 13 control patients without pain were analyzed to identify the differentially expressed genes (DEGs), followed by functional enrichment analysis and protein‑protein interaction (PPI) network construction. In addition, a transcriptional regulation network was constructed and functional gene clustering was performed. A total of 70 upregulated and 61 downregulated DEGs were identified in the PBMC samples from patients with NP. The upregulated and downregulated genes were significantly involved in different Gene Ontology terms and pathways, including focal adhesion, T cell receptor signaling pathway and mitochondrial function. Glycogen synthase kinase 3 β (GSK3B) was identified as a hub protein in the PPI network. In addition, ornithine decarboxylase 1 (ODC1) and ornithine aminotransferase (OAT) were regulated by additional transcription factors in the regulation network. GSK3B, OAT and ODC1 were significantly enriched in two functional gene clusters, the function of mitochondrial membrane and DNA binding. Focal adhesion and the T cell receptor signaling pathway may be significantly linked with NP, and GSK3B, OAT and ODC1 may be potential targets for the treatment of NP.

  8. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

  9. Cross-species transcriptomic approach reveals genes in hamster implantation sites.

    Science.gov (United States)

    Lei, Wei; Herington, Jennifer; Galindo, Cristi L; Ding, Tianbing; Brown, Naoko; Reese, Jeff; Paria, Bibhash C

    2014-12-01

    The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS. © 2014 Society for Reproduction and Fertility.

  10. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

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    Liang-Hui Chu

    Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

  11. Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum.

    Science.gov (United States)

    Rao, Soumya; Nandineni, Madhusudan R

    2017-01-01

    Colletotrichum truncatum, a major fungal phytopathogen, causes the anthracnose disease on an economically important spice crop chilli (Capsicum annuum), resulting in huge economic losses in tropical and sub-tropical countries. It follows a subcuticular intramural infection strategy on chilli with a short, asymptomatic, endophytic phase, which contrasts with the intracellular hemibiotrophic lifestyle adopted by most of the Colletotrichum species. However, little is known about the molecular determinants and the mechanism of pathogenicity in this fungus. A high quality whole genome sequence and gene annotation based on transcriptome data of an Indian isolate of C. truncatum from chilli has been obtained. Analysis of the genome sequence revealed a rich repertoire of pathogenicity genes in C. truncatum encoding secreted proteins, effectors, plant cell wall degrading enzymes, secondary metabolism associated proteins, with potential roles in the host-specific infection strategy, placing it next only to the Fusarium species. The size of genome assembly, number of predicted genes and some of the functional categories were similar to other sequenced Colletotrichum species. The comparative genomic analyses with other species and related fungi identified some unique genes and certain highly expanded gene families of CAZymes, proteases and secondary metabolism associated genes in the genome of C. truncatum. The draft genome assembly and functional annotation of potential pathogenicity genes of C. truncatum provide an important genomic resource for understanding the biology and lifestyle of this important phytopathogen and will pave the way for designing efficient disease control regimens.

  12. Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

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    Qiang Zhang

    Full Text Available The Nkrp1 (Klrb1-Clr (Clec2 genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b, Nkrp1c (Klrb1c, and Clr-c (Clec2f genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d and Clr-g (Clec2i showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells, as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

  13. Gene expression profiling of mucolipidosis type IV fibroblasts reveals deregulation of genes with relevant functions in lysosome physiology.

    Science.gov (United States)

    Bozzato, Andrea; Barlati, Sergio; Borsani, Giuseppe

    2008-04-01

    Mucolipidosis type IV (MLIV, MIM 252650) is an autosomal recessive lysosomal storage disorder that causes mental and motor retardation as well as visual impairment. The lysosomal storage defect in MLIV is consistent with abnormalities of membrane traffic and organelle dynamics in the late endocytic pathway. MLIV is caused by mutations in the MCOLN1 gene, which codes for mucolipin-1 (MLN1), a member of the large family of transient receptor potential (TRP) cation channels. Although a number of studies have been performed on mucolipin-1, the pathological mechanisms underlying MLIV are not fully understood. To identify genes that characterize pathogenic changes in mucolipidosis type IV, we compared the expression profiles of three MLIV and three normal skin fibroblasts cell lines using oligonucleotide microarrays. Genes that were differentially expressed in patients' cells were identified. 231 genes were up-regulated, and 116 down-regulated. Real-Time RT-PCR performed on selected genes in six independent MLIV fibroblasts cell lines was generally consistent with the microarray findings. This study allowed to evidence the modulation at the transcriptional level of a discrete number of genes relevant in biological processes which are altered in the disease such as endosome/lysosome trafficking, lysosome biogenesis, organelle acidification and lipid metabolism.

  14. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

    Science.gov (United States)

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

    2016-07-15

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. © 2016 Sato et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Identification of horizontally transferred genes in the genus Colletotrichum reveals a steady tempo of bacterial to fungal gene transfer.

    Science.gov (United States)

    Jaramillo, Vinicio D Armijos; Sukno, Serenella A; Thon, Michael R

    2015-01-02

    Horizontal gene transfer (HGT) is the stable transmission of genetic material between organisms by means other than vertical inheritance. HGT has an important role in the evolution of prokaryotes but is relatively rare in eukaryotes. HGT has been shown to contribute to virulence in eukaryotic pathogens. We studied the importance of HGT in plant pathogenic fungi by identifying horizontally transferred genes in the genomes of three members of the genus Colletotrichum. We identified eleven HGT events from bacteria into members of the genus Colletotrichum or their ancestors. The HGT events include genes involved in amino acid, lipid and sugar metabolism as well as lytic enzymes. Additionally, the putative minimal dates of transference were calculated using a time calibrated phylogenetic tree. This analysis reveals a constant flux of genes from bacteria to fungi throughout the evolution of subphylum Pezizomycotina. Genes that are typically transferred by HGT are those that are constantly subject to gene duplication and gene loss. The functions of some of these genes suggest roles in niche adaptation and virulence. We found no evidence of a burst of HGT events coinciding with major geological events. In contrast, HGT appears to be a constant, albeit rare phenomenon in the Pezizomycotina, occurring at a steady rate during their evolution.

  16. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  17. Gene expression profiling in equine polysaccharide storage myopathy revealed inflammation, glycogenesis inhibition, hypoxia and mitochondrial dysfunctions

    Directory of Open Access Journals (Sweden)

    Benech Philippe

    2009-08-01

    Full Text Available Abstract Background Several cases of myopathies have been observed in the horse Norman Cob breed. Muscle histology examinations revealed that some families suffer from a polysaccharide storage myopathy (PSSM. It is assumed that a gene expression signature related to PSSM should be observed at the transcriptional level because the glycogen storage disease could also be linked to other dysfunctions in gene regulation. Thus, the functional genomic approach could be conducted in order to provide new knowledge about the metabolic disorders related to PSSM. We propose exploring the PSSM muscle fiber metabolic disorders by measuring gene expression in relationship with the histological phenotype. Results Genotypying analysis of GYS1 mutation revealed 2 homozygous (AA and 5 heterozygous (GA PSSM horses. In the PSSM muscles, histological data revealed PAS positive amylase resistant abnormal polysaccharides, inflammation, necrosis, and lipomatosis and active regeneration of fibers. Ultrastructural evaluation revealed a decrease of mitochondrial number and structural disorders. Extensive accumulation of an abnormal polysaccharide displaced and partially replaced mitochondria and myofibrils. The severity of the disease was higher in the two homozygous PSSM horses. Gene expression analysis revealed 129 genes significantly modulated (p Conclusion The main disorders observed in PSSM muscles could be related to mitochondrial dysfunctions, glycogenesis inhibition and the chronic hypoxia of the PSSM muscles.

  18. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

    Science.gov (United States)

    Lin, Yihan; Li, Ying; Crosson, Sean; Dinner, Aaron R; Scherer, Norbert F

    2012-01-01

    Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS).

  19. Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

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    Keerthi Krishnan

    Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

  20. Opto-acoustic microscopy reveals adhesion mechanics of single cells.

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Z c , as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZ c reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, K m , that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, S r /S t . We show that K m can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while S r /S t is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  1. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

    Science.gov (United States)

    Schoggins, John W.; MacDuff, Donna A.; Imanaka, Naoko; Gainey, Maria D.; Shrestha, Bimmi; Eitson, Jennifer L.; Mar, Katrina B.; Richardson, R. Blake; Ratushny, Alexander V.; Litvak, Vladimir; Dabelic, Rea; Manicassamy, Balaji; Aitchison, John D.; Aderem, Alan; Elliott, Richard M.; García-Sastre, Adolfo; Racaniello, Vincent; Snijder, Eric J.; Yokoyama, Wayne M.; Diamond, Michael S.; Virgin, Herbert W.; Rice, Charles M.

    2014-01-01

    The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

  2. Optimizing autologous cell grafts to improve stem cell gene therapy.

    Science.gov (United States)

    Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia

    2016-07-01

    Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  3. QTL mapping and transcriptome analysis of cowpea reveals candidate genes for root-knot nematode resistance.

    Science.gov (United States)

    Santos, Jansen Rodrigo Pereira; Ndeve, Arsenio Daniel; Huynh, Bao-Lam; Matthews, William Charles; Roberts, Philip Alan

    2018-01-01

    Cowpea is one of the most important food and forage legumes in drier regions of the tropics and subtropics. However, cowpea yield worldwide is markedly below the known potential due to abiotic and biotic stresses, including parasitism by root-knot nematodes (Meloidogyne spp., RKN). Two resistance genes with dominant effect, Rk and Rk2, have been reported to provide resistance against RKN in cowpea. Despite their description and use in breeding for resistance to RKN and particularly genetic mapping of the Rk locus, the exact genes conferring resistance to RKN remain unknown. In the present work, QTL mapping using recombinant inbred line (RIL) population 524B x IT84S-2049 segregating for a newly mapped locus and analysis of the transcriptome changes in two cowpea near-isogenic lines (NIL) were used to identify candidate genes for Rk and the newly mapped locus. A major QTL, designated QRk-vu9.1, associated with resistance to Meloidogyne javanica reproduction, was detected and mapped on linkage group LG9 at position 13.37 cM using egg production data. Transcriptome analysis on resistant and susceptible NILs 3 and 9 days after inoculation revealed up-regulation of 109 and 98 genes and down-regulation of 110 and 89 genes, respectively, out of 19,922 unique genes mapped to the common bean reference genome. Among the differentially expressed genes, four and nine genes were found within the QRk-vu9.1 and QRk-vu11.1 QTL intervals, respectively. Six of these genes belong to the TIR-NBS-LRR family of resistance genes and three were upregulated at one or more time-points. Quantitative RT-PCR validated gene expression to be positively correlated with RNA-seq expression pattern for eight genes. Future functional analysis of these cowpea genes will enhance our understanding of Rk-mediated resistance and identify the specific gene responsible for the resistance.

  4. QTL mapping and transcriptome analysis of cowpea reveals candidate genes for root-knot nematode resistance.

    Directory of Open Access Journals (Sweden)

    Jansen Rodrigo Pereira Santos

    Full Text Available Cowpea is one of the most important food and forage legumes in drier regions of the tropics and subtropics. However, cowpea yield worldwide is markedly below the known potential due to abiotic and biotic stresses, including parasitism by root-knot nematodes (Meloidogyne spp., RKN. Two resistance genes with dominant effect, Rk and Rk2, have been reported to provide resistance against RKN in cowpea. Despite their description and use in breeding for resistance to RKN and particularly genetic mapping of the Rk locus, the exact genes conferring resistance to RKN remain unknown. In the present work, QTL mapping using recombinant inbred line (RIL population 524B x IT84S-2049 segregating for a newly mapped locus and analysis of the transcriptome changes in two cowpea near-isogenic lines (NIL were used to identify candidate genes for Rk and the newly mapped locus. A major QTL, designated QRk-vu9.1, associated with resistance to Meloidogyne javanica reproduction, was detected and mapped on linkage group LG9 at position 13.37 cM using egg production data. Transcriptome analysis on resistant and susceptible NILs 3 and 9 days after inoculation revealed up-regulation of 109 and 98 genes and down-regulation of 110 and 89 genes, respectively, out of 19,922 unique genes mapped to the common bean reference genome. Among the differentially expressed genes, four and nine genes were found within the QRk-vu9.1 and QRk-vu11.1 QTL intervals, respectively. Six of these genes belong to the TIR-NBS-LRR family of resistance genes and three were upregulated at one or more time-points. Quantitative RT-PCR validated gene expression to be positively correlated with RNA-seq expression pattern for eight genes. Future functional analysis of these cowpea genes will enhance our understanding of Rk-mediated resistance and identify the specific gene responsible for the resistance.

  5. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

    Directory of Open Access Journals (Sweden)

    B Alex Merrick

    Full Text Available Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1, a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL. Paired-end reads were mapped to the rat genome (Rn4 with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005 compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c

  6. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

    Science.gov (United States)

    Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

    2013-01-01

    Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the

  7. Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

    Science.gov (United States)

    Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

    2003-08-14

    The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

  8. Global gene expression response to telomerase in bovine adrenocortical cells

    International Nuclear Information System (INIS)

    Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H.

    2005-01-01

    The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state

  9. Iron homeostasis in Arabidopsis thaliana: transcriptomic analyses reveal novel FIT-regulated genes, iron deficiency marker genes and functional gene networks.

    Science.gov (United States)

    Mai, Hans-Jörg; Pateyron, Stéphanie; Bauer, Petra

    2016-10-03

    FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is the central regulator of iron uptake in Arabidopsis thaliana roots. We performed transcriptome analyses of six day-old seedlings and roots of six week-old plants using wild type, a fit knock-out mutant and a FIT over-expression line grown under iron-sufficient or iron-deficient conditions. We compared genes regulated in a FIT-dependent manner depending on the developmental stage of the plants. We assembled a high likelihood dataset which we used to perform co-expression and functional analysis of the most stably iron deficiency-induced genes. 448 genes were found FIT-regulated. Out of these, 34 genes were robustly FIT-regulated in root and seedling samples and included 13 novel FIT-dependent genes. Three hundred thirty-one genes showed differential regulation in response to the presence and absence of FIT only in the root samples, while this was the case for 83 genes in the seedling samples. We assembled a virtual dataset of iron-regulated genes based on a total of 14 transcriptomic analyses of iron-deficient and iron-sufficient wild-type plants to pinpoint the best marker genes for iron deficiency and analyzed this dataset in depth. Co-expression analysis of this dataset revealed 13 distinct regulons part of which predominantly contained functionally related genes. We could enlarge the list of FIT-dependent genes and discriminate between genes that are robustly FIT-regulated in roots and seedlings or only in one of those. FIT-regulated genes were mostly induced, few of them were repressed by FIT. With the analysis of a virtual dataset we could filter out and pinpoint new candidates among the most reliable marker genes for iron deficiency. Moreover, co-expression and functional analysis of this virtual dataset revealed iron deficiency-induced and functionally distinct regulons.

  10. Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility

    Science.gov (United States)

    Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

    2014-01-01

    A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis. PMID:24997565

  11. Gene expression profiling reveals candidate genes related to residual feed intake in duodenum of laying ducks.

    Science.gov (United States)

    Zeng, T; Huang, L; Ren, J; Chen, L; Tian, Y; Huang, Y; Zhang, H; Du, J; Lu, L

    2017-12-01

    Feed represents two-thirds of the total costs of poultry production, especially in developing countries. Improvement in feed efficiency would reduce the amount of feed required for production (growth or laying), the production cost, and the amount of nitrogenous waste. The most commonly used measures for feed efficiency are feed conversion ratio (FCR) and residual feed intake (RFI). As a more suitable indicator assessing feed efficiency, RFI is defined as the difference between observed and expected feed intake based on maintenance and growth or laying. However, the genetic and biological mechanisms regulating RFI are largely unknown. Identifying molecular mechanisms explaining divergence in RFI in laying ducks would lead to the development of early detection methods for the selection of more efficient breeding poultry. The objective of this study was to identify duodenum genes and pathways through transcriptional profiling in 2 extreme RFI phenotypes (HRFI and LRFI) of the duck population. Phenotypic aspects of feed efficiency showed that RFI was strongly positive with FCR and feed intake (FI). Transcriptomic analysis identified 35 differentially expressed genes between LRFI and HRFI ducks. These genes play an important role in metabolism, digestibility, secretion, and innate immunity including (), (), (), β (), and (). These results improve our knowledge of the biological basis underlying RFI, which would be useful for further investigations of key candidate genes for RFI and for the development of biomarkers.

  12. Global Analysis of miRNA Gene Clusters and Gene Families Reveals Dynamic and Coordinated Expression

    Directory of Open Access Journals (Sweden)

    Li Guo

    2014-01-01

    Full Text Available To further understand the potential expression relationships of miRNAs in miRNA gene clusters and gene families, a global analysis was performed in 4 paired tumor (breast cancer and adjacent normal tissue samples using deep sequencing datasets. The compositions of miRNA gene clusters and families are not random, and clustered and homologous miRNAs may have close relationships with overlapped miRNA species. Members in the miRNA group always had various expression levels, and even some showed larger expression divergence. Despite the dynamic expression as well as individual difference, these miRNAs always indicated consistent or similar deregulation patterns. The consistent deregulation expression may contribute to dynamic and coordinated interaction between different miRNAs in regulatory network. Further, we found that those clustered or homologous miRNAs that were also identified as sense and antisense miRNAs showed larger expression divergence. miRNA gene clusters and families indicated important biological roles, and the specific distribution and expression further enrich and ensure the flexible and robust regulatory network.

  13. Gene Expression Correlated with Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease.

    Science.gov (United States)

    Modena, Brian D; Bleecker, Eugene R; Busse, William W; Erzurum, Serpil C; Gaston, Benjamin M; Jarjour, Nizar N; Meyers, Deborah A; Milosevic, Jadranka; Tedrow, John R; Wu, Wei; Kaminski, Naftali; Wenzel, Sally E

    2017-06-01

    Severe asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals. Identify networks of genes reflective of underlying biological processes that define SA. Airway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders. Weighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12-21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA. In this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes.

  14. Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

    Directory of Open Access Journals (Sweden)

    Davey Jennifer C

    2007-12-01

    Full Text Available Abstract Background Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT. The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for

  15. Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

    Directory of Open Access Journals (Sweden)

    Kahlem Pascal

    2006-06-01

    Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

  16. Unexpected functional similarities between gatekeeper tumour suppressor genes and proto-oncogenes revealed by systems biology.

    Science.gov (United States)

    Zhao, Yongzhong; Epstein, Richard J

    2011-05-01

    Familial tumor suppressor genes comprise two subgroups: caretaker genes (CTs) that repair DNA, and gatekeeper genes (GKs) that trigger cell death. Since GKs may also induce cell cycle delay and thus enhance cell survival by facilitating DNA repair, we hypothesized that the prosurvival phenotype of GKs could be selected during cancer progression, and we used a multivariable systems biology approach to test this. We performed multidimensional data analysis, non-negative matrix factorization and logistic regression to compare the features of GKs with those of their putative antagonists, the proto-oncogenes (POs), as well as with control groups of CTs and functionally unrelated congenital heart disease genes (HDs). GKs and POs closely resemble each other, but not CTs or HDs, in terms of gene structure (Pexpression level and breadth (Pimplied suggest a common functional attribute that is strongly negatively selected-that is, a shared phenotype that enhances cell survival. The counterintuitive finding of similar evolutionary pressures affecting GKs and POs raises an intriguing possibility: namely, that cancer microevolution is accelerated by an epistatic cascade in which upstream suppressor gene defects subvert the normal bifunctionality of wild-type GKs by constitutively shifting the phenotype away from apoptosis towards survival. If correct, this interpretation would explain the hitherto unexplained phenomenon of frequent wild-type GK (for example, p53) overexpression in tumors.

  17. Gene expression of circulating tumour cells in breast cancer patients

    Directory of Open Access Journals (Sweden)

    Bölke E

    2009-09-01

    Full Text Available Abstract Background The diagnostic tools to predict the prognosis in patients suffering from breast cancer (BC need further improvements. New technological achievements like the gene profiling of circulating tumour cells (CTC could help identify new prognostic markers in the clinical setting. Furthermore, gene expression patterns of CTC might provide important informations on the mechanisms of tumour cell metastasation. Materials and methods We performed realtime-PCR and multiplex-PCR analyses following immunomagnetic separation of CTC. Peripheral blood (PB samples of 63 patients with breast cancer of various stages were analyzed and compared to a control group of 14 healthy individuals. After reverse-transcription, we performed multiplex PCR using primers for the genes ga733.3, muc-1 and c-erbB2. Mammaglobin1, spdef and c-erbB2 were analyzed applying realtime-PCR. Results ga733.2 overexpression was found in 12.7% of breast cancer cases, muc-1 in 15.9%, mgb1 in 9.1% and spdef in 12.1%. In this study, c-erbB2 did not show any significant correlation to BC, possibly due to a highly ambient expression. Besides single gene analyses, gene profiles were additionally evaluated. Highly significant correlations to BC were found in single gene analyses of ga733.2 and muc-1 and in gene profile analyses of ga733.3*muc-1 and GA7 ga733.3*muc-1*mgb1*spdef. Conclusion Our study reveals that the single genes ga733.3, muc-1 and the gene profiles ga733.3*muc-1 and ga733.3*3muc-1*mgb1*spdef can serve as markers for the detection of CTC in BC. The multigene analyses found highly positive levels in BC patients. Our study indicates that not single gene analyses but subtle patterns of multiple genes lead to rising accuracy and low loss of specificity in detection of breast cancer cases.

  18. Mapping of gene expression reveals CYP27A1 as a susceptibility gene for sporadic ALS.

    Directory of Open Access Journals (Sweden)

    Frank P Diekstra

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive, neurodegenerative disease characterized by loss of upper and lower motor neurons. ALS is considered to be a complex trait and genome-wide association studies (GWAS have implicated a few susceptibility loci. However, many more causal loci remain to be discovered. Since it has been shown that genetic variants associated with complex traits are more likely to be eQTLs than frequency-matched variants from GWAS platforms, we conducted a two-stage genome-wide screening for eQTLs associated with ALS. In addition, we applied an eQTL analysis to finemap association loci. Expression profiles using peripheral blood of 323 sporadic ALS patients and 413 controls were mapped to genome-wide genotyping data. Subsequently, data from a two-stage GWAS (3,568 patients and 10,163 controls were used to prioritize eQTLs identified in the first stage (162 ALS, 207 controls. These prioritized eQTLs were carried forward to the second sample with both gene-expression and genotyping data (161 ALS, 206 controls. Replicated eQTL SNPs were then tested for association in the second-stage GWAS data to find SNPs associated with disease, that survived correction for multiple testing. We thus identified twelve cis eQTLs with nominally significant associations in the second-stage GWAS data. Eight SNP-transcript pairs of highest significance (lowest p = 1.27 × 10(-51 withstood multiple-testing correction in the second stage and modulated CYP27A1 gene expression. Additionally, we show that C9orf72 appears to be the only gene in the 9p21.2 locus that is regulated in cis, showing the potential of this approach in identifying causative genes in association loci in ALS. This study has identified candidate genes for sporadic ALS, most notably CYP27A1. Mutations in CYP27A1 are causal to cerebrotendinous xanthomatosis which can present as a clinical mimic of ALS with progressive upper motor neuron loss, making it a plausible

  19. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    Energy Technology Data Exchange (ETDEWEB)

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  20. Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development.

    Science.gov (United States)

    Foerst-Potts, L; Sadler, T W

    1997-05-01

    In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

  1. RNA Sequencing Reveals the Alteration of the Expression of Novel Genes in Ethanol-Treated Embryoid Bodies.

    Science.gov (United States)

    Mandal, Chanchal; Kim, Sun Hwa; Chai, Jin Choul; Oh, Seon Mi; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2016-01-01

    Fetal alcohol spectrum disorder is a collective term representing fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not well characterized. In this present study, our aim is to profile important genes that regulate cellular development during fetal development. Human embryonic carcinoma cells (NCCIT) are cultured to form embryoid bodies and then treated in the presence and absence of ethanol (50 mM). We employed RNA sequencing to profile differentially expressed genes in the ethanol-treated embryoid bodies from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH data sets. A total of 632, 205 and 517 differentially expressed genes were identified from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. Functional annotation using bioinformatics tools reveal significant enrichment of differential cellular development and developmental disorders. Furthermore, a group of 42, 15 and 35 transcription factor-encoding genes are screened from all of the differentially expressed genes obtained from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. We validated relative gene expression levels of several transcription factors from these lists by quantitative real-time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanism underlying the pathology of alcohol-mediated anomalies and ease further research.

  2. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms

    DEFF Research Database (Denmark)

    Svingen, T; Jørgensen, Anne; Rajpert-De Meyts, E

    2014-01-01

    to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers......, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis...... and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further...

  3. Biofilm growth program and architecture revealed by single-cell live imaging

    Science.gov (United States)

    Yan, Jing; Sabass, Benedikt; Stone, Howard; Wingreen, Ned; Bassler, Bonnie

    Biofilms are surface-associated bacterial communities. Little is known about biofilm structure at the level of individual cells. We image living, growing Vibrio cholerae biofilms from founder cells to ten thousand cells at single-cell resolution, and discover the forces underpinning the architectural evolution of the biofilm. Mutagenesis, matrix labeling, and simulations demonstrate that surface-adhesion-mediated compression causes V. cholerae biofilms to transition from a two-dimensional branched morphology to a dense, ordered three-dimensional cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture, and this growth pattern is controlled by a single gene. Competition analyses reveal the advantages of the dense growth mode in providing the biofilm with superior mechanical properties. We will further present continuum theory to model the three-dimensional growth of biofilms at the solid-liquid interface as well as solid-air interface.

  4. Gene expression profiling, pathway analysis and subtype classification reveal molecular heterogeneity in hepatocellular carcinoma and suggest subtype specific therapeutic targets.

    Science.gov (United States)

    Agarwal, Rahul; Narayan, Jitendra; Bhattacharyya, Amitava; Saraswat, Mayank; Tomar, Anil Kumar

    2017-10-01

    A very low 5-year survival rate among hepatocellular carcinoma (HCC) patients is mainly due to lack of early stage diagnosis, distant metastasis and high risk of postoperative recurrence. Hence ascertaining novel biomarkers for early diagnosis and patient specific therapeutics is crucial and urgent. Here, we have performed a comprehensive analysis of the expression data of 423 HCC patients (373 tumors and 50 controls) downloaded from The Cancer Genome Atlas (TCGA) followed by pathway enrichment by gene ontology annotations, subtype classification and overall survival analysis. The differential gene expression analysis using non-parametric Wilcoxon test revealed a total of 479 up-regulated and 91 down-regulated genes in HCC compared to controls. The list of top differentially expressed genes mainly consists of tumor/cancer associated genes, such as AFP, THBS4, LCN2, GPC3, NUF2, etc. The genes over-expressed in HCC were mainly associated with cell cycle pathways. In total, 59 kinases associated genes were found over-expressed in HCC, including TTK, MELK, BUB1, NEK2, BUB1B, AURKB, PLK1, CDK1, PKMYT1, PBK, etc. Overall four distinct HCC subtypes were predicted using consensus clustering method. Each subtype was unique in terms of gene expression, pathway enrichment and median survival. Conclusively, this study has exposed a number of interesting genes which can be exploited in future as potential markers of HCC, diagnostic as well as prognostic and subtype classification may guide for improved and specific therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. DNA microarray revealed and RNAi plants confirmed key genes conferring low Cd accumulation in barley grains

    DEFF Research Database (Denmark)

    Sun, Hongyan; Chen, Zhong-Hua; Chen, Fei

    2015-01-01

    Background Understanding the mechanism of low Cd accumulation in crops is crucial for sustainable safe food production in Cd-contaminated soils. Results Confocal microscopy, atomic absorption spectrometry, gas exchange and chlorophyll fluorescence analyses revealed a distinct difference in Cd...... with a substantial difference between the two genotypes. Cd stress led to higher expression of genes involved in transport, carbohydrate metabolism and signal transduction in the low-grain-Cd-accumulating genotype. Novel transporter genes such as zinc transporter genes were identified as being associated with low Cd...... accumulation. Quantitative RT-PCR confirmed our microarray data. Furthermore, suppression of the zinc transporter genes HvZIP3 and HvZIP8 by RNAi silencing showed increased Cd accumulation and reduced Zn and Mn concentrations in barley grains. Thus, HvZIP3 and HvZIP8 could be candidate genes related to low...

  6. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  7. Low cytotoxicity effect of dendrosome as an efficient carrier for rotavirus VP2 gene transferring into a human lung cell line : dendrosome, as a novel intranasally gene porter.

    Science.gov (United States)

    Pourasgari, Farzaneh; Ahmadian, Shahin; Salmanian, Ali Hatef; Sarbolouki, Mohammad Nabi; Massumi, Mohammad

    2009-01-01

    The efficiency of dendrosome (a gene porter) was assessed in transferring recombinant human rotavirus VP2 cDNA into A549, a human lung cell line. After gene transferring, transmission electron microscopy showed core-like particles (CLPs) formation in the transfected cells both with dendrosome and lipofectamine porters. In addition, western blotting analysis showed that the expression of VP2 gene was almost equal in the dendrosome and lipofectamine-transfected cells. Also, the cytotoxicity studies revealed that dendrosome had a lower cytotoxicity than lipofectamine. Therefore, our study may introduce dendrosome as a possible carrier for gene transferring into the human lung cell line, especially, for intranasally administration of DNA vaccines.

  8. Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

    Science.gov (United States)

    Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

    2018-05-01

    The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of

  9. Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus.

    Science.gov (United States)

    Derjuga, Anna; Gourley, Tania S; Holm, Teresa M; Heng, Henry H Q; Shivdasani, Ramesh A; Ahmed, Rafi; Andrews, Nancy C; Blank, Volker

    2004-04-01

    Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(-/-) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice.

  10. Bioinformatics analysis of RNA-seq data revealed critical genes in colon adenocarcinoma.

    Science.gov (United States)

    Xi, W-D; Liu, Y-J; Sun, X-B; Shan, J; Yi, L; Zhang, T-T

    2017-07-01

    RNA-seq data of colon adenocarcinoma (COAD) were analyzed with bioinformatics tools to discover critical genes in the disease. Relevant small molecule drugs, transcription factors (TFs) and microRNAs (miRNAs) were also investigated. RNA-seq data of COAD were downloaded from The Cancer Genome Atlas (TCGA). Differential analysis was performed with package edgeR. False positive discovery (FDR) 1 were set as the cut-offs to screen out differentially expressed genes (DEGs). Gene coexpression network was constructed with package Ebcoexpress. GO enrichment analysis was performed for the DEGs in the gene coexpression network with DAVID. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed for the genes with KOBASS 2.0. Modules were identified with MCODE of Cytoscape. Relevant small molecules drugs were predicted by Connectivity map. Relevant miRNAs and TFs were searched by WebGestalt. A total of 457 DEGs, including 255 up-regulated and 202 down-regulated genes, were identified from 437 COAD and 39 control samples. A gene coexpression network was constructed containing 40 DEGs and 101 edges. The genes were mainly associated with collagen fibril organization, extracellular matrix organization and translation. Two modules were identified from the gene coexpression network, which were implicated in muscle contraction and extracellular matrix organization, respectively. Several critical genes were disclosed, such as MYH11, COL5A2 and ribosomal proteins. Nine relevant small molecule drugs were identified, such as scriptaid and STOCK1N-35874. Accordingly, a total of 17 TFs and 10 miRNAs related to COAD were acquired, such as ETS2, NFAT, AP4, miR-124A, MiR-9, miR-96 and let-7. Several critical genes and relevant drugs, TFs and miRNAs were revealed in COAD. These findings could advance the understanding of the disease and benefit therapy development.

  11. Temporal gene expression profiling reveals CEBPD as a candidate regulator of brain disease in prosaposin deficient mice

    Directory of Open Access Journals (Sweden)

    Ran Huimin

    2008-08-01

    Full Text Available Abstract Background Prosaposin encodes, in tandem, four small acidic activator proteins (saposins with specificities for glycosphingolipid (GSL hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian prosaposin deficiencies. Results Our hypomorphic prosaposin deficient mouse, PS-NA, exhibited 45% WT levels of brain saposins and showed neuropathology that included neuronal GSL storage and Purkinje cell loss. Impairment of neuronal function was observed as early as 6 wks as demonstrated by the narrow bridges tests. Temporal transcriptome microarray analyses of brain tissues were conducted with mRNA from three prosaposin deficient mouse models: PS-NA, prosaposin null (PS-/- and a V394L/V394L glucocerebrosidase mutation combined with PS-NA (4L/PS-NA. Gene expression alterations in cerebrum and cerebellum were detectable at birth preceding the neuronal deficits. Differentially expressed genes encompassed a broad spectrum of cellular functions. The number of down-regulated genes was constant, but up-regulated gene numbers increased with age. CCAAT/enhancer-binding protein delta (CEBPD was the only up-regulated transcription factor in these two brain regions of all three models. Network analyses revealed that CEBPD has functional relationships with genes in transcription, pro-inflammation, cell death, binding, myelin and transport. Conclusion These results show that: 1 Regionally specific gene expression abnormalities precede the brain histological and neuronal function changes, 2 Temporal gene expression profiles provide insights into the molecular mechanism during the GSL storage disease course, and 3 CEBPD is a candidate regulator of brain disease in prosaposin deficiency to participate in modulating disease acceleration or progression.

  12. CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Yue Hu

    Full Text Available We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC and adenocarcinoma (AC subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.

  13. Integrated analysis of microRNA and gene expression profiles reveals a functional regulatory module associated with liver fibrosis.

    Science.gov (United States)

    Chen, Wei; Zhao, Wenshan; Yang, Aiting; Xu, Anjian; Wang, Huan; Cong, Min; Liu, Tianhui; Wang, Ping; You, Hong

    2017-12-15

    Liver fibrosis, characterized with the excessive accumulation of extracellular matrix (ECM) proteins, represents the final common pathway of chronic liver inflammation. Ever-increasing evidence indicates microRNAs (miRNAs) dysregulation has important implications in the different stages of liver fibrosis. However, our knowledge of miRNA-gene regulation details pertaining to such disease remains unclear. The publicly available Gene Expression Omnibus (GEO) datasets of patients suffered from cirrhosis were extracted for integrated analysis. Differentially expressed miRNAs (DEMs) and genes (DEGs) were identified using GEO2R web tool. Putative target gene prediction of DEMs was carried out using the intersection of five major algorithms: DIANA-microT, TargetScan, miRanda, PICTAR5 and miRWalk. Functional miRNA-gene regulatory network (FMGRN) was constructed based on the computational target predictions at the sequence level and the inverse expression relationships between DEMs and DEGs. DAVID web server was selected to perform KEGG pathway enrichment analysis. Functional miRNA-gene regulatory module was generated based on the biological interpretation. Internal connections among genes in liver fibrosis-related module were determined using String database. MiRNA-gene regulatory modules related to liver fibrosis were experimentally verified in recombinant human TGFβ1 stimulated and specific miRNA inhibitor treated LX-2 cells. We totally identified 85 and 923 dysregulated miRNAs and genes in liver cirrhosis biopsy samples compared to their normal controls. All evident miRNA-gene pairs were identified and assembled into FMGRN which consisted of 990 regulations between 51 miRNAs and 275 genes, forming two big sub-networks that were defined as down-network and up-network, respectively. KEGG pathway enrichment analysis revealed that up-network was prominently involved in several KEGG pathways, in which "Focal adhesion", "PI3K-Akt signaling pathway" and "ECM

  14. Genome-wide characterization of pectin methyl esterase genes reveals members differentially expressed in tolerant and susceptible wheats in response to Fusarium graminearum.

    Science.gov (United States)

    Zega, Alessandra; D'Ovidio, Renato

    2016-11-01

    Pectin methyl esterase (PME) genes code for enzymes that are involved in structural modifications of the plant cell wall during plant growth and development. They are also involved in plant-pathogen interaction. PME genes belong to a multigene family and in this study we report the first comprehensive analysis of the PME gene family in bread wheat (Triticum aestivum L.). Like in other species, the members of the TaPME family are dispersed throughout the genome and their encoded products retain the typical structural features of PMEs. qRT-PCR analysis showed variation in the expression pattern of TaPME genes in different tissues and revealed that these genes are mainly expressed in flowering spikes. In our attempt to identify putative TaPME genes involved in wheat defense, we revealed a strong variation in the expression of the TaPME following Fusarium graminearum infection, the causal agent of Fusarium head blight (FHB). Particularly interesting was the finding that the expression profile of some PME genes was markedly different between the FHB-resistant wheat cultivar Sumai3 and the FHB-susceptible cultivar Bobwhite, suggesting a possible involvement of these PME genes in FHB resistance. Moreover, the expression analysis of the TaPME genes during F. graminearum progression within the spike revealed those genes that responded more promptly to pathogen invasion. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Molecular Subtyping of Primary Prostate Cancer Reveals Specific and Shared Target Genes of Different ETS Rearrangements

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    Paula Paulo

    2012-07-01

    Full Text Available This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa, namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1 and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2 was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

  16. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

    Science.gov (United States)

    Pei, Weike; Feyerabend, Thorsten B; Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

    2017-08-24

    Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

  17. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo

    Science.gov (United States)

    Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

    2017-01-01

    Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping1 has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites2, viral barcodes3, and strategies based on transposons4 and CRISPR/Cas9 genome editing5; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system6,7. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs8–10. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure. PMID:28813413

  18. Metallothionein gene expression in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Deeksha Pal

    2014-01-01

    Full Text Available Introduction: Metallothioneins (MTs are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1 the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2 apoptosis and (3 the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC. No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR analysis was done for the MT2A gene expression using b-actin as an internal control. All statistical calculations were performed using SPSS software. Results: The MT2A gene expression was found to be significantly increased (P < 0.01 in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05, while no change was observed in high-grade tumor (grade III and IV in comparison to adjacent normal renal tissue. Conclusion: The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.

  19. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas.

    Directory of Open Access Journals (Sweden)

    Priscila Daniele Ramos Cirilo

    Full Text Available Uterine Leiomyomas (ULs are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs. Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs.We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC and gene expression microarrays (SAM. The CONEXIC algorithm was applied to integrate the data.The integrated analysis identified the top 30 significant genes (P<0.01, which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively and IGFBP5 (P = 0.0002 and P = 0.006, respectively were up-regulated in the tumours when compared with the adjacent normal myometrium.The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs.

  20. Gain and loss of phototrophic genes revealed by comparison of two Citromicrobium bacterial genomes.

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    Qiang Zheng

    Full Text Available Proteobacteria are thought to have diverged from a phototrophic ancestor, according to the scattered distribution of phototrophy throughout the proteobacterial clade, and so the occurrence of numerous closely related phototrophic and chemotrophic microorganisms may be the result of the loss of genes for phototrophy. A widespread form of bacterial phototrophy is based on the photochemical reaction center, encoded by puf and puh operons that typically are in a 'photosynthesis gene cluster' (abbreviated as the PGC with pigment biosynthesis genes. Comparison of two closely related Citromicrobial genomes (98.1% sequence identity of complete 16S rRNA genes, Citromicrobium sp. JL354, which contains two copies of reaction center genes, and Citromicrobium strain JLT1363, which is chemotrophic, revealed evidence for the loss of phototrophic genes. However, evidence of horizontal gene transfer was found in these two bacterial genomes. An incomplete PGC (pufLMC-puhCBA in strain JL354 was located within an integrating conjugative element, which indicates a potential mechanism for the horizontal transfer of genes for phototrophy.

  1. Genes expressed in grapevine leaves reveal latent wood infection by the fungal pathogen Neofusicoccum parvum.

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    Stefan Czemmel

    Full Text Available Some pathogenic species of the Botryosphaeriaceae have a latent phase, colonizing woody tissues while perennial hosts show no apparent symptoms until conditions for disease development become favorable. Detection of these pathogens is often limited to the later pathogenic phase. The latent phase is poorly characterized, despite the need for non-destructive detection tools and effective quarantine strategies, which would benefit from identification of host-based markers in leaves. Neofusicoccum parvum infects the wood of grapevines and other horticultural crops, killing the fruit-bearing shoots. We used light microscopy and high-resolution computed tomography (HRCT to examine the spatio-temporal relationship between pathogen colonization and anatomical changes in stem sections. To identify differentially-expressed grape genes, leaves from inoculated and non-inoculated plants were examined using RNA-Seq. The latent phase occurred between 0 and 1.5 months post-inoculation (MPI, during which time the pathogen did not spread significantly beyond the inoculation site nor were there differences in lesion lengths between inoculated and non-inoculated plants. The pathogenic phase occurred between 1.5 and 2 MPI, when recovery beyond the inoculation site increased and lesion lengths of inoculated plants tripled. By 2 MPI, inoculated plants also had decreased starch content in xylem fibers and rays, and increased levels of gel-occluded xylem vessels, the latter of which HRCT revealed at a higher frequency than microscopy. RNA-Seq and screening of 21 grape expression datasets identified 20 candidate genes that were transcriptionally-activated by infection during the latent phase, and confirmed that the four best candidates (galactinol synthase, abscisic acid-induced wheat plasma membrane polypeptide-19 ortholog, embryonic cell protein 63, BURP domain-containing protein were not affected by a range of common foliar and wood pathogens or abiotic stresses

  2. Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

    Science.gov (United States)

    Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

    2013-01-01

    After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

  3. Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis.

    Science.gov (United States)

    Vargas-Bautista, Carol; Rahlwes, Kathryn; Straight, Paul

    2014-02-01

    Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305-310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis.

  4. An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data.

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    Daniel Ramsköld

    2009-12-01

    Full Text Available The parts of the genome transcribed by a cell or tissue reflect the biological processes and functions it carries out. We characterized the features of mammalian tissue transcriptomes at the gene level through analysis of RNA deep sequencing (RNA-Seq data across human and mouse tissues and cell lines. We observed that roughly 8,000 protein-coding genes were ubiquitously expressed, contributing to around 75% of all mRNAs by message copy number in most tissues. These mRNAs encoded proteins that were often intracellular, and tended to be involved in metabolism, transcription, RNA processing or translation. In contrast, genes for secreted or plasma membrane proteins were generally expressed in only a subset of tissues. The distribution of expression levels was broad but fairly continuous: no support was found for the concept of distinct expression classes of genes. Expression estimates that included reads mapping to coding exons only correlated better with qRT-PCR data than estimates which also included 3' untranslated regions (UTRs. Muscle and liver had the least complex transcriptomes, in that they expressed predominantly ubiquitous genes and a large fraction of the transcripts came from a few highly expressed genes, whereas brain, kidney and testis expressed more complex transcriptomes with the vast majority of genes expressed and relatively small contributions from the most expressed genes. mRNAs expressed in brain had unusually long 3'UTRs, and mean 3'UTR length was higher for genes involved in development, morphogenesis and signal transduction, suggesting added complexity of UTR-based regulation for these genes. Our results support a model in which variable exterior components feed into a large, densely connected core composed of ubiquitously expressed intracellular proteins.

  5. Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

    Science.gov (United States)

    Vargas-Bautista, Carol; Rahlwes, Kathryn

    2014-01-01

    Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis. PMID:24187085

  6. Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

    International Nuclear Information System (INIS)

    Hoever, Gerold; Vogel, Jens-Uwe; Lukashenko, Polina; Hofmann, Wolf-Karsten; Komor, Martina; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2005-01-01

    In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies

  7. Comparative transcriptome and gene co-expression network analysis reveal genes and signaling pathways adaptively responsive to varied adverse stresses in the insect fungal pathogen, Beauveria bassiana.

    Science.gov (United States)

    He, Zhangjiang; Zhao, Xin; Lu, Zhuoyue; Wang, Huifang; Liu, Pengfei; Zeng, Fanqin; Zhang, Yongjun

    2018-01-01

    Sensing, responding, and adapting to the surrounding environment are crucial for all living organisms to survive, proliferate, and differentiate in their biological niches. Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. The fungal pathogen unavoidably encounters a variety of adverse environmental stresses and defense response from the host insects during application of the fungal agents. However, few are known about the transcription response of the fungus to respond or adapt varied adverse stresses. Here, we comparatively analyzed the transcriptome of B. bassiana in globe genome under the varied stationary-phase stresses including osmotic agent (0.8 M NaCl), high temperature (32 °C), cell wall-perturbing agent (Congo red), and oxidative agents (H 2 O 2 or menadione). Total of 12,412 reads were obtained, and mapped to the 6767 genes of the B. bassiana. All of these stresses caused transcription responses involved in basal metabolism, cell wall construction, stress response or cell rescue/detoxification, signaling transduction and gene transcription regulation, and likely other cellular processes. An array of genes displayed similar transcription patterns in response to at least two of the five stresses, suggesting a shared transcription response to varied adverse stresses. Gene co-expression network analysis revealed that mTOR signaling pathway, but not HOG1 MAP kinase pathway, played a central role in regulation the varied adverse stress responses, which was verified by RNAi-mediated knockdown of TOR1. Our findings provided an insight of transcription response and gene co-expression network of B. bassiana in adaptation to varied environments. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

    Science.gov (United States)

    Reverón, Inés; Jiménez, Natalia; Curiel, José Antonio; Peñas, Elena; López de Felipe, Félix; de Las Rivas, Blanca; Muñoz, Rosario

    2017-04-01

    Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase ( lpdC , or lp_2945 ) is only 6.5 kb distant from the gene encoding inducible tannase ( L. plantarum tanB [ tanB Lp ], or lp_2956 ). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B ( lpdB , or lp_0271 ) and D ( lpdD , or lp_0272 ) of the gallate decarboxylase are cotranscribed, whereas subunit C ( lpdC , or lp_2945 ) is cotranscribed with a gene encoding a transport protein ( gacP , or lp_2943 ). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator ( lp_2942 ) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment. IMPORTANCE Lactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are

  9. Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

    Science.gov (United States)

    Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

    2009-01-01

    Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

  10. Collective Dynamics of Specific Gene Ensembles Crucial for Neutrophil Differentiation: The Existence of Genome Vehicles Revealed

    Science.gov (United States)

    Giuliani, Alessandro; Tomita, Masaru

    2010-01-01

    Cell fate decision remarkably generates specific cell differentiation path among the multiple possibilities that can arise through the complex interplay of high-dimensional genome activities. The coordinated action of thousands of genes to switch cell fate decision has indicated the existence of stable attractors guiding the process. However, origins of the intracellular mechanisms that create “cellular attractor” still remain unknown. Here, we examined the collective behavior of genome-wide expressions for neutrophil differentiation through two different stimuli, dimethyl sulfoxide (DMSO) and all-trans-retinoic acid (atRA). To overcome the difficulties of dealing with single gene expression noises, we grouped genes into ensembles and analyzed their expression dynamics in correlation space defined by Pearson correlation and mutual information. The standard deviation of correlation distributions of gene ensembles reduces when the ensemble size is increased following the inverse square root law, for both ensembles chosen randomly from whole genome and ranked according to expression variances across time. Choosing the ensemble size of 200 genes, we show the two probability distributions of correlations of randomly selected genes for atRA and DMSO responses overlapped after 48 hours, defining the neutrophil attractor. Next, tracking the ranked ensembles' trajectories, we noticed that only certain, not all, fall into the attractor in a fractal-like manner. The removal of these genome elements from the whole genomes, for both atRA and DMSO responses, destroys the attractor providing evidence for the existence of specific genome elements (named “genome vehicle”) responsible for the neutrophil attractor. Notably, within the genome vehicles, genes with low or moderate expression changes, which are often considered noisy and insignificant, are essential components for the creation of the neutrophil attractor. Further investigations along with our findings might

  11. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes

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    Elvezia Maria Paraboschi

    2015-09-01

    Full Text Available Abnormalities in RNA metabolism and alternative splicing (AS are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls, followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p = 0.0015 by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

  12. Global Gene Expression Analysis of Yeast Cells during Sake Brewing▿ †

    Science.gov (United States)

    Wu, Hong; Zheng, Xiaohong; Araki, Yoshio; Sahara, Hiroshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2006-01-01

    During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process. PMID:16997994

  13. Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1.

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    Guillaume Blanc

    Full Text Available Paramecium bursaria chlorella virus 1 (PBCV-1 is the prototype of the genus Chlorovirus (family Phycodnaviridae that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i., transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i. was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

  14. Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

    Science.gov (United States)

    Breuer, Rebecca J; Bandyopadhyay, Arpan; O'Brien, Sofie A; Barnes, Aaron M T; Hunter, Ryan C; Hu, Wei-Shou; Dunny, Gary M

    2017-07-01

    In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

  15. Integrated Metabolo-Transcriptomics Reveals Fusarium Head Blight Candidate Resistance Genes in Wheat QTL-Fhb2.

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    Dhananjay Dhokane

    Full Text Available Fusarium head blight (FHB caused by Fusarium graminearum not only causes severe losses in yield, but also reduces quality of wheat grain by accumulating mycotoxins. Breeding for host plant resistance is considered as the best strategy to manage FHB. Resistance in wheat to FHB is quantitative in nature, involving cumulative effects of many genes governing resistance. The poor understanding of genetics and lack of precise phenotyping has hindered the development of FHB resistant cultivars. Though more than 100 QTLs imparting FHB resistance have been reported, none discovered the specific genes localized within the QTL region, nor the underlying mechanisms of resistance.In our study recombinant inbred lines (RILs carrying resistant (R-RIL and susceptible (S-RIL alleles of QTL-Fhb2 were subjected to metabolome and transcriptome profiling to discover the candidate genes. Metabolome profiling detected a higher abundance of metabolites belonging to phenylpropanoid, lignin, glycerophospholipid, flavonoid, fatty acid, and terpenoid biosynthetic pathways in R-RIL than in S-RIL. Transcriptome analysis revealed up-regulation of several receptor kinases, transcription factors, signaling, mycotoxin detoxification and resistance related genes. The dissection of QTL-Fhb2 using flanking marker sequences, integrating metabolomic and transcriptomic datasets, identified 4-Coumarate: CoA ligase (4CL, callose synthase (CS, basic Helix Loop Helix (bHLH041 transcription factor, glutathione S-transferase (GST, ABC transporter-4 (ABC4 and cinnamyl alcohol dehydrogenase (CAD as putative resistance genes localized within the QTL-Fhb2 region.Some of the identified genes within the QTL region are associated with structural resistance through cell wall reinforcement, reducing the spread of pathogen through rachis within a spike and few other genes that detoxify DON, the virulence factor, thus eventually reducing disease severity. In conclusion, we report that the wheat

  16. Single-cell analysis reveals early manifestation of cancerous phenotype in pre-malignant esophageal cells.

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    Jiangxin Wang

    Full Text Available Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett's esophagus (BE as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

  17. Blood-gene expression reveals reduced circadian rhythmicity in individuals resistant to sleep deprivation.

    Science.gov (United States)

    Arnardottir, Erna S; Nikonova, Elena V; Shockley, Keith R; Podtelezhnikov, Alexei A; Anafi, Ron C; Tanis, Keith Q; Maislin, Greg; Stone, David J; Renger, John J; Winrow, Christopher J; Pack, Allan I

    2014-10-01

    To address whether changes in gene expression in blood cells with sleep loss are different in individuals resistant and sensitive to sleep deprivation. Blood draws every 4 h during a 3-day study: 24-h normal baseline, 38 h of continuous wakefulness and subsequent recovery sleep, for a total of 19 time-points per subject, with every 2-h psychomotor vigilance task (PVT) assessment when awake. Sleep laboratory. Fourteen subjects who were previously identified as behaviorally resistant (n = 7) or sensitive (n = 7) to sleep deprivation by PVT. Thirty-eight hours of continuous wakefulness. We found 4,481 unique genes with a significant 24-h diurnal rhythm during a normal sleep-wake cycle in blood (false discovery rate [FDR] sleep. After accounting for circadian effects, two genes (SREBF1 and CPT1A, both involved in lipid metabolism) exhibited small, but significant, linear changes in expression with the duration of sleep deprivation (FDR sleep deprivation was a reduction in the amplitude of the diurnal rhythm of expression of normally cycling probe sets. This reduction was noticeably higher in behaviorally resistant subjects than sensitive subjects, at any given P value. Furthermore, blood cell type enrichment analysis showed that the expression pattern difference between sensitive and resistant subjects is mainly found in cells of myeloid origin, such as monocytes. Individual differences in behavioral effects of sleep deprivation are associated with differences in diurnal amplitude of gene expression for genes that show circadian rhythmicity. © 2014 Associated Professional Sleep Societies, LLC.

  18. Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes.

    Science.gov (United States)

    Wuest, Samuel E; Vijverberg, Kitty; Schmidt, Anja; Weiss, Manuel; Gheyselinck, Jacqueline; Lohr, Miriam; Wellmer, Frank; Rahnenführer, Jörg; von Mering, Christian; Grossniklaus, Ueli

    2010-03-23

    The development of multicellular organisms is controlled by differential gene expression whereby cells adopt distinct fates. A spatially resolved view of gene expression allows the elucidation of transcriptional networks that are linked to cellular identity and function. The haploid female gametophyte of flowering plants is a highly reduced organism: at maturity, it often consists of as few as three cell types derived from a common precursor [1, 2]. However, because of its inaccessibility and small size, we know little about the molecular basis of cell specification and differentiation in the female gametophyte. Here we report expression profiles of all cell types in the mature Arabidopsis female gametophyte. Differentially expressed posttranscriptional regulatory modules and metabolic pathways characterize the distinct cell types. Several transcription factor families are overrepresented in the female gametophyte in comparison to other plant tissues, e.g., type I MADS domain, RWP-RK, and reproductive meristem transcription factors. PAZ/Piwi-domain encoding genes are upregulated in the egg, indicating a role of epigenetic regulation through small RNA pathways-a feature paralleled in the germline of animals [3]. A comparison of human and Arabidopsis egg cells for enrichment of functional groups identified several similarities that may represent a consequence of coevolution or ancestral gametic features. 2010 Elsevier Ltd. All rights reserved.

  19. Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

    Science.gov (United States)

    Salmond, G P; Lutkenhaus, J F; Donachie, W D

    1980-01-01

    We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

  20. Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes.

    Science.gov (United States)

    Noar, Roslyn D; Daub, Margaret E

    2016-08-30

    genes with higher expression in infected leaf tissue, suggesting that they may play a role in pathogenicity. For two other scaffolds, no transcripts were detected in either condition, and PCR assays support the hypothesis that at least one of these scaffolds corresponds to a dispensable chromosome that is not required for survival or pathogenicity. Our study revealed major changes in the transcriptome of Mycosphaerella fijiensis, when associating with its host compared to during saprophytic growth in medium. This analysis identified putative pathogenicity genes and also provides support for the existence of dispensable chromosomes in this fungus.

  1. Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

    Science.gov (United States)

    Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

    2015-01-01

    A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

  2. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.; Lovrics, Anna; Byrne, Helen M.; Jensen, Oliver E.; King, John R.; Shakesheff, Kevin M.; Buttery, Lee D. K.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2

  3. In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.

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    Chien-Chih Ke

    Full Text Available Mesenchymal stromal cells (MSCs are multipotent adult stem cells which are recruited to the tumor microenvironment (TME and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

  4. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.).

    Science.gov (United States)

    Dou, Junling; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhang, Lei; Ali, Aslam; Kuang, Hanhui; Liu, Wenge

    2018-04-01

    A 159 bp deletion in ClFS1 gene encoding IQD protein is responsible for fruit shape in watermelon. Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is known for its rich diversity in fruit size and shape. Fruit shape has been one of the major objectives of watermelon breeding. However, the candidate genes and the underlying genetic mechanism for such an important trait in watermelon are unknown. In this study, we identified a locus on chromosome 3 of watermelon genome controlling fruit shape. Segregation analysis in F 2 and BC 1 populations derived from a cross between two inbred lines "Duan125" (elongate fruit) and "Zhengzhouzigua" (spherical fruit) suggests that fruit shape of watermelon is controlled by a single locus and elongate fruit (OO) is incompletely dominant to spherical fruit (oo) with the heterozygote (Oo) being oval fruit. GWAS profiles among 315 accessions identified a major locus designated on watermelon chromosome 3, which was confirmed by BSA-seq mapping in the F 2 population. The candidate gene was mapped to a region 46 kb on chromosome 3. There were only four genes present in the corresponding region in the reference genome. Four candidate genes were sequenced in this region, revealing that the CDS of Cla011257 had a 159 bp deletion which resulted in the omission of 53 amino acids in elongate watermelon. An indel marker was derived from the 159 bp deletion to test the F 2 population and 105 watermelon accessions. The results showed that Cla011257 cosegregated with watermelon fruit shape. In addition, the Cla011257 expression was the highest at ovary formation stage. The predicted protein of the Cla011257 gene fitted in IQD protein family which was reported to have association with cell arrays and Ca 2+ -CaM signaling modules. Clear understanding of the genes facilitating the fruit shape along with marker association selection will be an effective way to develop new cultivars.

  5. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  6. Comprehensive gene expression profiling reveals synergistic functional networks in cerebral vessels after hypertension or hypercholesterolemia.

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    Wei-Yi Ong

    Full Text Available Atherosclerotic stenosis of cerebral arteries or intracranial large artery disease (ICLAD is a major cause of stroke especially in Asians, Hispanics and Africans, but relatively little is known about gene expression changes in vessels at risk. This study compares comprehensive gene expression profiles in the middle cerebral artery (MCA of New Zealand White rabbits exposed to two stroke risk factors i.e. hypertension and/or hypercholesterolemia, by the 2-Kidney-1-Clip method, or dietary supplementation with cholesterol. Microarray and Ingenuity Pathway Analyses of the MCA of the hypertensive rabbits showed up-regulated genes in networks containing the node molecules: UBC (ubiquitin, P38 MAPK, ERK, NFkB, SERPINB2, MMP1 and APP (amyloid precursor protein; and down-regulated genes related to MAPK, ERK 1/2, Akt, 26 s proteasome, histone H3 and UBC. The MCA of hypercholesterolemic rabbits showed differentially expressed genes that are surprisingly, linked to almost the same node molecules as the hypertensive rabbits, despite a relatively low percentage of 'common genes' (21 and 7% between the two conditions. Up-regulated common genes were related to: UBC, SERPINB2, TNF, HNF4A (hepatocyte nuclear factor 4A and APP, and down-regulated genes, related to UBC. Increased HNF4A message and protein were verified in the aorta. Together, these findings reveal similar nodal molecules and gene pathways in cerebral vessels affected by hypertension or hypercholesterolemia, which could be a basis for synergistic action of risk factors in the pathogenesis of ICLAD.

  7. Peripheral blood transcriptome sequencing reveals rejection-relevant genes in long-term heart transplantation.

    Science.gov (United States)

    Chen, Yan; Zhang, Haibo; Xiao, Xue; Jia, Yixin; Wu, Weili; Liu, Licheng; Jiang, Jun; Zhu, Baoli; Meng, Xu; Chen, Weijun

    2013-10-03

    Peripheral blood-based gene expression patterns have been investigated as biomarkers to monitor the immune system and rule out rejection after heart transplantation. Recent advances in the high-throughput deep sequencing (HTS) technologies provide new leads in transcriptome analysis. By performing Solexa/Illumina's digital gene expression (DGE) profiling, we analyzed gene expression profiles of PBMCs from 6 quiescent (grade 0) and 6 rejection (grade 2R&3R) heart transplant recipients at more than 6 months after transplantation. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in an independent validation cohort of 47 individuals from three rejection groups (ISHLT, grade 0,1R, 2R&3R). Through DGE sequencing and qPCR validation, 10 genes were identified as informative genes for detection of cardiac transplant rejection. A further clustering analysis showed that the 10 genes were not only effective for distinguishing patients with acute cardiac allograft rejection, but also informative for discriminating patients with renal allograft rejection based on both blood and biopsy samples. Moreover, PPI network analysis revealed that the 10 genes were connected to each other within a short interaction distance. We proposed a 10-gene signature for heart transplant patients at high-risk of developing severe rejection, which was found to be effective as well in other organ transplant. Moreover, we supposed that these genes function systematically as biomarkers in long-time allograft rejection. Further validation in broad transplant population would be required before the non-invasive biomarkers can be generally utilized to predict the risk of transplant rejection. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

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    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  9. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae

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    Ma Menggen

    2010-06-01

    Full Text Available Abstract Background Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. Results A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Conclusion Enriched background of transcription abundance

  10. Identification and characterization of parasitism genes from the pinewood nematode Bursaphelenchus xylophilus reveals a multilayered detoxification strategy.

    Science.gov (United States)

    Espada, Margarida; Silva, Ana Cláudia; Eves van den Akker, Sebastian; Cock, Peter J A; Mota, Manuel; Jones, John T

    2016-02-01

    The migratory endoparasitic nematode Bursaphelenchus xylophilus, which is the causal agent of pine wilt disease, has phytophagous and mycetophagous phases during its life cycle. This highly unusual feature distinguishes it from other plant-parasitic nematodes and requires profound changes in biology between modes. During the phytophagous stage, the nematode migrates within pine trees, feeding on the contents of parenchymal cells. Like other plant pathogens, B. xylophilus secretes effectors from pharyngeal gland cells into the host during infection. We provide the first description of changes in the morphology of these gland cells between juvenile and adult life stages. Using a comparative transcriptomics approach and an effector identification pipeline, we identify numerous novel parasitism genes which may be important for the mediation of interactions of B. xylophilus with its host. In-depth characterization of all parasitism genes using in situ hybridization reveals two major categories of detoxification proteins, those specifically expressed in either the pharyngeal gland cells or the digestive system. These data suggest that B. xylophilus incorporates effectors in a multilayer detoxification strategy in order to protect itself from host defence responses during phytophagy. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  11. Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation

    DEFF Research Database (Denmark)

    Kieffer-Kwon, Kyong-Rim; Tang, Zhonghui; Mathe, Ewy

    2013-01-01

    IA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which...... associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during...

  12. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

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    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  13. Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

    Science.gov (United States)

    Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

    2017-05-01

    Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8 + T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8 + T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8 + T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8 + T cell differentiation.

  14. Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

    Science.gov (United States)

    Starcevic, Antonio; Dunlap, Walter C; Cullum, John; Shick, J Malcolm; Hranueli, Daslav; Long, Paul F

    2010-11-12

    The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH) from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues. A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs), which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+)-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis. Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

  15. Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

    Directory of Open Access Journals (Sweden)

    Antonio Starcevic

    2010-11-01

    Full Text Available The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues.A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs, which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis.Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

  16. Gene expression analysis reveals new possible mechanisms of vancomycin-induced nephrotoxicity and identifies gene markers candidates.

    Science.gov (United States)

    Dieterich, Christine; Puey, Angela; Lin, Sylvia; Lyn, Sylvia; Swezey, Robert; Furimsky, Anna; Fairchild, David; Mirsalis, Jon C; Ng, Hanna H

    2009-01-01

    Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription-polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury.

  17. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

    Science.gov (United States)

    Vonk, Freek J; Casewell, Nicholas R; Henkel, Christiaan V; Heimberg, Alysha M; Jansen, Hans J; McCleary, Ryan J R; Kerkkamp, Harald M E; Vos, Rutger A; Guerreiro, Isabel; Calvete, Juan J; Wüster, Wolfgang; Woods, Anthony E; Logan, Jessica M; Harrison, Robert A; Castoe, Todd A; de Koning, A P Jason; Pollock, David D; Yandell, Mark; Calderon, Diego; Renjifo, Camila; Currier, Rachel B; Salgado, David; Pla, Davinia; Sanz, Libia; Hyder, Asad S; Ribeiro, José M C; Arntzen, Jan W; van den Thillart, Guido E E J M; Boetzer, Marten; Pirovano, Walter; Dirks, Ron P; Spaink, Herman P; Duboule, Denis; McGlinn, Edwina; Kini, R Manjunatha; Richardson, Michael K

    2013-12-17

    Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

  18. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

    Science.gov (United States)

    Vonk, Freek J.; Casewell, Nicholas R.; Henkel, Christiaan V.; Heimberg, Alysha M.; Jansen, Hans J.; McCleary, Ryan J. R.; Kerkkamp, Harald M. E.; Vos, Rutger A.; Guerreiro, Isabel; Calvete, Juan J.; Wüster, Wolfgang; Woods, Anthony E.; Logan, Jessica M.; Harrison, Robert A.; Castoe, Todd A.; de Koning, A. P. Jason; Pollock, David D.; Yandell, Mark; Calderon, Diego; Renjifo, Camila; Currier, Rachel B.; Salgado, David; Pla, Davinia; Sanz, Libia; Hyder, Asad S.; Ribeiro, José M. C.; Arntzen, Jan W.; van den Thillart, Guido E. E. J. M.; Boetzer, Marten; Pirovano, Walter; Dirks, Ron P.; Spaink, Herman P.; Duboule, Denis; McGlinn, Edwina; Kini, R. Manjunatha; Richardson, Michael K.

    2013-01-01

    Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection. PMID:24297900

  19. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Science.gov (United States)

    Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

    2012-01-01

    RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  20. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Directory of Open Access Journals (Sweden)

    Sara Kangaspeska

    Full Text Available RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60% of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  1. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

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    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  2. Laminar and dorsoventral molecular organization of the medial entorhinal cortex revealed by large-scale anatomical analysis of gene expression.

    Directory of Open Access Journals (Sweden)

    Helen L Ramsden

    2015-01-01

    Full Text Available Neural circuits in the medial entorhinal cortex (MEC encode an animal's position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. We propose a new molecular basis for distinguishing the deep layers of the MEC and show that their similarity to corresponding layers of neocortex is greater than that of superficial layers. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. We also reveal laminar organization of genes related to disease pathology and suggest that a high metabolic demand predisposes layer II to neurodegenerative pathology. In principle, our computational pipeline can be applied to high-throughput analysis of many forms of neuroanatomical data. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations.

  3. Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells

    International Nuclear Information System (INIS)

    Askmyr, M; Ågerstam, H; Lilljebjörn, H; Hansen, N; Karlsson, C; Palffy, S von; Landberg, N; Högberg, C; Lassen, C; Rissler, M; Richter, J; Ehinger, M; Järås, M; Fioretos, T

    2014-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34 + cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML

  4. Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

    Science.gov (United States)

    Zheng, Chunhong; Zheng, Liangtao; Yoo, Jae-Kwang; Guo, Huahu; Zhang, Yuanyuan; Guo, Xinyi; Kang, Boxi; Hu, Ruozhen; Huang, Julie Y; Zhang, Qiming; Liu, Zhouzerui; Dong, Minghui; Hu, Xueda; Ouyang, Wenjun; Peng, Jirun; Zhang, Zemin

    2017-06-15

    Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8 + T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8 + T cells and Tregs and represses the CD8 + T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Dissection of a locus on mouse chromosome 5 reveals arthritis promoting and inhibitory genes

    DEFF Research Database (Denmark)

    Lindvall, Therese; Karlsson, Jenny; Holmdahl, Rikard

    2009-01-01

    with Eae39 congenic- and sub-interval congenic mice, carrying RIIIS/J genes on the B10.RIII genetic background, revealed three loci within Eae39 that control disease and anti-collagen antibody titers. Two of the loci promoted disease and the third locus was protecting from collagen induced arthritis...... development. By further breeding of mice with small congenic fragments, we identified a 3.2 Megabasepair (Mbp) interval that regulates disease. CONCLUSIONS: Disease promoting- and protecting genes within the Eae39 locus on mouse chromosome 5, control susceptibility to collagen induced arthritis. A disease......-protecting locus in the telomeric part of Eae39 results in lower anti-collagen antibody responses. The study shows the importance of breeding sub-congenic mouse strains to reveal genetic effects on complex diseases....

  6. Stem cell and gene therapies for diabetes mellitus.

    Science.gov (United States)

    Calne, Roy Y; Gan, Shu Uin; Lee, Kok Onn

    2010-03-01

    In this Perspectives article, we comment on the progress in experimental stem cell and gene therapies that might one day become a clinical reality for the treatment of patients with diabetes mellitus. Research on the ability of human embryonic stem cells to differentiate into islet cells has defined the developmental stages and transcription factors involved in this process. However, the clinical applications of human embryonic stem cells are limited by ethical concerns, as well as the potential for teratoma formation. As a consequence, alternative forms of stem cell therapies, such as induced pluripotent stem cells and bone marrow-derived mesenchymal stem cells, have become an area of intense study. Finally, gene therapy shows some promise for the generation of insulin-producing cells. Here, we discuss two of the most frequently used approaches: in vitro gene delivery into cells which are then transplanted into the recipient and direct delivery of genes in vivo.

  7. Comprehensive Gene Expression Profiling Reveals Synergistic Functional Networks in Cerebral Vessels after Hypertension or Hypercholesterolemia

    Science.gov (United States)

    Ong, Wei-Yi; Ng, Mary Pei-Ern; Loke, Sau-Yeen; Jin, Shalai; Wu, Ya-Jun; Tanaka, Kazuhiro; Wong, Peter Tsun-Hon

    2013-01-01

    Atherosclerotic stenosis of cerebral arteries or intracranial large artery disease (ICLAD) is a major cause of stroke especially in Asians, Hispanics and Africans, but relatively little is known about gene expression changes in vessels at risk. This study compares comprehensive gene expression profiles in the middle cerebral artery (MCA) of New Zealand White rabbits exposed to two stroke risk factors i.e. hypertension and/or hypercholesterolemia, by the 2-Kidney-1-Clip method, or dietary supplementation with cholesterol. Microarray and Ingenuity Pathway Analyses of the MCA of the hypertensive rabbits showed up-regulated genes in networks containing the node molecules: UBC (ubiquitin), P38 MAPK, ERK, NFkB, SERPINB2, MMP1 and APP (amyloid precursor protein); and down-regulated genes related to MAPK, ERK 1/2, Akt, 26 s proteasome, histone H3 and UBC. The MCA of hypercholesterolemic rabbits showed differentially expressed genes that are surprisingly, linked to almost the same node molecules as the hypertensive rabbits, despite a relatively low percentage of ‘common genes’ (21 and 7%) between the two conditions. Up-regulated common genes were related to: UBC, SERPINB2, TNF, HNF4A (hepatocyte nuclear factor 4A) and APP, and down-regulated genes, related to UBC. Increased HNF4A message and protein were verified in the aorta. Together, these findings reveal similar nodal molecules and gene pathways in cerebral vessels affected by hypertension or hypercholesterolemia, which could be a basis for synergistic action of risk factors in the pathogenesis of ICLAD. PMID:23874591

  8. Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis.

    Science.gov (United States)

    Peng, Lu; Wang, Lei; Yang, Yi-Fan; Zou, Ming-Min; He, Wei-Yi; Wang, Yue; Wang, Qing; Vasseur, Liette; You, Min-Sheng

    2017-12-30

    As a specialized organ, the insect ovary performs valuable functions by ensuring fecundity and population survival. Oogenesis is the complex physiological process resulting in the production of mature eggs, which are involved in epigenetic programming, germ cell behavior, cell cycle regulation, etc. Identification of the genes involved in ovary development and oogenesis is critical to better understand the reproductive biology and screening for the potential molecular targets in Plutella xylostella, a worldwide destructive pest of economically major crops. Based on transcriptome sequencing, a total of 7.88Gb clean nucleotides was obtained, with 19,934 genes and 1861 new transcripts being identified. Expression profiling indicated that 61.7% of the genes were expressed (FPKM≥1) in the P. xylostella ovary. GO annotation showed that the pathways of multicellular organism reproduction and multicellular organism reproduction process, as well as gamete generation and chorion were significantly enriched. Processes that were most likely relevant to reproduction included the spliceosome, ubiquitin mediated proteolysis, endocytosis, PI3K-Akt signaling pathway, insulin signaling pathway, cAMP signaling pathway, and focal adhesion were identified in the top 20 'highly represented' KEGG pathways. Functional genes involved in oogenesis were further analyzed and validated by qRT-PCR to show their potential predominant roles in P. xylostella reproduction. Our newly developed P. xylostella ovary transcriptome provides an overview of the gene expression profiling in this specialized tissue and the functional gene network closely related to the ovary development and oogenesis. This is the first genome-wide transcriptome dataset of P. xylostella ovary that includes a subset of functionally activated genes. This global approach will be the basis for further studies on molecular mechanisms of P. xylostella reproduction aimed at screening potential molecular targets for integrated pest

  9. In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

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    Arase Sachiko

    2012-03-01

    Full Text Available Abstract Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

  10. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.

    Science.gov (United States)

    Singh, Upinder; Brewer, Jeremy L; Boothroyd, John C

    2002-05-01

    Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

  11. RNA-Seq Reveals Infection-Related Gene Expression Changes in Phytophthora capsici

    Science.gov (United States)

    Chen, Xiao-Ren; Xing, Yu-Ping; Li, Yan-Peng; Tong, Yun-Hui; Xu, Jing-You

    2013-01-01

    Phytophthora capsici is a soilborne plant pathogen capable of infecting a wide range of plants, including many solanaceous crops. However, genetic resistance and fungicides often fail to manage P. capsici due to limited knowledge on the molecular biology and basis of P. capsici pathogenicity. To begin to rectify this situation, Illumina RNA-Seq was used to perform massively parallel sequencing of three cDNA samples derived from P. capsici mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). Over 11 million reads were generated for each cDNA library analyzed. After read mapping to the gene models of P. capsici reference genome, 13,901, 14,633 and 14,695 putative genes were identified from the reads of the MY, ZO and GC libraries, respectively. Comparative analysis between two of samples showed major differences between the expressed gene content of MY, ZO and GC stages. A large number of genes associated with specific stages and pathogenicity were identified, including 98 predicted effector genes. The transcriptional levels of 19 effector genes during the developmental and host infection stages of P. capsici were validated by RT-PCR. Ectopic expression in Nicotiana benthamiana showed that P. capsici RXLR and Crinkler effectors can suppress host cell death triggered by diverse elicitors including P. capsici elicitin and NLP effectors. This study provides a first look at the transcriptome and effector arsenal of P. capsici during the important pre-infection stages. PMID:24019970

  12. Transcript profiling reveals rewiring of iron assimilation gene expression in Candida albicans and C. dubliniensis.

    LENUS (Irish Health Repository)

    Moran, Gary P

    2012-12-01

    Hyphal growth is repressed in Candida albicans and Candida dubliniensis by the transcription factor Nrg1. Transcript profiling of a C. dubliniensis NRG1 mutant identified a common group of 28 NRG1-repressed genes in both species, including the hypha-specific genes HWP1, ECE1 and the regulator of cell elongation UME6. Unexpectedly, C. dubliniensis NRG1 was required for wild-type levels of expression of 10 genes required for iron uptake including seven ferric reductases, SIT1, FTR1 and RBT5. However, at alkaline pH and during filamentous growth in 10% serum, most of these genes were highly induced in C. dubliniensis. Conversely, RBT5, PGA10, FRE10 and FRP1 did not exhibit induction during hyphal growth when NRG1 is downregulated, indicating that in C. dubliniensis NRG1 is also required for optimal expression of these genes in alkaline environments. In iron-depleted medium at pH 4.5, reduced growth of the NRG1 mutant relative to wild type was observed; however, growth was restored to wild-type levels or greater at pH 6.5, indicating that alkaline induction of iron assimilation gene expression could rescue this phenotype. These data indicate that transcriptional control of iron assimilation and pseudohypha formation has been separated in C. albicans, perhaps promoting growth in a wider range of niches.

  13. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    Crescenzi, M.; Pulciani, S.; Carbonari, M.; Tedesco, L.; Russo, G.; Gaetano, C.; Fiorilli, M.

    1986-01-01

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

  14. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

    Directory of Open Access Journals (Sweden)

    Tsutomu Motohashi

    2016-03-01

    Full Text Available Neural crest cells (NC cells are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+ cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

  15. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kejun; Chen, Yaoming; Dai, Zongming; Bi, Yuan; Cai, Tongjian [Department of Occupational and Environmental Health, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Hou, Lichao [Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Chai, Yubo; Song, Qinghe; Chen, Sumin [Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Luo, Wenjing, E-mail: luowenj@fmmu.edu.cn [Department of Occupational and Environmental Health, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China); Chen, Jingyuan, E-mail: jy_chen@fmmu.edu.cn [Department of Occupational and Environmental Health, Fourth Military Medical University, Xi' an 710032, Shaanxi Province (China)

    2010-01-01

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  16. Epigenetic landscapes reveal transcription factors regulating CD8+ T cell differentiation

    Science.gov (United States)

    Yu, Bingfei; Zhang, Kai; Milner, J. Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P.; Pereira, Renata M.; Crotty, Shane; Chang, John T.; Pipkin, Matthew E.; Wang, Wei; Goldrath, Ananda W.

    2017-01-01

    Dynamic changes in the expression of transcription factors (TFs) can influence specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TF among differentially-fated precursor cells suggests additional underlying mechanisms. Here, we profiled genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that TF expression and binding contributed to establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal novel TFs influencing the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector and memory-precursor cell-fates, respectively. Our data define the epigenetic landscape of differentiation intermediates, facilitating identification of TFs with previously unappreciated roles in CD8+ T cell differentiation. PMID:28288100

  17. Complete genome sequence of Fer-de-Lance Virus reveals a novel gene in reptilian Paramyxoviruses

    Science.gov (United States)

    Kurath, G.; Batts, W.N.; Ahne, W.; Winton, J.R.

    2004-01-01

    The complete RNA genome sequence of the archetype reptilian paramyxovirus, Fer-de-Lance virus (FDLV), has been determined. The genome is 15,378 nucleotides in length and consists of seven nonoverlapping genes in the order 3??? N-U-P-M-F-HN-L 5???, coding for the nucleocapsid, unknown, phospho-, matrix, fusion, hemagglutinin-neuraminidase, and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and tri-nucleotide intergenic regions similar to those of other Paramyxoviridae. The FDLV P gene expression strategy is like that of rubulaviruses, which express the accessory V protein from the primary transcript and edit a portion of the mRNA to encode P and I proteins. There is also an overlapping open reading frame potentially encoding a small basic protein in the P gene. The gene designated U (unknown), encodes a deduced protein of 19.4 kDa that has no counterpart in other paramyxoviruses and has no similarity with sequences in the National Center for Biotechnology Information database. Active transcription of the U gene in infected cells was demonstrated by Northern blot analysis, and bicistronic N-U mRNA was also evident. The genomes of two other snake paramyxovirus genotypes were also found to have U genes, with 11 to 16% nucleotide divergence from the FDLV U gene. Pairwise comparisons of amino acid identities and phylogenetic analyses of all deduced FDLV protein sequences with homologous sequences from other Paramyxoviridae indicate that FDLV represents a new genus within the subfamily Paramyxovirinae. We suggest the name Ferlavirus for the new genus, with FDLV as the type species.

  18. Chemoresistance acquisition induces a global shift of expression of aniogenesis-associated genes and increased pro-angogenic activity in neuroblastoma cells

    NARCIS (Netherlands)

    Michaelis, Martin; Klassert, Denise; Barth, Susanne; Suhan, Tatyana; Breitling, Rainer; Mayer, Bernd; Hinsch, Nora; Doerr, Hans W.; Cinatl, Jaroslav; Cinatl, Jindrich; Cinatl jr., Jindrich

    2009-01-01

    Background: Chemoresistance acquisition may influence cancer cell biology. Here, bioinformatics analysis of gene expression data was used to identify chemoresistance-associated changes in neuroblastoma biology. Results: Bioinformatics analysis of gene expression data revealed that expression of

  19. Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

    Science.gov (United States)

    Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J.; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F.; Emili, Andrew

    2011-01-01

    As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. PMID:22125496

  20. Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2011-11-01

    Full Text Available As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium and prototrophic (minimal medium culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens and an important target.

  1. Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics.

    Directory of Open Access Journals (Sweden)

    Andreas Sagner

    2018-02-01

    Full Text Available During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We addressed the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we defined the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.

  2. Analysis of gene expression during odontogenic differentiation of cultured human dental pulp cells

    Directory of Open Access Journals (Sweden)

    Min-Seock Seo

    2012-08-01

    Full Text Available Objectives We analyzed gene-expression profiles after 14 day odontogenic induction of human dental pulp cells (DPCs using a DNA microarray and sought candidate genes possibly associated with mineralization. Materials and Methods Induced human dental pulp cells were obtained by culturing DPCs in odontogenic induction medium (OM for 14 day. Cells exposed to normal culture medium were used as controls. Total RNA was extracted from cells and analyzed by microarray analysis and the key results were confirmed selectively by reverse-transcriptase polymerase chain reaction (RT-PCR. We also performed a gene set enrichment analysis (GSEA of the microarray data. Results Six hundred and five genes among the 47,320 probes on the BeadChip differed by a factor of more than two-fold in the induced cells. Of these, 217 genes were upregulated, and 388 were down-regulated. GSEA revealed that in the induced cells, genes implicated in Apoptosis and Signaling by wingless MMTV integration (Wnt were significantly upregulated. Conclusions Genes implicated in Apoptosis and Signaling by Wnt are highly connected to the differentiation of dental pulp cells into odontoblast.

  3. Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana.

    Science.gov (United States)

    Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe; Probst, Aline V

    2018-04-06

    Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization.

  4. Potential translational targets revealed by linking mouse grooming behavioral phenotypes to gene expression using public databases.

    Science.gov (United States)

    Roth, Andrew; Kyzar, Evan J; Cachat, Jonathan; Stewart, Adam Michael; Green, Jeremy; Gaikwad, Siddharth; O'Leary, Timothy P; Tabakoff, Boris; Brown, Richard E; Kalueff, Allan V

    2013-01-10

    Rodent self-grooming is an important, evolutionarily conserved behavior, highly sensitive to pharmacological and genetic manipulations. Mice with aberrant grooming phenotypes are currently used to model various human disorders. Therefore, it is critical to understand the biology of grooming behavior, and to assess its translational validity to humans. The present in-silico study used publicly available gene expression and behavioral data obtained from several inbred mouse strains in the open-field, light-dark box, elevated plus- and elevated zero-maze tests. As grooming duration differed between strains, our analysis revealed several candidate genes with significant correlations between gene expression in the brain and grooming duration. The Allen Brain Atlas, STRING, GoMiner and Mouse Genome Informatics databases were used to functionally map and analyze these candidate mouse genes against their human orthologs, assessing the strain ranking of their expression and the regional distribution of expression in the mouse brain. This allowed us to identify an interconnected network of candidate genes (which have expression levels that correlate with grooming behavior), display altered patterns of expression in key brain areas related to grooming, and underlie important functions in the brain. Collectively, our results demonstrate the utility of large-scale, high-throughput data-mining and in-silico modeling for linking genomic and behavioral data, as well as their potential to identify novel neural targets for complex neurobehavioral phenotypes, including grooming. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Transcriptome analysis of paired primary colorectal carcinoma and liver metastases reveals fusion transcripts and similar gene expression profiles in primary carcinoma and liver metastases

    International Nuclear Information System (INIS)

    Lee, Ja-Rang; Kwon, Chae Hwa; Choi, Yuri; Park, Hye Ji; Kim, Hyun Sung; Jo, Hong-Jae; Oh, Nahmgun; Park, Do Youn

    2016-01-01

    Despite the clinical significance of liver metastases, the difference between molecular and cellular changes in primary colorectal cancers (CRC) and matched liver metastases is poorly understood. In order to compare gene expression patterns and identify fusion genes in these two types of tumors, we performed high-throughput transcriptome sequencing of five sets of quadruple-matched tissues (primary CRC, liver metastases, normal colon, and liver). The gene expression patterns in normal colon and liver were successfully distinguished from those in CRCs; however, RNA sequencing revealed that the gene expression between primary CRCs and their matched liver metastases is highly similar. We identified 1895 genes that were differentially expressed in the primary carcinoma and liver metastases, than that in the normal colon tissues. A major proportion of the transcripts, identified by gene expression profiling as significantly enriched in the primary carcinoma and metastases, belonged to gene ontology categories involved in the cell cycle, mitosis, and cell division. Furthermore, we identified gene fusion events in primary carcinoma and metastases, and the fusion transcripts were experimentally confirmed. Among these, a chimeric transcript resulting from the fusion of RNF43 and SUPT4H1 was found to occur frequently in primary colorectal carcinoma. In addition, knockdown of the expression of this RNF43-SUPT4H1 chimeric transcript was found to have a growth-inhibitory effect in colorectal cancer cells. The present study reports a high concordance of gene expression in the primary carcinoma and liver metastases, and reveals potential new targets, such as fusion genes, against primary and metastatic colorectal carcinoma. The online version of this article (doi:10.1186/s12885-016-2596-3) contains supplementary material, which is available to authorized users

  6. Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Zhifu Sun

    Full Text Available We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+ and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A, and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

  7. Signature gene expression reveals novel clues to the molecular mechanisms of dimorphic transition in Penicillium marneffei.

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    Ence Yang

    2014-10-01

    Full Text Available Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition

  8. Fine Mapping and Transcriptome Analysis Reveal Candidate Genes Associated with Hybrid Lethality in Cabbage (Brassica Oleracea).

    Science.gov (United States)

    Xiao, Zhiliang; Hu, Yang; Zhang, Xiaoli; Xue, Yuqian; Fang, Zhiyuan; Yang, Limei; Zhang, Yangyong; Liu, Yumei; Li, Zhansheng; Liu, Xing; Liu, Zezhou; Lv, Honghao; Zhuang, Mu

    2017-06-05

    Hybrid lethality is a deleterious phenotype that is vital to species evolution. We previously reported hybrid lethality in cabbage ( Brassica oleracea ) and performed preliminary mapping of related genes. In the present study, the fine mapping of hybrid lethal genes revealed that BoHL1 was located on chromosome C1 between BoHLTO124 and BoHLTO130, with an interval of 101 kb. BoHL2 was confirmed to be between insertion-deletion (InDels) markers HL234 and HL235 on C4, with a marker interval of 70 kb. Twenty-eight and nine annotated genes were found within the two intervals of BoHL1 and BoHL2 , respectively. We also applied RNA-Seq to analyze hybrid lethality in cabbage. In the region of BoHL1 , seven differentially expressed genes (DEGs) and five resistance (R)-related genes (two in common, i.e., Bo1g153320 and Bo1g153380 ) were found, whereas in the region of BoHL2 , two DEGs and four R-related genes (two in common, i.e., Bo4g173780 and Bo4g173810 ) were found. Along with studies in which R genes were frequently involved in hybrid lethality in other plants, these interesting R-DEGs may be good candidates associated with hybrid lethality. We also used SNP/InDel analyses and quantitative real-time PCR to confirm the results. This work provides new insight into the mechanisms of hybrid lethality in cabbage.

  9. Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

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    Gerasimos F Kremmydas

    Full Text Available Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ, and two genes (sup5 and sup6 which seem to be organized in a putative operon. This operon (named supX consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon.

  10. Advances of reporter gene imaging monitoring stem cell therapy

    International Nuclear Information System (INIS)

    Pei Zhijun; Zhang Yongxue

    2010-01-01

    Stem cell transplantation in the treatment of various tissue damage or degenerative diseases are research hotspots both at home and abroad. However, ignorance of the homing, differentiation and functional expression of the stem cell in vivo influence the further development of stem cell therapy. As an important component of molecular imaging technology, reporter gene imaging dynamically monitors the change of stem cell in vivo via monitoring the expression of transfected reporter gene. This paper briefly describes the latest research progress and the future development trend of the monitoring of reporter gene imaging in stem cell therapy in vivo. (authors)

  11. Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis.

    Science.gov (United States)

    Cochain, Clément; Vafadarnejad, Ehsan; Arampatzi, Panagiota; Jaroslav, Pelisek; Winkels, Holger; Ley, Klaus; Wolf, Dennis; Saliba, Antoine-Emmanuel; Zernecke, Alma

    2018-03-15

    Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they ha ve been defined by the expression of a restricted number of markers. Objective: We have applied single-cell RNA-seq as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Methods and Results: We performed single-cell RNA sequencing of total aortic CD45 + cells extracted from the non-diseased (chow fed) and atherosclerotic (11 weeks of high fat diet) aorta of Ldlr -/- mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, Resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortae, whereas monocytes, monocyte-derived dendritic cells (MoDC), and two populations of macrophages were almost exclusively detectable in atherosclerotic aortae, comprising Inflammatory macrophages showing enrichment in I l1b , and previously undescribed TREM2 hi macrophages. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these three macrophage subsets and MoDC, and uncovered putative functions of each cell type. Notably, TREM2 hi macrophages appeared to be endowed with specialized functions in lipid metabolism and catabolism, and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe -/- aortae, indicating relevance of our findings in different stages of atherosclerosis and mouse models. Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic

  12. Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.

    Science.gov (United States)

    Glessner, Joseph T; Wang, Kai; Cai, Guiqing; Korvatska, Olena; Kim, Cecilia E; Wood, Shawn; Zhang, Haitao; Estes, Annette; Brune, Camille W; Bradfield, Jonathan P; Imielinski, Marcin; Frackelton, Edward C; Reichert, Jennifer; Crawford, Emily L; Munson, Jeffrey; Sleiman, Patrick M A; Chiavacci, Rosetta; Annaiah, Kiran; Thomas, Kelly; Hou, Cuiping; Glaberson, Wendy; Flory, James; Otieno, Frederick; Garris, Maria; Soorya, Latha; Klei, Lambertus; Piven, Joseph; Meyer, Kacie J; Anagnostou, Evdokia; Sakurai, Takeshi; Game, Rachel M; Rudd, Danielle S; Zurawiecki, Danielle; McDougle, Christopher J; Davis, Lea K; Miller, Judith; Posey, David J; Michaels, Shana; Kolevzon, Alexander; Silverman, Jeremy M; Bernier, Raphael; Levy, Susan E; Schultz, Robert T; Dawson, Geraldine; Owley, Thomas; McMahon, William M; Wassink, Thomas H; Sweeney, John A; Nurnberger, John I; Coon, Hilary; Sutcliffe, James S; Minshew, Nancy J; Grant, Struan F A; Bucan, Maja; Cook, Edwin H; Buxbaum, Joseph D; Devlin, Bernie; Schellenberg, Gerard D; Hakonarson, Hakon

    2009-05-28

    Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with approximately 550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 x 10(-3)). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 x 10(-3)). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 x 10(-6)). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD.

  13. Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells

    International Nuclear Information System (INIS)

    Zhao, Gang; Chen, Jiawei; Deng, Yanqiu; Gao, Feng; Zhu, Jiwei; Feng, Zhenzhong; Lv, Xiuhong; Zhao, Zheng

    2011-01-01

    Highlights: → NDRG1 was knockdown in cervical and ovarian cancer cell lines by shRNA technology. → NDRG1 knockdown resulted in increased cell invasion activities. → Ninety-six common deregulated genes in both cell lines were identified by cDNA microarray. → Eleven common NDRG1-regulated genes might enhance cell invasive activity. → Regulation of invasion by NDRG1 is an indirect and complicated process. -- Abstract: N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.

  14. [Prostatic granulomas revealing a peripheral T-cell lymphoma].

    Science.gov (United States)

    Foguem, C; Curlier, E; Rouamba, M-M; Regent, A; Philippe, P

    2009-02-01

    The presence of granulomas on tissue biopsie has been reported in a wide range of disorders. The clinical presentation and the diagnostic work-up of granulomatosis can be difficult as it is illustrated in the following report. A 59-year-old patient was referred in 2002 for a granulomatous prostatitis. Physical examination was normal. Except for the increase of prostate-specific antigen (which motivated a biopsy), the laboratory results were normal. Thoracic CT-scan disclosed mediastinal lymph nodes. A minor salivary gland biopsy was consistent with the diagnosis of sarcoidosis. In 2004, the patient presented an epidermal necrolysis, and in 2005 the deterioration of general status raised suspicion of a lymphoproliferative disorder. Liver and bone marrow biopsies revealed a granulomatous process. Despite steroid therapy, the patient died. Autopsy discloses a anaplasic T cell lymphoma. This report illustrates the relationship between sarcoidosis and lymphoma as a mode of presentation, a complication, or an accidental but misleading association? The association between anaplastic lymphoma and sarcoidosis is exceptional.

  15. RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature

    Science.gov (United States)

    Viollet, Coralie; Davis, David A.; Tekeste, Shewit S.; Reczko, Martin; Pezzella, Francesco; Ragoussis, Jiannis

    2017-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases. PMID:28046107

  16. Analysis of bone marrow stromal cell transferred bacterial {beta}-galactosidase gene by PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Kumakawa, Toshiro [Tokyo Metropolitan Geriatric Hospital, Tokyo (Japan). Dept. of Blood Transfusion and Hematology; Hibino, Hitoshi; Tani, Kenzaburo; Asano, Shigetaka; Futatugawa, Shouji; Sera, Kouichiro

    1997-12-31

    PIXE, Particle Induced X-ray Emission, is a powerful, multi-elemental analysis method which has many distinguishing features and has been used in varies research fields. Recently the method of applying baby cyclotrons for nuclear medicine to PIXE has been developed. This enables us to study biomedical phenomena from the physical point of view. Mouse bone marrow stromal cells were transferred bacterial {beta}-galactosidase gene (LacZ gene) by murine retroviral vectors. Analysis of the bone marrow stromal cells with the LacZ gene by PIXE revealed remarkable changes of intracellular trace elements compared with the normal control cells. These results indicate that gene transfer by retroviral vectors may bring about a dynamic change of intracellular circumstances of the target cell. (author)

  17. Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Xuewei Wang

    Full Text Available The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD.

  18. Gene expression profiling reveals new potential players of gonad differentiation in the chicken embryo.

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    Gwenn-Aël Carré

    Full Text Available BACKGROUND: In birds as in mammals, a genetic switch determines whether the undifferentiated gonad develops into an ovary or a testis. However, understanding of the molecular pathway(s involved in gonad differentiation is still incomplete. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of improving characterization of the molecular pathway(s involved in gonad differentiation in the chicken embryo, we developed a large scale real time reverse transcription polymerase chain reaction approach on 110 selected genes for evaluation of their expression profiles during chicken gonad differentiation between days 5.5 and 19 of incubation. Hierarchical clustering analysis of the resulting datasets discriminated gene clusters expressed preferentially in the ovary or the testis, and/or at early or later periods of embryonic gonad development. Fitting a linear model and testing the comparisons of interest allowed the identification of new potential actors of gonad differentiation, such as Z-linked ADAMTS12, LOC427192 (corresponding to NIM1 protein and CFC1, that are upregulated in the developing testis, and BMP3 and Z-linked ADAMTSL1, that are preferentially expressed in the developing ovary. Interestingly, the expression patterns of several members of the transforming growth factor β family were sexually dimorphic, with inhibin subunits upregulated in the testis, and bone morphogenetic protein subfamily members including BMP2, BMP3, BMP4 and BMP7, upregulated in the ovary. This study also highlighted several genes displaying asymmetric expression profiles such as GREM1 and BMP3 that are potentially involved in different aspects of gonad left-right asymmetry. CONCLUSION/SIGNIFICANCE: This study supports the overall conservation of vertebrate sex differentiation pathways but also reveals some particular feature of gene expression patterns during gonad development in the chicken. In particular, our study revealed new candidate genes which may be potential actors

  19. Gene Expression Profiling Reveals New Potential Players of Gonad Differentiation in the Chicken Embryo

    Science.gov (United States)

    Carré, Gwenn-Aël; Couty, Isabelle; Hennequet-Antier, Christelle; Govoroun, Marina S.

    2011-01-01

    Background In birds as in mammals, a genetic switch determines whether the undifferentiated gonad develops into an ovary or a testis. However, understanding of the molecular pathway(s) involved in gonad differentiation is still incomplete. Methodology/Principal Findings With the aim of improving characterization of the molecular pathway(s) involved in gonad differentiation in the chicken embryo, we developed a large scale real time reverse transcription polymerase chain reaction approach on 110 selected genes for evaluation of their expression profiles during chicken gonad differentiation between days 5.5 and 19 of incubation. Hierarchical clustering analysis of the resulting datasets discriminated gene clusters expressed preferentially in the ovary or the testis, and/or at early or later periods of embryonic gonad development. Fitting a linear model and testing the comparisons of interest allowed the identification of new potential actors of gonad differentiation, such as Z-linked ADAMTS12, LOC427192 (corresponding to NIM1 protein) and CFC1, that are upregulated in the developing testis, and BMP3 and Z-linked ADAMTSL1, that are preferentially expressed in the developing ovary. Interestingly, the expression patterns of several members of the transforming growth factor β family were sexually dimorphic, with inhibin subunits upregulated in the testis, and bone morphogenetic protein subfamily members including BMP2, BMP3, BMP4 and BMP7, upregulated in the ovary. This study also highlighted several genes displaying asymmetric expression profiles such as GREM1 and BMP3 that are potentially involved in different aspects of gonad left-right asymmetry. Conclusion/Significance This study supports the overall conservation of vertebrate sex differentiation pathways but also reveals some particular feature of gene expression patterns during gonad development in the chicken. In particular, our study revealed new candidate genes which may be potential actors of chicken gonad

  20. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms.

    Science.gov (United States)

    Svingen, T; Jørgensen, A; Rajpert-De Meyts, E

    2014-08-01

    The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Dynamic changes in protein functional linkage networks revealed by integration with gene expression data.

    Directory of Open Access Journals (Sweden)

    Shubhada R Hegde

    2008-11-01

    Full Text Available Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume, proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein:protein interactions might yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile of protein:protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data.

  2. The Him gene reveals a balance of inputs controlling muscle differentiation in Drosophila.

    Science.gov (United States)

    Liotta, David; Han, Jun; Elgar, Stuart; Garvey, Clare; Han, Zhe; Taylor, Michael V

    2007-08-21

    Tissue development requires the controlled regulation of cell-differentiation programs. In muscle, the Mef2 transcription factor binds to and activates the expression of many genes and has a major positive role in the orchestration of differentiation. However, little is known about how Mef2 activity is regulated in vivo during development. Here, we characterize a gene, Holes in muscle (Him), which our results indicate is part of this control in Drosophila. Him expression rapidly declines as embryonic muscle differentiates, and consistent with this, Him overexpression inhibits muscle differentiation. This inhibitory effect is suppressed by mef2, implicating Him in the mef2 pathway. We then found that Him downregulates the transcriptional activity of Mef2 in both cell culture and in vivo. Furthermore, Him protein binds Groucho, a conserved, transcriptional corepressor, through a WRPW motif and requires this motif and groucho function to inhibit both muscle differentiation and Mef2 activity during development. Together, our results identify a mechanism that can inhibit muscle differentiation in vivo. We conclude that a balance of positive and negative inputs, including Mef2, Him, and Groucho, controls muscle differentiation during Drosophila development and suggest that one outcome is to hold developing muscle cells in a state with differentiation genes poised to be expressed.

  3. A model of gene expression based on random dynamical systems reveals modularity properties of gene regulatory networks.

    Science.gov (United States)

    Antoneli, Fernando; Ferreira, Renata C; Briones, Marcelo R S

    2016-06-01

    Here we propose a new approach to modeling gene expression based on the theory of random dynamical systems (RDS) that provides a general coupling prescription between the nodes of any given regulatory network given the dynamics of each node is modeled by a RDS. The main virtues of this approach are the following: (i) it provides a natural way to obtain arbitrarily large networks by coupling together simple basic pieces, thus revealing the modularity of regulatory networks; (ii) the assumptions about the stochastic processes used in the modeling are fairly general, in the sense that the only requirement is stationarity; (iii) there is a well developed mathematical theory, which is a blend of smooth dynamical systems theory, ergodic theory and stochastic analysis that allows one to extract relevant dynamical and statistical information without solving the system; (iv) one may obtain the classical rate equations form the corresponding stochastic version by averaging the dynamic random variables (small noise limit). It is important to emphasize that unlike the deterministic case, where coupling two equations is a trivial matter, coupling two RDS is non-trivial, specially in our case, where the coupling is performed between a state variable of one gene and the switching stochastic process of another gene and, hence, it is not a priori true that the resulting coupled system will satisfy the definition of a random dynamical system. We shall provide the necessary arguments that ensure that our coupling prescription does indeed furnish a coupled regulatory network of random dynamical systems. Finally, the fact that classical rate equations are the small noise limit of our stochastic model ensures that any validation or prediction made on the basis of the classical theory is also a validation or prediction of our model. We illustrate our framework with some simple examples of single-gene system and network motifs. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    International Nuclear Information System (INIS)

    Robinson, Claire; Kolb, Andreas F.

    2009-01-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A β-galactosidase reporter gene was inserted in place of the β-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the β-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal β-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the β-casein gene

  5. Estimating immunoregulatory gene networks in human herpesvirus type 6-infected T cells

    International Nuclear Information System (INIS)

    Takaku, Tomoiku; Ohyashiki, Junko H.; Zhang, Yu; Ohyashiki, Kazuma

    2005-01-01

    The immune response to viral infection involves complex network of dynamic gene and protein interactions. We present here the dynamic gene network of the host immune response during human herpesvirus type 6 (HHV-6) infection in an adult T-cell leukemia cell line. Using a pathway-focused oligonucleotide DNA microarray, we found a possible association between chemokine genes regulating Th1/Th2 balance and genes regulating T-cell proliferation during HHV-6B infection. Gene network analysis using an integrated comprehensive workbench, VoyaGene, revealed that a gene encoding a TEC-family kinase, ITK, might be a putative modulator in the host immune response against HHV-6B infection. We conclude that Th2-dominated inflammatory reaction in host cells may play an important role in HHV-6B-infected T cells, thereby suggesting the possibility that ITK might be a therapeutic target in diseases related to dysregulation of Th1/Th2 balance. This study describes a novel approach to find genes related with the complex host-virus interaction using microarray data employing the Bayesian statistical framework

  6. Gene expression profiling in cells with enhanced gamma-secretase activity.

    Directory of Open Access Journals (Sweden)

    Alexandra I Magold

    2009-09-01

    Full Text Available Processing by gamma-secretase of many type-I membrane protein substrates triggers signaling cascades by releasing intracellular domains (ICDs that, following nuclear translocation, modulate the transcription of different genes regulating a diverse array of cellular and biological processes. Because the list of gamma-secretase substrates is growing quickly and this enzyme is a cancer and Alzheimer's disease therapeutic target, the mapping of gamma-secretase activity susceptible gene transcription is important for sharpening our view of specific affected genes, molecular functions and biological pathways.To identify genes and molecular functions transcriptionally affected by gamma-secretase activity, the cellular transcriptomes of Chinese hamster ovary (CHO cells with enhanced and inhibited gamma-secretase activity were analyzed and compared by cDNA microarray. The functional clustering by FatiGO of the 1,981 identified genes revealed over- and under-represented groups with multiple activities and functions. Single genes with the most pronounced transcriptional susceptibility to gamma-secretase activity were evaluated by real-time PCR. Among the 21 validated genes, the strikingly decreased transcription of PTPRG and AMN1 and increased transcription of UPP1 potentially support data on cell cycle disturbances relevant to cancer, stem cell and neurodegenerative diseases' research. The mapping of interactions of proteins encoded by the validated genes exclusively relied on evidence-based data and revealed broad effects on Wnt pathway members, including WNT3A and DVL3. Intriguingly, the transcription of TERA, a gene of unknown function, is affected by gamma-secretase activity and was significantly altered in the analyzed human Alzheimer's disease brain cortices.Investigating the effects of gamma-secretase activity on gene transcription has revealed several affected clusters of molecular functions and, more specifically, 21 genes that hold significant

  7. Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

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    Chung Jae

    2009-06-01

    Full Text Available Abstract Background Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition. Methods To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis. Results Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99 between the total number of hypermethylated CGIs and GI50 values (i.e., increased drug resistance following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells. Conclusion Selective epigenetic disruption of distinct biological

  8. Genomic characterisation of Wongabel virus reveals novel genes within the Rhabdoviridae.

    Science.gov (United States)

    Gubala, Aneta J; Proll, David F; Barnard, Ross T; Cowled, Chris J; Crameri, Sandra G; Hyatt, Alex D; Boyle, David B

    2008-06-20

    Viruses belonging to the family Rhabdoviridae infect a variety of different hosts, including insects, vertebrates and plants. Currently, there are approximately 200 ICTV-recognised rhabdoviruses isolated around the world. However, the majority remain poorly characterised and only a fraction have been definitively assigned to genera. The genomic and transcriptional complexity displayed by several of the characterised rhabdoviruses indicates large diversity and complexity within this family. To enable an improved taxonomic understanding of this family, it is necessary to gain further information about the poorly characterised members of this family. Here we present the complete genome sequence and predicted transcription strategy of Wongabel virus (WONV), a previously uncharacterised rhabdovirus isolated from biting midges (Culicoides austropalpalis) collected in northern Queensland, Australia. The 13,196 nucleotide genome of WONV encodes five typical rhabdovirus genes N, P, M, G and L. In addition, the WONV genome contains three genes located between the P and M genes (U1, U2, U3) and two open reading frames overlapping with the N and G genes (U4, U5). These five additional genes and their putative protein products appear to be novel, and their functions are unknown. Predictive analysis of the U5 gene product revealed characteristics typical of viroporins, and indicated structural similarities with the alpha-1 protein (putative viroporin) of viruses in the genus Ephemerovirus. Phylogenetic analyses of the N and G proteins of WONV indicated closest similarity with the avian-associated Flanders virus; however, the genomes of these two viruses are significantly diverged. WONV displays a novel and unique genome structure that has not previously been described for any animal rhabdovirus.

  9. Comparative transcriptome analysis reveals differentially expressed genes associated with sex expression in garden asparagus (Asparagus officinalis).

    Science.gov (United States)

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Gao, Wu-Jun

    2017-08-22

    Garden asparagus (Asparagus officinalis) is a highly valuable vegetable crop of commercial and nutritional interest. It is also commonly used to investigate the mechanisms of sex determination and differentiation in plants. However, the sex expression mechanisms in asparagus remain poorly understood. De novo transcriptome sequencing via Illumina paired-end sequencing revealed more than 26 billion bases of high-quality sequence data from male and female asparagus flower buds. A total of 72,626 unigenes with an average length of 979 bp were assembled. In comparative transcriptome analysis, 4876 differentially expressed genes (DEGs) were identified in the possible sex-determining stage of female and male/supermale flower buds. Of these DEGs, 433, including 285 male/supermale-biased and 149 female-biased genes, were annotated as flower related. Of the male/supermale-biased flower-related genes, 102 were probably involved in anther development. In addition, 43 DEGs implicated in hormone response and biosynthesis putatively associated with sex expression and reproduction were discovered. Moreover, 128 transcription factor (TF)-related genes belonging to various families were found to be differentially expressed, and this finding implied the essential roles of TF in sex determination or differentiation in asparagus. Correlation analysis indicated that miRNA-DEG pairs were also implicated in asparagus sexual development. Our study identified a large number of DEGs involved in the sex expression and reproduction of asparagus, including known genes participating in plant reproduction, plant hormone signaling, TF encoding, and genes with unclear functions. We also found that miRNAs might be involved in the sex differentiation process. Our study could provide a valuable basis for further investigations on the regulatory networks of sex determination and differentiation in asparagus and facilitate further genetic and genomic studies on this dioecious species.

  10. Transcriptome and proteome data reveal candidate genes for pollinator attraction in sexually deceptive orchids.

    Science.gov (United States)

    Sedeek, Khalid E M; Qi, Weihong; Schauer, Monica A; Gupta, Alok K; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P; Schlüter, Philipp M

    2013-01-01

    Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of

  11. Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells.

    Science.gov (United States)

    Jung, Ha-Na; Zerin, Tamanna; Podder, Biswajit; Song, Ho-Yeon; Kim, Yong-Sik

    2014-06-01

    In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 μg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 μg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 μg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding antioxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intracellular reactive oxygen species and alteration of gene expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

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    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  13. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

    Science.gov (United States)

    Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

    2011-01-01

    Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  14. Live cell CRISPR-imaging in plants reveals dynamic telomere movements

    KAUST Repository

    Dreissig, Steven

    2017-05-16

    Elucidating the spatio-temporal organization of the genome inside the nucleus is imperative to understand the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies which reveal genomic information and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm within 30 minutes during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for imaging of multiple genomic loci in live plants cells. CRISPR-imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

  15. Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila.

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    Bruno Zeitouni

    2007-10-01

    Full Text Available Drosophila provides a powerful system for defining the complex genetic programs that drive organogenesis. Under control of the steroid hormone ecdysone, the adult heart in Drosophila forms during metamorphosis by a remodelling of the larval cardiac organ. Here, we evaluated the extent to which transcriptional signatures revealed by genomic approaches can provide new insights into the molecular pathways that underlie heart organogenesis. Whole-genome expression profiling at eight successive time-points covering adult heart formation revealed a highly dynamic temporal map of gene expression through 13 transcript clusters with distinct expression kinetics. A functional atlas of the transcriptome profile strikingly points to the genomic transcriptional response of the ecdysone cascade, and a sharp regulation of key components belonging to a few evolutionarily conserved signalling pathways. A reverse genetic analysis provided evidence that these specific signalling pathways are involved in discrete steps of adult heart formation. In particular, the Wnt signalling pathway is shown to participate in inflow tract and cardiomyocyte differentiation, while activation of the PDGF-VEGF pathway is required for cardiac valve formation. Thus, a detailed temporal map of gene expression can reveal signalling pathways responsible for specific developmental programs and provides here substantial grasp into heart formation.

  16. Circuit-wide Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulating Depression Susceptibility.

    Science.gov (United States)

    Bagot, Rosemary C; Cates, Hannah M; Purushothaman, Immanuel; Lorsch, Zachary S; Walker, Deena M; Wang, Junshi; Huang, Xiaojie; Schlüter, Oliver M; Maze, Ian; Peña, Catherine J; Heller, Elizabeth A; Issler, Orna; Wang, Minghui; Song, Won-Min; Stein, Jason L; Liu, Xiaochuan; Doyle, Marie A; Scobie, Kimberly N; Sun, Hao Sheng; Neve, Rachael L; Geschwind, Daniel; Dong, Yan; Shen, Li; Zhang, Bin; Nestler, Eric J

    2016-06-01

    Depression is a complex, heterogeneous disorder and a leading contributor to the global burden of disease. Most previous research has focused on individual brain regions and genes contributing to depression. However, emerging evidence in humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here, we performed RNA sequencing on four brain regions from control animals and those susceptible or resilient to chronic social defeat stress at multiple time points. We employed an integrative network biology approach to identify transcriptional networks and key driver genes that regulate susceptibility to depressive-like symptoms. Further, we validated in vivo several key drivers and their associated transcriptional networks that regulate depression susceptibility and confirmed their functional significance at the levels of gene transcription, synaptic regulation, and behavior. Our study reveals novel transcriptional networks that control stress susceptibility and offers fundamentally new leads for antidepressant drug discovery. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Reliable and rapid characterization of functional FCN2 gene variants reveals diverse geographical patterns

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    Ojurongbe Olusola

    2012-05-01

    Full Text Available Abstract Background Ficolin-2 coded by FCN2 gene is a soluble serum protein and an innate immune recognition element of the complement system. FCN2 gene polymorphisms reveal distinct geographical patterns and are documented to alter serum ficolin levels and modulate disease susceptibility. Methods We employed a real-time PCR based on Fluorescence Resonance Energy Transfer (FRET method to genotype four functional SNPs including -986 G > A (#rs3124952, -602 G > A (#rs3124953, -4A > G (#rs17514136 and +6424 G > T (#rs7851696 in the ficolin-2 (FCN2 gene. We characterized the FCN2 variants in individuals representing Brazilian (n = 176, Nigerian (n = 180, Vietnamese (n = 172 and European Caucasian ethnicity (n = 165. Results We observed that the genotype distribution of three functional SNP variants (−986 G > A, -602 G > A and -4A > G differ significantly between the populations investigated (p p  Conclusions The observed distribution of the FCN2 functional SNP variants may likely contribute to altered serum ficolin levels and this may depend on the different disease settings in world populations. To conclude, the use of FRET based real-time PCR especially for FCN2 gene will benefit a larger scientific community who extensively depend on rapid, reliable method for FCN2 genotyping.

  18. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

    OpenAIRE

    Soucie, E.L.; Weng, Z.; Geirsdottir, L.; Molawi, K.; Maurizio, J.; Fenouil, R.; Mossadegh-Keller, N.; Gimenez, G.; VanHille, L.; Beniazza, M.; Favret, J.; Berruyer, C.; Perrin, P.; Hacohen, N.; Andrau, J.C.

    2016-01-01

    Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down...

  19. Stem cell collection and gene transfer in Fanconi anemia.

    Science.gov (United States)

    Kelly, Patrick F; Radtke, Susan; von Kalle, Christof; Balcik, Brenden; Bohn, Kimberley; Mueller, Robin; Schuesler, Todd; Haren, Moira; Reeves, Lilith; Cancelas, Jose A; Leemhuis, Thomas; Harris, Richard; Auerbach, Arleen D; Smith, Franklin O; Davies, Stella M; Williams, David A

    2007-01-01

    Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34(+) stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34(+) cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34(+) cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

  20. Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

    Science.gov (United States)

    Judelson, Howard S; Ah-Fong, Audrey M V; Aux, George; Avrova, Anna O; Bruce, Catherine; Cakir, Cahid; da Cunha, Luis; Grenville-Briggs, Laura; Latijnhouwers, Maita; Ligterink, Wilco; Meijer, Harold J G; Roberts, Samuel; Thurber, Carrie S; Whisson, Stephen C; Birch, Paul R J; Govers, Francine; Kamoun, Sophien; van West, Pieter; Windass, John

    2008-04-01

    Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

  1. Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential

    Science.gov (United States)

    Hurley, Jennifer M.; Dasgupta, Arko; Emerson, Jillian M.; Zhou, Xiaoying; Ringelberg, Carol S.; Knabe, Nicole; Lipzen, Anna M.; Lindquist, Erika A.; Daum, Christopher G.; Barry, Kerrie W.; Grigoriev, Igor V.; Smith, Kristina M.; Galagan, James E.; Bell-Pedersen, Deborah; Freitag, Michael; Cheng, Chao; Loros, Jennifer J.; Dunlap, Jay C.

    2014-01-01

    Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation–based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter–luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level. PMID:25362047

  2. Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells.

    Science.gov (United States)

    Shi, Ming-Zhu; Xie, De-Yu

    2011-04-01

    We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC-MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1-GL3/TT8-PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

  3. Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    Science.gov (United States)

    2013-01-01

    Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also

  4. Spinal cord injury reveals multilineage differentiation of ependymal cells.

    Directory of Open Access Journals (Sweden)

    Konstantinos Meletis

    2008-07-01

    Full Text Available Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.

  5. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

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    Charalampos Rallis

    2014-01-01

    Target of rapamycin complex 1 (TORC1, which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

  6. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

    Directory of Open Access Journals (Sweden)

    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  7. Sexual selection, genetic conflict, selfish genes, and the atypical patterns of gene expression in spermatogenic cells.

    Science.gov (United States)

    Kleene, Kenneth C

    2005-01-01

    This review proposes that the peculiar patterns of gene expression in spermatogenic cells are the consequence of powerful evolutionary forces known as sexual selection. Sexual selection is generally characterized by intense competition of males for females, an enormous variety of the strategies to maximize male reproductive success, exaggerated male traits at all levels of biological organization, co-evolution of sexual traits in males and females, and conflict between the sexual advantage of the male trait and the reproductive fitness of females and the individual fitness of both sexes. In addition, spermatogenesis is afflicted by selfish genes that promote their transmission to progeny while causing deleterious effects. Sexual selection, selfish genes, and genetic conflict provide compelling explanations for many atypical features of gene expression in spermatogenic cells including the gross overexpression of certain mRNAs, transcripts encoding truncated proteins that cannot carry out basic functions of the proteins encoded by the same genes in somatic cells, the large number of gene families containing paralogous genes encoding spermatogenic cell-specific isoforms, the large number of testis-cancer-associated genes that are expressed only in spermatogenic cells and malignant cells, and the overbearing role of Sertoli cells in regulating the number and quality of spermatozoa.

  8. Geometry of the Gene Expression Space of Individual Cells.

    Directory of Open Access Journals (Sweden)

    Yael Korem

    2015-07-01

    Full Text Available There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a

  9. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu

    2010-01-01

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  10. Network Analysis Reveals Putative Genes Affecting Meat Quality in Angus Cattle.

    Science.gov (United States)

    Mateescu, Raluca G; Garrick, Dorian J; Reecy, James M

    2017-01-01

    Improvements in eating satisfaction will benefit consumers and should increase beef demand which is of interest to the beef industry. Tenderness, juiciness, and flavor are major determinants of the palatability of beef and are often used to reflect eating satisfaction. Carcass qualities are used as indicator traits for meat quality, with higher quality grade carcasses expected to relate to more tender and palatable meat. However, meat quality is a complex concept determined by many component traits making interpretation of genome-wide association studies (GWAS) on any one component challenging to interpret. Recent approaches combining traditional GWAS with gene network interactions theory could be more efficient in dissecting the genetic architecture of complex traits. Phenotypic measures of 23 traits reflecting carcass characteristics, components of meat quality, along with mineral and peptide concentrations were used along with Illumina 54k bovine SNP genotypes to derive an annotated gene network associated with meat quality in 2,110 Angus beef cattle. The efficient mixed model association (EMMAX) approach in combination with a genomic relationship matrix was used to directly estimate the associations between 54k SNP genotypes and each of the 23 component traits. Genomic correlated regions were identified by partial correlations which were further used along with an information theory algorithm to derive gene network clusters. Correlated SNP across 23 component traits were subjected to network scoring and visualization software to identify significant SNP. Significant pathways implicated in the meat quality complex through GO term enrichment analysis included angiogenesis, inflammation, transmembrane transporter activity, and receptor activity. These results suggest that network analysis using partial correlations and annotation of significant SNP can reveal the genetic architecture of complex traits and provide novel information regarding biological mechanisms

  11. Network analysis of ChIP-Seq data reveals key genes in prostate cancer.

    Science.gov (United States)

    Zhang, Yu; Huang, Zhen; Zhu, Zhiqiang; Liu, Jianwei; Zheng, Xin; Zhang, Yuhai

    2014-09-03

    Prostate cancer (PC) is the second most common cancer among men in the United States, and it imposes a considerable threat to human health. A deep understanding of its underlying molecular mechanisms is the premise for developing effective targeted therapies. Recently, deep transcriptional sequencing has been used as an effective genomic assay to obtain insights into diseases and may be helpful in the study of PC. In present study, ChIP-Seq data for PC and normal samples were compared, and differential peaks identified, based upon fold changes (with P-values calculated with t-tests). Annotations of these peaks were performed. Protein-protein interaction (PPI) network analysis was performed with BioGRID and constructed with Cytoscape, following which the highly connected genes were screened. We obtained a total of 5,570 differential peaks, including 3,726 differentially enriched peaks in tumor samples and 1,844 differentially enriched peaks in normal samples. There were eight significant regions of the peaks. The intergenic region possessed the highest score (51%), followed by intronic (31%) and exonic (11%) regions. The analysis revealed the top 35 highly connected genes, which comprised 33 differential genes (such as YWHAQ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein and θ polypeptide) from ChIP-Seq data and 2 differential genes retrieved from the PPI network: UBA52 (ubiquitin A-52 residue ribosomal protein fusion product (1) and SUMO2 (SMT3 suppressor of mif two 3 homolog (2) . Our findings regarding potential PC-related genes increase the understanding of PC and provides direction for future research.

  12. Genes affecting β-cell function in type 1 diabetes

    DEFF Research Database (Denmark)

    Fløyel, Tina; Kaur, Simranjeet; Pociot, Flemming

    2015-01-01

    Type 1 diabetes (T1D) is a multifactorial disease resulting from an immune-mediated destruction of the insulin-producing pancreatic β cells. Several environmental and genetic risk factors predispose to the disease. Genome-wide association studies (GWAS) have identified around 50 genetic regions...... that affect the risk of developing T1D, but the disease-causing variants and genes are still largely unknown. In this review, we discuss the current status of T1D susceptibility loci and candidate genes with focus on the β cell. At least 40 % of the genes in the T1D susceptibility loci are expressed in human...... islets and β cells, where they according to recent studies modulate the β-cell response to the immune system. As most of the risk variants map to noncoding regions of the genome, i.e., promoters, enhancers, intergenic regions, and noncoding genes, their possible involvement in T1D pathogenesis as gene...

  13. Genome-Wide RNAi Ionomics Screen Reveals New Genes and Regulation of Human Trace Element Metabolism

    OpenAIRE

    Malinouski, Mikalai; Hasan, Nesrin M.; Zhang, Yan; Seravalli, Javier; Lin, Jie; Avanesov, Andrei; Lutsenko, Svetlana; Gladyshev, Vadim N.

    2017-01-01

    Trace elements are essential for human metabolism and dysregulation of their homeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundam...

  14. Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

    Science.gov (United States)

    Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

    1999-07-01

    The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

  15. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis

    DEFF Research Database (Denmark)

    Størling, Joachim; Pociot, Flemming

    2017-01-01

    (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D...... susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes...

  16. Early Gene Expression in Wounded Human Keratinocytes Revealed by DNA Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Pascal Barbry

    2006-04-01

    Full Text Available Wound healing involves several steps: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical ‘scratch’ method. The two aims of the present study were: (a to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor α-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.

  17. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    International Nuclear Information System (INIS)

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1996-01-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation mediated by UV radiation. In these experiments, we investigated the effects of exposure to 254 nm radiation on cell cycle progression in the rabbit lens epithelial cell line N/N1003A. The RNA was harvested at various times following exposure to UV (254 nm) radiation and analyzed by dot-blot and northern blot hybridizations. These results revealed that during the first 6 h following exposure of the cells to UV, there was, associated with decreasing dose, a decrease in accumulation of transcripts specific for histones H3 and H4 and an increase in the mRNA encoding protein kinase C and β- and γ-actin. Using flow cytometry, we detected an accumulation of cells in G1/S phase of the cell cycle 1 h following exposure to 254 nm radiation. The observed changes in gene expression, especially the decreased accumulation of histone transcripts reported here, may play a role in UV-induced inhibition of cell cycle progression. (Author)

  18. Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

    LENUS (Irish Health Repository)

    Perry, Antoinette S

    2013-04-15

    Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2\\/20) (SFRP1), 64.86% (48\\/74) (SFRP2), 0% (0\\/20) (SFRP4) and 60% (12\\/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6\\/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7\\/69), p < 0.0001) and BPH (11.43% (4\\/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

  19. Lineage-specific evolution of the vertebrate Otopetrin gene family revealed by comparative genomic analyses

    Directory of Open Access Journals (Sweden)

    Ryan Joseph F

    2011-01-01

    Full Text Available Abstract Background Mutations in the Otopetrin 1 gene (Otop1 in mice and fish produce an unusual bilateral vestibular pathology that involves the absence of otoconia without hearing impairment. The encoded protein, Otop1, is the only functionally characterized member of the Otopetrin Domain Protein (ODP family; the extended sequence and structural preservation of ODP proteins in metazoans suggest a conserved functional role. Here, we use the tools of sequence- and cytogenetic-based comparative genomics to study the Otop1 and the Otop2-Otop3 genes and to establish their genomic context in 25 vertebrates. We extend our evolutionary study to include the gene mutated in Usher syndrome (USH subtype 1G (Ush1g, both because of the head-to-tail clustering of Ush1g with Otop2 and because Otop1 and Ush1g mutations result in inner ear phenotypes. Results We established that OTOP1 is the boundary gene of an inversion polymorphism on human chromosome 4p16 that originated in the common human-chimpanzee lineage more than 6 million years ago. Other lineage-specific evolutionary events included a three-fold expansion of the Otop genes in Xenopus tropicalis and of Ush1g in teleostei fish. The tight physical linkage between Otop2 and Ush1g is conserved in all vertebrates. To further understand the functional organization of the Ushg1-Otop2 locus, we deduced a putative map of binding sites for CCCTC-binding factor (CTCF, a mammalian insulator transcription factor, from genome-wide chromatin immunoprecipitation-sequencing (ChIP-seq data in mouse and human embryonic stem (ES cells combined with detection of CTCF-binding motifs. Conclusions The results presented here clarify the evolutionary history of the vertebrate Otop and Ush1g families, and establish a framework for studying the possible interaction(s of Ush1g and Otop in developmental pathways.

  20. Effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells.

    Directory of Open Access Journals (Sweden)

    Lode Godderis

    Full Text Available Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes, we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose-response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti- apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control.

  1. Knockin of Cre Gene at Ins2 Locus Reveals No Cre Activity in Mouse Hypothalamic Neurons.

    Science.gov (United States)

    Li, Ling; Gao, Lin; Wang, Kejia; Ma, Xianhua; Chang, Xusheng; Shi, Jian-Hui; Zhang, Ye; Yin, Kai; Liu, Zhimin; Shi, Yuguang; Xie, Zhifang; Zhang, Weiping J

    2016-02-02

    The recombination efficiency and cell specificity of Cre driver lines are critical for exploring pancreatic β cell biology with the Cre/LoxP approach. Some commonly used Cre lines are based on the short Ins2 promoter fragment and show recombination activity in hypothalamic neurons; however, whether this stems from endogenous Ins2 promoter activity remains controversial. In this study, we generated Ins2-Cre knockin mice with a targeted insertion of IRES-Cre at the Ins2 locus and demonstrated with a cell lineage tracing study that the Ins2 gene is not transcriptionally active in the hypothalamus. The Ins2-Cre driver line displayed robust Cre expression and activity in pancreatic β cells without significant alterations in insulin expression. In the brain, Cre activity was mainly restricted to the choroid plexus, without significant recombination detected in the hippocampus or hypothalamus by the LacZ or fluorescent tdTomato reporters. Furthermore, Ins2-Cre mice exhibited normal glucose tolerance and insulin secretion upon glucose stimulation in vivo. In conclusion, this Ins2-Cre driver line allowed high-fidelity detection of endogenous Ins2 promoter activity in vivo, and the negative activity in the hypothalamus demonstrated that this system is a promising alternative tool for studying β cell biology.

  2. Ultrastructural observations reveal the presence of channels between cork cells.

    Science.gov (United States)

    Teixeira, Rita Teresa; Pereira, Helena

    2009-12-01

    The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

  3. Ontogeny of hepatic energy metabolism genes in mice as revealed by RNA-sequencing.

    Directory of Open Access Journals (Sweden)

    Helen J Renaud

    Full Text Available The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age. The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5-Day 5 (perinatal-enriched, Day 10-Day 20 (pre-weaning-enriched, and Day 25-Day 60 (adolescence/adulthood-enriched. Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty

  4. Spatiotemporal network motif reveals the biological traits of developmental gene regulatory networks in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Kim Man-Sun

    2012-05-01

    Full Text Available Abstract Background Network motifs provided a “conceptual tool” for understanding the functional principles of biological networks, but such motifs have primarily been used to consider static network structures. Static networks, however, cannot be used to reveal time- and region-specific traits of biological systems. To overcome this limitation, we proposed the concept of a “spatiotemporal network motif,” a spatiotemporal sequence of network motifs of sub-networks which are active only at specific time points and body parts. Results On the basis of this concept, we analyzed the developmental gene regulatory network of the Drosophila melanogaster embryo. We identified spatiotemporal network motifs and investigated their distribution pattern in time and space. As a result, we found how key developmental processes are temporally and spatially regulated by the gene network. In particular, we found that nested feedback loops appeared frequently throughout the entire developmental process. From mathematical simulations, we found that mutual inhibition in the nested feedback loops contributes to the formation of spatial expression patterns. Conclusions Taken together, the proposed concept and the simulations can be used to unravel the design principle of developmental gene regulatory networks.

  5. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Science.gov (United States)

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-07-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  6. The Cell Cycle–Regulated Genes of Schizosaccharomyces pombe

    Science.gov (United States)

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know. PMID:15966770

  7. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Anna Oliva

    2005-07-01

    Full Text Available Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast. The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  8. Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

    Science.gov (United States)

    Tsang, Jason C H; Yu, Yong; Burke, Shannon; Buettner, Florian; Wang, Cui; Kolodziejczyk, Aleksandra A; Teichmann, Sarah A; Lu, Liming; Liu, Pentao

    2015-09-21

    Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a (+/+) and Bcl11a (-/-)). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a (-/-) HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a (-/-) HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a (-/-) HSCs are myeloerythroid-restricted and defective in self-renewal. Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.

  9. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  10. ISC1-dependent metabolic adaptation reveals an indispensable role for mitochondria in induction of nuclear genes during the diauxic shift in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kitagaki, Hiroshi; Cowart, L Ashley; Matmati, Nabil; Montefusco, David; Gandy, Jason; de Avalos, Silvia Vaena; Novgorodov, Sergei A; Zheng, Jim; Obeid, Lina M; Hannun, Yusuf A

    2009-04-17

    Growth of Saccharomyces cerevisiae following glucose depletion (the diauxic shift) depends on a profound metabolic adaptation accompanied by a global reprogramming of gene expression. In this study, we provide evidence for a heretofore unsuspected role for Isc1p in mediating this reprogramming. Initial studies revealed that yeast cells deleted in ISC1, the gene encoding inositol sphingolipid phospholipase C, which resides in mitochondria in the post-diauxic phase, showed defective aerobic respiration in the post-diauxic phase but retained normal intrinsic mitochondrial functions, including intact mitochondrial DNA, normal oxygen consumption, and normal mitochondrial polarization. Microarray analysis revealed that the Deltaisc1 strain failed to up-regulate genes required for nonfermentable carbon source metabolism during the diauxic shift, thus suggesting a mechanism for the defective supply of respiratory substrates into mitochondria in the post-diauxic phase. This defect in regulating nuclear gene induction in response to a defect in a mitochondrial enzyme raised the possibility that mitochondria may initiate diauxic shift-associated regulation of nucleus-encoded genes. This was established by demonstrating that in respiratory-deficient petite cells these genes failed to be up-regulated across the diauxic shift in a manner similar to the Deltaisc1 strain. Isc1p- and mitochondrial function-dependent genes significantly overlapped with Adr1p-, Snf1p-, and Cat8p-dependent genes, suggesting some functional link among these factors. However, the retrograde response was not activated in Deltaisc1, suggesting that the response of Deltaisc1 cannot be simply attributed to mitochondrial dysfunction. These results suggest a novel role for Isc1p in allowing the reprogramming of gene expression during the transition from anaerobic to aerobic metabolism.

  11. Redistribution of cell cycle by arsenic trioxide is associated with demethylation and expression changes of cell cycle related genes in acute promyelocytic leukemia cell line (NB4).

    Science.gov (United States)

    Hassani, Saeed; Khaleghian, Ali; Ahmadian, Shahin; Alizadeh, Shaban; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

    2018-01-01

    PML-RARα perturbs the normal epigenetic setting, which is essential to oncogenic transformation in acute promyelocytic leukemia (APL). Transcription induction and recruitment of DNA methyltransferases (DNMTs) by PML-RARα and subsequent hypermethylation are components of this perturbation. Arsenic trioxide (ATO), an important drug in APL therapy, concurrent with degradation of PML-RARα induces cell cycle change and apoptosis. How ATO causes cell cycle alteration has remained largely unexplained. Here, we investigated DNA methylation patterns of cell cycle regulatory genes promoters, the effects of ATO on the methylated genes and cell cycle distribution in an APL cell line, NB4. Analysis of promoter methylation status of 22 cell cycle related genes in NB4 revealed that CCND1, CCNE1, CCNF, CDKN1A, GADD45α, and RBL1 genes were methylated 60.7, 84.6, 58.6, 8.7, 33.4, and 73.7%, respectively, that after treatment with 2 μM ATO for 48 h, turn into 0.6, 13.8, 0.1, 6.6, 10.7, and 54.5% methylated. ATO significantly reduced the expression of DNMT1, 3A, and 3B. ATO induced the expression of CCND1, CCNE1, and GADD45α genes, suppressed the expression of CCNF and CDKN1A genes, which were consistent with decreased number of cells in G1 and S phases and increased number of cells in G2/M phase. In conclusion, demethylation and alteration in the expression level of the cell cycle related genes may be possible mechanisms in ATO-induced cell cycle arrest in APL cells. It may suggest that ATO by demethylation of CCND1 and CCNE1 and their transcriptional activation accelerates G1 and S transition into the G2/M cell cycle arrest.

  12. Genome Binding and Gene Regulation by Stem Cell Transcription Factors

    NARCIS (Netherlands)

    J.H. Brandsma (Johan)

    2016-01-01

    markdownabstractNearly all cells of an individual organism contain the same genome. However, each cell type transcribes a different set of genes due to the presence of different sets of cell type-specific transcription factors. Such transcription factors bind to regulatory regions such as promoters

  13. Bone marrow transplantations to study gene function in hematopoietic cells

    NARCIS (Netherlands)

    de Winther, Menno P. J.; Heeringa, Peter

    2011-01-01

    Immune cells are derived from hematopoietic stem cells in the bone marrow. Experimental replacement of bone marrow offers the unique possibility to replace immune cells, to study gene function in mouse models of disease. Over the past decades, this technique has been used extensively to study, for

  14. Biophysical characteristics reveal neural stem cell differentiation potential.

    Directory of Open Access Journals (Sweden)

    Fatima H Labeed

    Full Text Available Distinguishing human neural stem/progenitor cell (huNSPC populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.We used dielectrophoresis (DEP to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

  15. Identification of Hematopoietic Stem Cell Engraftment Genes in Gene Therapy Studies.

    Science.gov (United States)

    Powers, John M; Trobridge, Grant D

    2013-09-01

    Hematopoietic stem cell (HSC) therapy using replication-incompetent retroviral vectors is a promising approach to provide life-long correction for genetic defects. HSC gene therapy clinical studies have resulted in functional cures for several diseases, but in some studies clonal expansion or leukemia has occurred. This is due to the dyregulation of endogenous host gene expression from vector provirus insertional mutagenesis. Insertional mutagenesis screens using replicating retroviruses have been used extensively to identify genes that influence oncogenesis. However, retroviral mutagenesis screens can also be used to determine the role of genes in biological processes such as stem cell engraftment. The aim of this review is to describe the potential for vector insertion site data from gene therapy studies to provide novel insights into mechanisms of HSC engraftment. In HSC gene therapy studies dysregulation of host genes by replication-incompetent vector proviruses may lead to enrichment of repopulating clones with vector integrants near genes that influence engraftment. Thus, data from HSC gene therapy studies can be used to identify novel candidate engraftment genes. As HSC gene therapy use continues to expand, the vector insertion site data collected will be of great interest to help identify novel engraftment genes and may ultimately lead to new therapies to improve engraftment.

  16. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

    Science.gov (United States)

    Soucie, Erinn L; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J-C; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H

    2016-02-12

    Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. Copyright © 2016, American Association for the Advancement of Science.

  17. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

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    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  18. Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Putnik, Milica, E-mail: milica.putnik@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden); Zhao, Chunyan, E-mail: chunyan.zhao@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden); Gustafsson, Jan-Ake, E-mail: jan-ake.gustafsson@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden); Department of Biology and Biochemistry, Science and Engineering Research Center Bldg, University of Houston, Houston, TX 77204-5056 (United States); Dahlman-Wright, Karin, E-mail: karin.dahlman-wright@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Estrogen signaling and demethylation can both control gene expression in breast cancers. Black-Right-Pointing-Pointer Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. Black-Right-Pointing-Pointer 137 genes are influenced by both 17{beta}-estradiol and demethylating agent 5-aza-2 Prime -deoxycytidine. Black-Right-Pointing-Pointer A set of genes is identified as targets of both estrogen signaling and demethylation. Black-Right-Pointing-Pointer There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17{beta}-estradiol (E2) and a demethylating agent 5-aza-2 Prime -deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of

  19. Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Putnik, Milica; Zhao, Chunyan; Gustafsson, Jan-Åke; Dahlman-Wright, Karin

    2012-01-01

    Highlights: ► Estrogen signaling and demethylation can both control gene expression in breast cancers. ► Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. ► 137 genes are influenced by both 17β-estradiol and demethylating agent 5-aza-2′-deoxycytidine. ► A set of genes is identified as targets of both estrogen signaling and demethylation. ► There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17β-estradiol (E2) and a demethylating agent 5-aza-2′-deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment

  20. Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Davis Paul

    2006-12-01

    Full Text Available Abstract Background Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. Aim This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1 and human glossal squamous cell carcinoma cell line (SCC25. Methods HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. Results HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p Conclusion We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control.

  1. Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells

    International Nuclear Information System (INIS)

    Anisowicz, A.; Bardwell, L.; Sager, R.

    1987-01-01

    Comparison by subtractive hybridization of mRNAs revealed a moderately abundant message in highly tumorigenic CHEF/16 cells present at very low levels in closely related nontumorigenic CHEF/18 cells. After cloning and sequencing the corresponding cDNA, computer comparison showed closest homology with the human connective tissue-activating peptide III (CTAP III). The human tumor cell cDNA hybridizing with the Chinese hamster clone was isolated, sequenced, and found to have closer similarity to the Chinese hamster gene than to CTAP III. Thus, the cloned cDNAs from Chinese hamster and human cells represent a different gene, named gro. Studies of its transcriptional regulation have shown that expression is tightly regulated by growth status in normal Chinese hamster and human cells and relaxed in the tumorigenic cells so far examined

  2. Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.

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    Christian Frezza

    Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

  3. Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

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    Schilling William P

    2002-01-01

    Full Text Available Abstract Background Maitotoxin (MTX initiates cell death by sequentially activating 1 Ca2+ influx via non-selective cation channels, 2 uptake of vital dyes via formation of large pores, and 3 release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. Results Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. Conclusions Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms.

  4. Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes

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    Deyholos Michael K

    2006-10-01

    Full Text Available Abstract Background Roots are an attractive system for genomic and post-genomic studies of NaCl responses, due to their primary importance to agriculture, and because of their relative structural and biochemical simplicity. Excellent genomic resources have been established for the study of Arabidopsis roots, however, a comprehensive microarray analysis of the root transcriptome following NaCl exposure is required to further understand plant responses to abiotic stress and facilitate future, systems-based analyses of the underlying regulatory networks. Results We used microarrays of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes to identify root transcripts that changed in relative abundance following 6 h, 24 h, or 48 h of hydroponic exposure to 150 mM NaCl. Enrichment analysis identified groups of structurally or functionally related genes whose members were statistically over-represented among up- or down-regulated transcripts. Our results are consistent with generally observed stress response themes, and highlight potentially important roles for underappreciated gene families, including: several groups of transporters (e.g. MATE, LeOPT1-like; signalling molecules (e.g. PERK kinases, MLO-like receptors, carbohydrate active enzymes (e.g. XTH18, transcription factors (e.g. members of ZIM, WRKY, NAC, and other proteins (e.g. 4CL-like, COMT-like, LOB-Class 1. We verified the NaCl-inducible expression of selected transcription factors and other genes by qRT-PCR. Conclusion Micorarray profiling of NaCl-treated Arabidopsis roots revealed dynamic changes in transcript abundance for at least 20% of the genome, including hundreds of transcription factors, kinases/phosphatases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this stress response. Our identification of these transcriptional responses, and groups of evolutionarily related genes with either similar or divergent

  5. Transcriptomic analyses reveal novel genes with sexually dimorphic expression in the zebrafish gonad and brain.

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    Rajini Sreenivasan

    Full Text Available BACKGROUND: Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs, 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and 'rest-of-body' from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4 has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. CONCLUSIONS/SIGNIFICANCE: We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar

  6. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

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    Shewmaker Christine K

    2010-10-01

    Full Text Available Abstract Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD 2 and fatty acid elongase (FAE 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general

  7. Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

    Science.gov (United States)

    Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

    2009-01-01

    The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-??